这项研究得到了美国国立卫生研究院的支持, 该计划的机构间协议 AOD13015-001。我们要感谢斯蒂芬妮 Froberg 和彼得赫斯特在视频制作方面的努力和专长。

1. 细胞培养维护
<ol><li>生长细胞线 SV40-HCEC 在37°c, 5% CO2, 90% 湿度 DMEM F-12 与15% 胎牛血清 (血清), 1% l-谷氨酰胺, 10 µg/升表皮生长因子 (EGF), 5 毫克/升胰岛素。</li>
	<li>通过细胞线每3到4天 (取决于播种密度), 以确保融合从来没有超过80% 在文化维护。</li>
	<li>使用分离溶液 (每150厘米2瓶) 将细胞从烧瓶中取出, 并在37摄氏度处孵化不超过8分钟 (14 毫升溶液)。</li>
	<li>用相同容积的细胞培养培养基中和分离液, 在 160 x g 处离心7分钟, 将50毫升锥形管中的细胞颗粒化。</li>
	<li>在培养基中重新悬浮细胞颗粒 (每150厘米2瓶20毫升)。</li>
	<li>使用自动手持单元格计数器和新烧瓶中的种子进行计数, 使其在不超过80% 融合的情况下生长3到4天。</li>
</ol>2. 实验用平板细胞
<ol><li>用相衬光镜检查 SV40-HCECs 的烧瓶, 以确保融合小于 80%, 并评估一般细胞的健康状况。</li>
	<li>分离, 颗粒, 并用重悬和计数 SV40-HCECs 从烧瓶中所述的步骤1.3 至1.6 的细胞培养维护。使用含有0.5 µg/毫升松 (HCORT 培养基) 的 SV40-HCEC 培养基并用重悬细胞。</li>
	<li>在一次性培养基瓶中, 在预热后的 HCORT 培养基中, 准备悬浮 SV40-HCECs 17857 细胞/毫升。<ol>
<li>为要播种的盘子数量准备足够数量的细胞悬浮。</li>
		<li>种子至少 14 x 96-井板与细胞为每个 siRNA 库板块进行研究。</li>
	</ol>
</li>
	<li>旋转瓶子均匀悬浮细胞, 将足够数量的细胞悬浮液倒入一个自动液体处理器的轨道振动筛巢中的储层, 并将含有细胞悬浮物的瓶子贮存在37摄氏度暖板上。过程。</li>
	<li>使用自动液体处理程序将单元格添加到每井1000年细胞密度 (5000 个细胞/厘米2) 的板上, 在70µL 的96井板中。对所有步骤使用50µL/秒吹打速度。<ol>
<li>在播种过程中不断地运行 100 rpm 的轨道振动筛。</li>
		<li>使用自动液体处理器将电池悬浮液混合三次 (140 µL 混合容积)。</li>
		<li>吸入140µL 细胞悬浮液, 并将70µL 放入两个细胞培养板的每一个井中。</li>
		<li>重复步骤2.5.3 和 2.5.4, 直到所有的盘子都被细胞所播种。</li>
		<li>在播种过程中需要重新填充细胞悬浮液。</li>
	</ol>
</li>
	<li>在将盘子播种后, 将它们从自动液体处理器中取出, 在室温下孵化30分钟, 然后将它们转移到细胞培养孵化器。</li>
	<li>第二天早上, 用一个自动成像系统在细胞培养孵化器中评估每块井的细胞密度, 并排除研究中没有15% 和22% 汇合的内部井的任何板块。</li>
</ol>3. 染细胞与 siRNA
<ol><li>从供应商处获取 siRNA 库板, 预配置为包含每板 80 siRNA 目标 (见图 4), 列1和12为空。</li>
	<li>根据制造商的说明19, 将 siRNA 库板与 siRNA 缓冲器重建为 2 pmol/µL 的最终浓度。</li>
	<li>染24小时后, 4 pmol/井 siRNA 和0.3 µL/井转染试剂。按照转染试剂制造商的协议执行所有步骤 (参见图 4中的板布局)20。<ol>
<li>使用自动液体处理程序执行所有 transfections, 并对所有步骤使用25µL/秒吹打速度。使用预配置的提示框只处理将被转染的水井。</li>
		<li>使用库板的上半部分染一组六个被称为 "顶部板集" 的复制板 (每 siRNA 六个复制, n=6)。</li>
		<li>使用库板的下半部分染不同的六个复制板, 称为 "底板集" (每 siRNA 六个复制, n=6)。</li>
		<li>使用 "全盘集" 一词来描述已转染图书馆 siRNA 的12个细胞板块。</li>
		<li>染井 B2-B6 所有板块的全板组与负池 siRNA 后, 所有顶部和底部板集已转染图书馆 siRNA。</li>
		<li>也染井 B2-B6 另外两个板材, 不接收图书馆 siRNA 并且将担当未曝光的控制 (一为顶部板集和一个为底部板设置), 与消极水池 siRNA。</li>
	</ol>
</li>
	<li>在细胞培养孵化器中孵化所有细胞板块4小时后, 所有的转染混合添加和混合通过温和的攻丝每小时。</li>
	<li>使用自动液体处理程序冲洗两次板的所有水井, 预热 HCORT 介质稀释1:5 在 PBS。对所有步骤使用50µL/秒吹打速度。<ol>
<li>从每个井中抽出100µL, 并将其丢弃在废物库中。</li>
		<li>添加到每个井100µL/井预热 HCORT 介质稀释1:5 在 PBS, 这是包含在一个不同的水库在足够数量的板块数量。</li>
		<li>对每个盘子重复步骤3.5.1 和3.5.2。<ol>
<li>根据需要清空废物库。</li>
			<li>根据需要, 在 PBS 中 HCORT 介质稀释1:5。</li>
		</ol>
</li>
		<li>从每个井中抽出100µL, 并将其丢弃在废物库中。</li>
	</ol>
</li>
	<li>Refeed 所有井的板材与100µL 或好预热的 HCORT 媒介, 在一个不同的水库包含在一个充足的容量介质为板材的数量。<ol>
<li>在需要的介质中重新填充储层。</li>
	</ol>
</li>
	<li>将所有板块的井 C2-C6 为未转染的控制。</li>
	<li>保持水井 B7-B11 和 C7-C11 的所有板块未转染, 用作药物和车辆控制毒物暴露。</li>
</ol>4. 第二天 Refeed 细胞
<ol><li>使用自动液体处理程序 refeed 所有油井的板材。对所有步骤使用50µL/秒吹打速度。<ol>
<li>从每个井中抽出100µL, 并将其丢弃在废物库中。</li>
		<li>Refeed 每个井与100µL/井预热 HCORT 介质, 这是包含在不同的水库, 并有足够的介质数量的板块是 refed。</li>
		<li>根据需要重复步骤4.1.1 和 4.1.2 refeed 所有细胞板。<ol>
<li>根据需要清空废物库。</li>
			<li>在需要的介质中重新填充储层。</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. 积极控制加法
<ol><li>转染两天后, 暴露前两个小时, 准备一个62.5 µM 解决方案的积极控制小豆蔻明在 HCORT 介质中用于高放暴露和增加 B 排的8行稀释水库。<ol>
<li>对于 CP 曝光, 准备和使用 SKF 86002 的25µM 溶液。</li>
		<li>准备一个足够数量的正控制量, 以测试的板材数量。</li>
	</ol>
</li>
	<li>在 HCORT 介质 (车辆控制) 上准备0.625% 的亚甲基亚砜溶液, 并加入同一储层的 C 排。<ol>
<li>对于 CP 暴露, 准备和使用0.5% 溶液的亚砜。</li>
		<li>准备足够的车辆控制量的车牌数量的测试。</li>
	</ol>
</li>
	<li>使用自动液体处理程序添加正面和车辆控制, 以井 B7-B11 和 C7-C11 的每个细胞板, 并使用50µL/秒吹打速度的所有步骤。使用预先配置的提示框, 只处理将接收到正面和车辆控制的水井。<ol>
<li>从 B7-B11 和 C7-C11 中去除每个细胞板的10µL 介质, 将其丢弃到8排稀释油藏的 G 和 H 行中。</li>
		<li>将正面和车辆控制的10µL 转移到井中, 混合3次。</li>
	</ol>
</li>
	<li>把这些电池板还给孵化器。</li>
	<li>重复步骤5.3 至 5.4, 直到所有电池板都收到正面和车辆控制。</li>
</ol>6. 培养细胞的 HF 暴露
注意: 高放具有腐蚀性和剧毒。
<ol><li>使用双腈手套, 实验室涂层, 一次性聚乙烯套筒保护器和安全眼镜, 在化学油烟机中执行所有化学接触操作。<ol>
<li>获取高放作为48% 解决方案。</li>
		<li>稀释高放到1% 与超纯水和存储在5毫升整除数10毫升厚壁聚乙烯瓶, 以提高安全性。</li>
		<li>在危险废物流处置之前, 对与高放和任何残留液体高放有2.5% 葡萄糖酸钙接触过的吸管提示和水库进行净化。</li>
	</ol>
</li>
	<li>对于每个 siRNA 库板块, 请在150毫升瓶中加入288µL 1% 高放到80毫升预热 HCORT 培养基, 准备一个0.0036% 高放介质溶液。<ol>
<li>旋流瓶和孵化稀释高放在37°c 孵化器在化学油烟机10分钟。</li>
	</ol>
</li>
	<li>通过旋转瓶子, 再将高放介质溶液混合, 并将高放介质溶液添加到板上较暖和的试剂库中。</li>
	<li>与两名技术员一起工作, 进行接触。<ol>
<li>让技术员在正确的100µL 从每一个细胞板的内部井使用12通道 pipettor, 然后把盘子传给技术员在左边。</li>
		<li>在左边的技术员增加100µL 每井高放中等解答。</li>
		<li>重复步骤6.4.1 和6.4.2 所有的细胞板, 将暴露于毒物。</li>
		<li>还重复步骤6.4.1 和6.4.2 未曝光的控制板, 利用新鲜的 HCORT 介质, 而不是高放介质溶液。</li>
	</ol>
</li>
	<li>将板块放在化学油烟机孵化器中20分钟, 然后将它们归还细胞培养孵化器。</li>
	<li>重复正面控制加法步骤 (步骤5.1 至 5.5), 如前所示, 一旦所有板块被暴露, 并返回到细胞培养孵化器。</li>
</ol>7. 培养细胞的 CP 暴露
注意: CP 是剧毒和刺激性。
<ol><li>使用双腈手套, 实验室涂层, 一次性聚乙烯套筒保护器和安全眼镜, 在化学油烟机中执行所有化学接触操作。<ol>
<li>获取 CP。</li>
		<li>稀释 CP 到5% 在亚砜和存储在10毫升整除数10毫升闪烁瓶, 以提高安全性。</li>
		<li>在危险废物流中处置之前, 用2.5% 亚硫酸氢钠与 cp 和任何残留液体 cp 接触的吸管尖和储层进行净化。</li>
	</ol>
</li>
	<li>准备足够数量的预热 HCORT 培养基, 含1x 支笔/链球菌和预热的 PBS, 以供暴露的板材数量。</li>
	<li>对于每个顶板设置或底部板集, 添加8.04 µL 5% CP 在亚砜到50毫升整除预热 HCORT 培养基和混合良好的最终 CP 浓度0.0008%。将管子紧紧地盖住, 在化学油烟机的37°c 孵化器中孵化出1小时的溶液。<ol>
<li>时间增加 cp 到50毫升整除数的介质, 使每个暴露的顶板设置和底部板集收到毒物恰好1小时后, CP 被添加到50毫升的整除培养基。</li>
	</ol>
</li>
	<li>从细胞培养孵化器中取出一个顶板和1个未曝光的控制板, 并将它们放在靠近孵化期结束的化学油烟罩中。</li>
	<li>混合 CP 溶液通过反转管, 然后醒酒它到试剂库在1小时的孵化期结束。</li>
	<li>与两名技术员一起工作, 进行接触。<ol>
<li>让技术员在正确的100µL 从每一个细胞板的内部井使用12通道 pipettor, 然后把盘子传给技术员在左边。</li>
		<li>让技术人员在左侧添加100µL 的 CP 介质解决方案。</li>
		<li>重复步骤7.6.1 和7.6.2 的所有细胞板块的顶部板设置要暴露于毒物。</li>
		<li>还重复步骤7.6.1 和7.6.2 未曝光的控制板, 利用新鲜的 HCORT 介质, 而不是 CP 介质溶液。</li>
	</ol>
</li>
	<li>将盘子放在化学油烟机的37摄氏度孵化器中, 10 分钟。</li>
	<li>添加足够数量的 PBS 和 HCORT 培养基, 含有1x 笔/链球菌, 以试剂库近10分钟孵化期结束。</li>
	<li>使用12通道 pipettor 从单元格中取出 CP 解决方案, 并在10分钟孵化期结束时将其替换为 PBS 的100µL。</li>
	<li>立即从细胞板中取出 PBS, 用含有1x 笔/链球菌的 HCORT 介质中的100µL 来替换它。</li>
	<li>对于未曝光的控制板, 也重复步骤7.9 和7.10。</li>
	<li>将电池板返回标准细胞培养孵化器。</li>
	<li>对底部板集重复步骤7.3 到7.12。</li>
	<li>重复的积极控制添加步骤 (步骤5.1 至 5.5), 如前所述, 一旦所有板块已经暴露, 并返回到细胞培养孵化器。</li>
</ol>8. 样品收集和细胞活性测定
<ol><li>暴露后二十四小时, 在 PBS 中准备0.5 毫克/毫升 MTT 基板的溶液, 含10克/升葡萄糖, 暖至37摄氏度21。为每个板材准备10毫升的基板进行化验。</li>
	<li>对于要测定的板材数量, 在自动液体处理程序中添加足够数量的 MTT 基板溶液到储层上。</li>
	<li>使用自动液体处理程序收集样本, 并添加 MTT 基板使用50µL/秒吹打速度为所有步骤。进行预配置提示框, 以解决只有那些水井要化验。<ol>
<li>从细胞板内部60井中抽出95µL, 并将42.5 µL 存入两个384阱的储存板中。</li>
		<li>立即增加100µL 每井的 MTT 基质溶液的细胞板块, 并孵化他们在37°c 在细胞培养孵化器1.5 小时。</li>
		<li>根据需要, 用 MTT 衬底溶液重新填充储层。</li>
	</ol>
</li>
	<li>在细胞培养孵化器中孵育37摄氏度的细胞板块, 1 小时。</li>
	<li>密封384井存储板, 并存储在-80 °c, 以供后续分析。</li>
	<li>对所有顶部和底部板集以及相关的未公开控件重复步骤8.3 到8.5。<ol>
<li>在复制之间重复使用提示 (如果需要), 但在添加 MTT 基板解决方案和在板集之间切换时清洗它们。</li>
	</ol>
</li>
	<li>在1小时孵化后, 在自动液体处理程序中添加一个足够数量的亚砜到一个水库。</li>
	<li>使用自动液体处理程序添加亚砜, 并对所有步骤使用50µL/秒吹打速度。<ol>
<li>从每个单元的内部井中抽出100µL, 并将 MTT 基板溶液丢弃在废弃的储层中。<ol>
<li>根据需要清空废物库。</li>
		</ol>
</li>
		<li>覆盖每个井与100µL 亚砜。<ol>
<li>根据需要重新填充亚砜水库。</li>
		</ol>
</li>
	</ol>
</li>
	<li>在平板振动筛上摇动盘子3分钟。</li>
	<li>用平板光谱仪测量570和 690 nm 的吸光度。</li>
	<li>通过将暴露的吸光度值除以未曝光的控件, 减去背景并计算% 细胞的生存能力。使用平均未公开的负池 siRNA 控制来计算所有目标的% 细胞生存能力。</li>
</ol>9. 测定细胞培养中的 IL-8 浓度上清液
<ol><li>根据制造商的说明22, 测量细胞培养上清液中 IL-8 的浓度。创建一个检测板布局, 以适应样品和标准曲线。</li>
	<li>从-80 °c 冷藏柜中取出384井的储物板, 室温解冻, 并简单地将板材离心在油井底部收集样品。</li>
	<li>对于要运行的检测板的数量, 在试剂盒中制作足够数量的 anti-IL-8 受体珠和生物素化 anti-IL-8 抗体。使用50µL 的 anti-IL8 受体珠和50µL 生物素化 anti-IL8 抗体的比例9.9 毫升的检测缓冲器。<ol>
<li>使用多通道 pipettor, 添加珠/抗体混合物的黑色384井存储板。<ol>
<li>根据检测板布局, 将受体珠/抗体添加到用于样品和标准曲线的井中。</li>
			<li>为要运行的检测板数量添加足够数量的每井容积。</li>
		</ol>
</li>
	</ol>
</li>
	<li>用含有354µM 氯化钠的细胞培养培养基重建 IL-8 标准到 1000 pg/毫升。为要运行的检测板数量制作足够的容积。</li>
	<li>制作标准曲线的八个双重串行稀释。</li>
	<li>根据检测板的布局, 将标准曲线加成三个, 在黑色384井存储板的适当井中加入。为要运行的检测板数量添加足够数量的每井容积。<ol>
<li>同样, 添加含有354µM 氯化钠的细胞培养培养基, 不 IL-8 背景测量。</li>
	</ol>
</li>
	<li>使用自动液体处理程序来执行化验。使用预配置的提示框只处理由检测板布局指定的水井。对所有步骤使用50µL/秒吹打速度。<ol>
<li>将受体珠抗体混合物的8µL 转移到白色384井浅层测井板中。<ol>
<li>根据检测板的布局, 仅添加到指定样品和标准曲线的井中。</li>
		</ol>
</li>
		<li>根据检测板布局, 将标准曲线的2µL 转移到浅层试片的适当井中。</li>
		<li>准备一个3.54 米氯化钠溶液, 并增加一个足够的数量的板块将被化验到一个浅水库上的自动化液体处理。</li>
		<li>通过将氯化钠溶液的4.5 µL 转移到黑色384井存储板中的样品, 调整样品的盐浓度, 使其与标准曲线相匹配。</li>
		<li>将样品混合三次, 使用30µL 的混合容积, 并根据检测板的布局将样品的2µL 转移到白色浅层试片中。<ol>
<li>在样品板之间更改提示。</li>
		</ol>
</li>
		<li>室温下在黑暗中孵化1小时。</li>
		<li>对于要运行的检测板数量, 在检测缓冲区中制作足够数量的受体珠子。使用200µL 的二次受体珠的比例到12.3 毫升的化验缓冲器。</li>
		<li>使用多通道 pipettor, 将二次珠添加到黑色的384井存储板上。<ol>
<li>根据检测板布局, 将二次珠添加到指定用于样品和标准曲线的井中。</li>
			<li>为要运行的检测板数量添加足够数量的每井容积。</li>
		</ol>
</li>
		<li>使用自动液体处理程序, 将二次珠的10µL 转移到白色384井浅层测井板上。<ol>
<li>只添加到接受样品和标准曲线的水井, 根据化验板布局, 并清洗每个板块之间的提示。</li>
		</ol>
</li>
		<li>在黑暗中的室温下孵化一个小时。</li>
	</ol>
</li>
	<li>用透明的印版密封和扫描用与该检测相兼容的平板读卡器来密封检测板。使用0.2 毫米的板和探测器之间的距离, 180 毫秒的励磁时间, 和550毫秒测量时间的扫描。</li>
	<li>将原始数据从 IL-8 检测导入电子表格。</li>
	<li>使用 IL-8 检测标准曲线的自动曲线拟合, 然后将原始数据转换为每个样本的 pg/mL。</li>
</ol>

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作者没有什么可透露的。
免责声明: 本文所表达的观点是作者的意见, 不反映陆军部、国防部或美国政府的官方政策。这项研究得到了 NIH/NIAID 与 USAMRICD 之间的一项跨部门协议的支持, 部分是由橡树管理的美国陆军医学研究所的研究生研究参与计划所提供的。通过美国能源部和 USAMRMC 之间的跨部门协议, 为科学和教育脊研究所。

在此, 我们描述了我们的方法和结果, 开发的高通量角膜上皮细胞筛选模型的研究 HF 和 CP 损伤。我们还从主 siRNA 屏幕上显示 HF 损伤的结果。研究 TIC 损伤的高温超导模型的发展面临着许多挑战。我们在文献中发现的与 HF、高放或 CP 在细胞培养模型中的研究有关的方法是没有帮助的。大多数的离体研究氟离子涉及口服细胞和使用氟化钠, 而不是 HF 或高放 (一些例子, 见28,29)。Pesonen 了支气管上皮细胞体外暴露于 CP 的方法. 30. 最终, 我们在有关 HF、高放或 CP 的高温超导研究的文献中没有发现具体的方法, 而与高温超导研究这些毒物有关的绝大多数挑战和变数都需要发展。特别注意的是, 为了实现高温超导研究所必需的高度曝光一致性和许多筛板。
遇到的一种挑战是与毒物在细胞培养系统中的应用有关。有些有毒物质要么是反应性的, 要么是不稳定的水溶液。我们选择用于研究的毒物不被视为有这些不稳定问题。一项关于 photohydrolysis 的农业研究表明, 0.001 米溶液中 cp 在水中的半衰期为31.1 小时, 当暴露在1100莱克丝的氙光源 (阴天的阳光强度) 10 天, 12 小时每天31。当溶液在黑暗中储存时, 同样的10天内没有可测量的水解。高放的质子和氟离子在水中明显稳定。然而, 在细胞培养培养基中的毒物稀释方面还有其他的稳定性考虑。我们发现, 在某种程度上, 当有毒物质在细胞培养基中首次稀释后, 在短暂的一段时间后稳定下来时, 细胞毒性的初步改变。我们认为这是由于毒物结合或与培养基中的成分的络合。细胞培养基中含有钙、镁、蛋白质等已知与氟化物离子32、33的相互作用。CP 是已知的氧化反应与生物硫醇, 但它也可能绑定 DNA, 蛋白质和其他亲核试剂34,35。一旦这些反应和相互作用完成或达到平衡, 可用毒物的数量稳定, 因而明显的细胞毒性稳定。我们初步探讨了使用盐水和水稀释的有毒物质用于接触的工作储存, 以避免有毒的相互作用, 在工作稀释中的成分。盐水的使用也将保留高放损伤的酸组分为媒介交换进行的暴露。我们发现, 在水和盐水稀释中对毒物的细胞死亡反应比使用培养基交换的细胞培养培养基稀释的方法更具变数 (未显示数据)。对于高放的尖峰曝光法, 可变性可能是由于在油井中的穗分散不一致, 导致细胞目标与培养基成分争夺可用氟化物离子。高放盐水稀释中高放暴露的变异原因是完全不清楚的。在任何情况下, 使用盐水稀释高放的暴露方法被放弃了, 并没有探索为 CP。我们高放稀释在培养基中的毒性可能与氟化钠密切相关, 因为细胞介质在我们的高放稀释中缓冲自由质子。
我们遇到的其他挑战与改进高温超导细胞培养模型有关。我们发现有必要修改细胞培养培养基成分的实验, 特别是氢化可的松和抗生素。在暴露研究期间, 我们使用了低剂量的氢化可松 (0.5 µg/毫升), 以抑制在天真的 SV40-HCEC 中出现的细胞因子产生的偶然激增。这种低量的氢化物没有对细胞对毒物的反应产生不利影响, 并且低于通常用来抑制培养细胞分泌细胞因子的水平, 以应对兴奋剂36,37。为什么天真的 SV40-HCEC 细胞偶尔产生过量细胞因子的确切原因是未知的, 但它可能是对细胞传代和电镀过程中发生的小的压力变敏感。许多实验室常规地包括细胞培养维护培养基中的抗生素, 但一些抗生素, 特别是四环素, 已显示出抗炎作用的培养细胞, 包括角膜上皮细胞38。一般来说, 我们的实验室避免使用抗生素, 只要可能, 因为这些可能会混淆研究。我们还研究了在细胞中多通道的表型漂移的文化。这是常见的永生细胞接受遗传和/或表型漂移超过多个通道, 如果不维持在指数阶段, 如果允许达到融合, 或如果暴露于某些环境压力源39,40 ,41。因此, 在高吞吐量屏幕中, 单元格线得到正确维护, 并且使用维护已知表型响应的单元格通道数完成屏幕是非常关键的。多井板块边缘效应是多井板块外围井中生长的细胞通常不响应相同或与内部井42生长的细胞一致的现象。有统计演习, 如中位波兰语, 可用于最小化边缘效应的数据集43。但是, 如果在一个特定的目标或控件上总是在边缘井中进行测试, 则不存在能够消除边缘效应的统计练习, 并且在复制之间改变板块布局以移动目标或控制边缘井的距离不是后勤上的。实用或成本效益。我们认为, 在基于细胞的高温超导检测中包括使用边缘井的决定应谨慎进行。我们发现有必要消除在边缘井中生长的细胞用于控制和目标的使用。
采用高温超导细胞模型和体外暴露方法, 成功完成了3120个靶的主 siRNA 筛。SSMD 被用于命中选择, 因为它是专门用来测量一个控制和一个 siRNA27之间的差异的大小。我们对细胞生存能力的研究结果表明, 在 SSMD 和褶皱变化之间的双手电筒图中, 几乎所有的目标都存在线性关系。出于这个原因, 我们使用了 SSMD 的单一标准来对此端点进行命中选择。IL-8 数据不同的是, 有些目标有微弱但一致的效果, 而另一些则表现出强烈但不一致的影响。因此, 我们同时使用了折变和 SSMD 对此端点的命中选择条件。由于下限介于1和 1.645 (或-1 和-1.65) 之间, 因此选择了 SSMD 值, 用于对细胞生存能力和 IL-8 数据进行命中选择, 这是因为它实现了低错误发现和非发现率44。在细胞生存能力和 IL-8 数据中都有一些目标被击中。在这些情况下, 将优先考虑纳入反褶积库, 使这些目标在改善总体健康 (改善生存能力和减少 IL-8) 或加重伤害 (增加细胞死亡和增加 IL-8)。总体上, 我们选择了250个目标之间的细胞生存能力和 IL-8 端点, 以弥补反褶积库的 HF 损伤。体外CP 损伤的主屏幕正在进行中。
总之, 我们已经开发出适合于高频和 CP 对眼部损伤的高温超导研究的暴露和培养条件。对暴露和培养条件下的几个变量进行了细化和优化, 并通过 Z 因子分析验证了模型的正确性。通过在 HF 模型中完成 siRNA 库的主屏幕, 我们进一步证明了这些模型的实用性。有许多伤害和疾病是没有或有限的兴趣的制药或生物技术产业, 毒物伤害由 CP 和 HF 包括在内, 台式机器人的出现促进了高温超导研究在几乎任何实验室的伤害和疾病.高通量基因组数据集可以大大有助于理解特定基因在损伤或疾病中所起的作用, 这有助于在体外或体内研究中更有效地关注后续工作。

毒物性眼外伤是一种真正的眼部急症, 因为化学物质有可能迅速造成严重的组织损伤。不幸的是, 对毒物引起的角膜损伤的治疗只是普遍支持, 因为没有具体的治疗方法来治疗这些损伤。目前的治疗策略是非特定的, 主要包括局部治疗治疗, 如润滑剂, 抗生素, cycloplegics 后, 炎 (如类固醇), 一旦角膜重新表皮1 ,2。尽管现有最佳治疗方案可供选择, 但长期预后通常较差, 因为渐进性角膜混浊和新生血管2,3。
动物模型历来被用来研究化学毒性和理解损伤机制。然而, 动物研究是费时和昂贵的。还有努力减少动物试验。例如, 在欧洲联盟中达成立法 (欧共体 1907/2006) 有旨在减少动物试验的规定。这些规定包括要求公司共享数据, 以便在对动物进行建议的试验之前避免动物试验并获得欧洲化学品机构的批准。根据达成的规定, 动物试验应该是最后的手段。还有欧洲化妆品条例 (EC 1223/2009), 逐步淘汰动物化妆品的测试。当进行动物研究时, 它们遵循3Rs 的原则 (提炼、减少和替换), 这为执行更人性化的动物研究、减少使用的动物数量和使用非动物替代品提供了框架。尽可能。出于这些原因, 毒理学领域试图采用体外检测, 可以提供对毒性分子机制的洞察力, 并可以在更高的吞吐量4中进行。这是一种功能毒物学方法, 毒物是由它们的功能定义的, 而不是完全由它们的化学组成。更进一步, 功能致毒学寻求了解特定基因在毒物5的作用中所起的作用。随着 siRNA 技术的应用, 筛查在分子和细胞对毒物反应中的基因功能可以在高通量的情况下进行。siRNA 是双链 RNA 分子, 是19-25 核苷酸长, 利用后转录基因沉默路径存在的所有哺乳动物细胞6。这些都是综合制作和设计的目标一个特定的基因。当引入细胞时, siRNA 被处理, 一个链, 导链, 被加载到 RNA 诱导的沉默复杂 (RISC)。siRNA 将 risc 定向到 mrna 分子中的互补区域, risc 会降低 mrna 的降解。这导致了特定基因表达的减少。因此, 在毒物暴露的细胞中, 可以对特定基因表达的减少进行研究, 以确定该基因在细胞对毒物的反应中的作用。这种方法已被用来进一步了解蓖麻毒素的敏感性机制和 AHR 依赖性诱导 CYP1A17,8。
《化学恐怖主义风险评估 (CTRA) 清单》和《有毒工业化学品 (TIC) 》列出了根据其毒性和可能在恐怖主义、战争或工业事故事件9中释放的可能性对化学品进行分项的分类。我们正在应用一种 siRNA 高通量筛选 (高温超导) toxicogenomic 方法研究 CTRA 清单毒物, 这些有毒物质被认定在恐怖事件中有很高的使用风险。传统毒理学试图了解化学品对生物体的有害影响;然而, 我们还希望了解伤害机制, 以便向治疗和治疗方法的发展提供通知, 并有可能发现可作为疗法发展目标的分子。这种努力在某种程度上可以被认为类似于在药物发现过程中使用高通量 siRNA 筛选和细胞检测方法10。一个主要的区别是, 药物发现通常寻求一个单一的治疗发现的目标, 而在我们的方法, 它是有点不太可能有一个单一的目标, 具有较高的治疗价值的治疗毒物暴露。我们预计, 任何有效的毒物暴露治疗范式都需要一种多层面的方法来达到高治疗价值, toxicogenomic 数据可能会非常有效地告知一种行之有效的治疗范式。
台式自动化为制药或生物技术行业以外的实验室带来了高吞吐量方法。在我院的体外研究历来是传统的化验, 是低吞吐量11,12,13。在过去的几年里, 我们的实验室已经过渡到使用台式机器人进行高通量 siRNA 筛选。在此, 我们提出了眼部细胞模型的细化和对氟化氢 (HF) 和氯化苦 (CP) 的体外暴露方法的发展, 适合高通量 siRNA 筛查。我们的目标是确定分子, 以应对这些有毒物质来调节细胞损伤。我们选择的 siRNA 库的目标包括 G 蛋白耦合受体、蛋白激酶、蛋白酶、磷酸、离子通道和其他潜在的 druggable 靶。HF 和 CP 被选择为研究通过交叉引用 CTRA 名单代理与 ToxNet 报告的工业事故, 以找到那些目前最危险的眼睛伤害通过蒸气暴露9,14。CP (化学配方 Cl3CNO2, CAS 号 76-06-2) 最初被用作催泪瓦斯在一战15。它目前被用作农业熏蒸和杀线虫剂、杀菌剂和杀虫剂16的功能。氟化氢 (HF) 用于炼油厂的烷基化反应和有机化合物的电化学氟化物17。hf (化学方程式 hf, CAS 号 62778-11-4) 是一种气体, 但它的水形式是氢氟酸 (高放, cas 号 7664-39-3)。因此, 我们选择使用高放在我们的细胞暴露模型。选择 SV40 大 T 抗原永生化人角膜上皮细胞系 SV40-HCEC 进行研究。细胞活力和炎症标记 IL-8 被选择为终点, 因为参与细胞损伤的靶点应反映在细胞死亡和炎症反应中。具体地说, 如果目标在毒物暴露中起到保护作用, 当目标表达被 siRNA 抑制时, 细胞死亡和/或炎症细胞因子的产生就会增加。对于那些发挥消极作用的目标, 恰恰相反。此外, 慢性炎症在角膜暴露后的病理中似乎起到了作用, 而细胞死亡通路的干预可能会改善临床结局2,18。

<table border="0" cellpadding="0" cellspacing="0" style="width:879px;" width="880">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo liquid handing platform</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">G5409A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo plate shaker</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96LT disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 178</td>
			<td>96-channel large tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 177</td>
			<td>96-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384ST disposable tip head</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">Option 179</td>
			<td>384-channel small tip pipetting head unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 96 250 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19477-022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 30 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Bravo 384 70 &#956;L sterile barrier tips</td>
			<td style="width:247px;">Agilent or equivalent</td>
			<td style="width:119px;">19133-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">EnSpire multimode plate reader</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">2300-0000</td>
			<td>AlphaLISA assay detector with high power laser excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IL-8 (human) AlphaLISA Detection Kit&#160;</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td style="width:119px;">AL224F</td>
			<td>no-wash bead-based assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">ProxiPlate-384 Plus white 384-shallow well microplates</td>
			<td style="width:247px;">Perkin Elmer or equivalent</td>
			<td>6008359</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Lipofectamine RNAiMAX</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">13778500</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Opti-MEM 1 Reduced Serum Medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">TrypLE Express</td>
			<td style="width:247px;">Gibco or equivalent</td>
			<td style="width:119px;">12605010</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">IncuCyte Zoom</td>
			<td style="width:247px;">Essen Instruments or equivalent</td>
			<td style="width:119px;">ESSEN BIOSCI 4473</td>
			<td>Incubator-housed automated microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chloropicrin</td>
			<td style="width:247px;">Trinity Manufacturing or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Acute toxicity and irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMEM-F12 cell culture medium</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>11330-057</td>
			<td>Contains HEPES</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fetal bovine serum</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>1891471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Human epidermal growth factor (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>E9644-.2MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Recombinant human insulin (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>12585-014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Penicillin-Streptomycin solution (cell culture grade)</td>
			<td style="width:247px;">Invitrogen or equivalent</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Hydrocortisone (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td>H0888-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Glucose&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">PBS&#160; (cell culture grade)</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">P5493</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA</td>
			<td style="width:247px;">Dharmacon or equivalent</td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazolyl blue tetrazolium bromide</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">M5655</td>
			<td>MTT assay substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">siRNA buffer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">B002000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96-well cell culture plates</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">T150 cell culture flasks</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">BSL-2 cell culture hood</td>
			<td style="width:247px;">Nuaire or equivalent</td>
			<td style="width:119px;">NU-540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">300 mL robotic reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">12-565-572&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">96 baffled automation reservoirs</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">1064-15-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">500 mL sterile disposable storage bottles</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">CLS430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealer</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-1443A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Microplate heat sealing foil</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">AB-0475</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Cardamonin</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2509</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">SKF 86002&#160;</td>
			<td style="width:247px;">Tocris or equivalent</td>
			<td style="width:119px;">2008</td>
			<td>Anti-inflammatory, used as positive control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DMSO</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">48% hydrofluoric acid</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">339261</td>
			<td>Corrosive and acute toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L Single channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">1000 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">200 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">20 &#956;L 12-channel pipettors</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17013808</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 1000 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 200 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17014294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Pipettor tips 20 &#956;L</td>
			<td style="width:247px;">Rainin or equivalent</td>
			<td style="width:119px;">17002928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemical fume hood</td>
			<td style="width:247px;">Jamestown Metal Products</td>
			<td style="width:119px;">MHCO_229</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">384-well sample storage plates</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">262261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sodium chloride</td>
			<td style="width:247px;">Sigma or equivalent</td>
			<td style="width:119px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">50 mL conical tubes</td>
			<td style="width:247px;">Thermo or equivalent</td>
			<td style="width:119px;">14-959-49A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 50 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 25 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 10 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Serological pipettes 5 mL</td>
			<td style="width:247px;">Corning or equivalent</td>
			<td style="width:119px;">07-200-573</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">SV40-HCEC immortalized human corneal epithelial cells</td>
			<td style="width:247px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Sceptor Handheld Automated Cell Counter</td>
			<td style="width:247px;">Millipore or equivalent</td>
			<td style="width:119px;">PHCC20060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">GeneTitan Multi-Channel (MC) Instrument</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">00-0372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Affymetrix 24- and 96-array plates</td>
			<td style="width:247px;">Affymetrix or equivalent</td>
			<td style="width:119px;">901257; 901434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube HF</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger tube CP</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">8103421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Draegger pump</td>
			<td style="width:247px;">Draeger or equivalent</td>
			<td style="width:119px;">6400000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Clear Plate seals</td>
			<td style="width:247px;">Resesarch Products International or Equivalent</td>
			<td style="width:119px;">202502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Reagent reservoirs</td>
			<td style="width:247px;">VistaLab Technologies or equivalent</td>
			<td style="width:119px;">3054-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Xlfit</td>
			<td style="width:247px;">IDBS or equivalent</td>
			<td style="width:119px;">N/A</td>
			<td>Excel add-in used for automated curve fitting</td>
		</tr>
	</tbody>
</table>

high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury

毒物性眼外伤是一种真正的眼部急症, 因为化学物质有可能迅速造成严重的组织损伤。毒物性角膜损伤的治疗通常是支持的, 因为没有具体的治疗方法来治疗这些损伤。在努力开发治疗和治疗, 以照顾暴露, 这是很重要的了解分子和细胞机制的这些伤害。我们建议利用高通量小抑制 RNA (siRNA) 筛查可以是一个重要的工具, 有助于更迅速地阐明分子机制的化学角膜上皮损伤。siRNA 是双链 RNA 分子, 是19-25 核苷酸长, 利用后转录基因沉默途径降解 mRNA, 有同源性的 siRNA。因此, 在毒物暴露的细胞中, 可以对特定基因表达的减少进行研究, 以确定该基因在细胞对毒物的反应中的作用。本文介绍了氟化氢 (HF) 和氯化苦 (CP) 致眼损伤的高吞吐量筛查模型和方法的建立和验证。尽管我们选择了这两种毒物, 但我们的方法也适用于对有毒暴露协议进行细微修改的其他毒物的研究。选择 SV40 大 T 抗原永生化人角膜上皮细胞系 SV40-HCEC 进行研究。在筛选协议中选择了细胞活力和 IL-8 生产作为终点。介绍了与高温超导研究相适应的毒物暴露和细胞培养方法的一些挑战。这些毒物的高温超导模型的建立, 使进一步的研究, 以更好地了解损伤机制和筛查潜在治疗化学性眼外伤。

曝光方法开发
我们对人角膜上皮细胞系 SV40-HCEC 在高温超导研究中的适用性进行了改进和评价。SV40-HCEC 是永生化使用 SV-40 大 T 抗原, 是一个礼物从 Dhanajay Pal23。在曝光方法论的发展中, 有太多的变数被探索出来, 因此, 只有一些我们认为可以更广泛地适用于多种毒物的结果样本。
用穗或中交换法接触毒物
我们最初试图揭露细胞通过5µL 的工作浓度的有毒物质稀释在水, 生理盐水, 或中等到95µL 的介质已经存在到每个井的细胞在96井板, 以达到最终预期的接触浓度。SV40-HCEC 被暴露了使用此尖峰曝光/refeed 方法 1 X ld50的高放 24 h (1 X ld50 = 0.02% 高放用这种方法时, 高放穗被稀释的培养基), 和水井 refed 与新鲜培养基10分钟后。在24小时后, 细胞的覆盖与 MTT 基板, 如上文所述的细胞活力测定。在 formazan 晶体与亚砜的增溶之前, photomicroscopy 捕获了图像。对水井的仔细检查表明, 在一个地方有很大程度的细胞死亡, 这是尽管该板块在十五年代被添加到整个板块后立即被震动 (图 1a)。这一现象被观察到, 当钉被添加到井底的井或当添加在半月板。使用相位对比度较高的这个区域的放大图像显示, 细胞仍然存在于无瑕的区域中 (图 1b)。我们还测试了介质交换暴露/refeed 方法, 其中介质从井中移除, 用含有 1 x ld50高放 (1 x ld50 = 0.035% 高放) 的细胞培养培养基取代, 而水井则 refed 新鲜介质10分钟后。24小时后, 细胞被覆盖的 MTT 基板, 如上文所述。这种方法在每一个井中都有明显的细胞死亡反应 (图 1c 和 1d)。未公开的控制被提供供参考, 并且不显示细胞死亡的局部区域 (图 1e 和 1f)。对于 CP 暴露, 穗法并没有导致大程度的细胞死亡本地化到一个地点在每个井;但是, 使用中交换曝光方法 (未显示的数据), 从一次迭代到下一次的细胞死亡响应更加一致。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57372/57372fig1.jpg"> 图 1: 由穗和中交换暴露方法引起的细胞死亡.在加入 MTT 基片后, 所有图像均被捕获1.5 小时。面板 (a) 是 SV40-HCEC 暴露于高放尖峰曝光/refeed 方法的明亮的场图像。面板 (b) 是在面板 (a) 中使用相对比度较高的放大倍数。面板 (c) 是 SV40-HCEC 曝光/refeed 方法暴露的明亮的场图像。面板 (d) 是一个更高的放大倍数的相同井在面板 (c) 使用相对比。面板 (e) 和 (f) 是从未曝光的井, 明亮的领域和相对比更高的放大率, 分别。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
介质中的毒物稳定性
观察到, 一旦在培养基中稀释, HF 和 CP 的明显细胞毒性就会改变, 然后稳定下来。高放被稀释在中等到0.0036% 和允许孵化在37°c 1-60 分钟之前暴露 SV40-HCECs 如上所述。对于高放, 细胞毒性增加的前5分钟内, 高放稀释的培养基, 然后稳定至少一个小时 (图 2a)。一个方法方差分析后, Dunnett 的多项比较试验表明,% 细胞生存能力在所有时间点是显著不同的1分钟时间点。2.5 至60分钟时间点之间没有显著差异。CP 是有机的, 首先稀释到5% 在亚砜。然后它被稀释在中等到0.0008% 并且允许在37摄氏度孵化5-120 分钟之前暴露 SV40-HCECs 如上所述。一旦在培养基中稀释, CP 的细胞毒性降低60分钟以上, 然后稳定至少下 h (图 2b)。一个方法方差分析后, Dunnett 的多项比较试验表明,% 细胞生存能力在所有时间点是显著不同的5分钟时间点。60至120分钟时间点之间没有显著差异。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57372/57372fig2.jpg"> 图 2: 中稀释毒性损失.数据表示为生存率的平均值 n=6。面板 (a) 显示0.0036% 高放细胞毒性的稳定性, 当稀释在培养基中, 并在室温下孵化1-60 分钟之前, 暴露 SV40-HCECs 如上所述。面板 (b) 显示 0.0008% CP 细胞毒性的稳定性, 当稀释在培养基中, 并允许孵化在37摄氏度5-120 分钟之前, 暴露 SV40-HCECs 如上所述。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
仅公开/Refeed 或公开
暴露方法研究的另一个因素是: A) 添加毒物10分钟, 洗涤, refeed, 从化学油烟机中取出盘子, 并在标准细胞培养孵化器中孵化24小时 (暴露/refeed 方法);或 B) 添加毒物, 留在上面, 从化学油烟机中取出盘子, 在标准细胞培养孵化器中孵化24小时 (仅公开方法)。安全是一个重要的因素, 因为它可能不允许删除含有稀释毒物从化学油烟机的细胞, 因为有毒物质将关闭气体和暴露人员。一个相关的考虑是在一个给定的时间内暴露的板块数量, 因为有更多的板块, 将有一个更大的体积脱气。在我们的高温超导方法中, 我们在给定的研究日暴露出 48 x 96 井板。我们把暴露的细胞培养板放入一个密封袋中, 然后用比色气体检测器管来评估 CP 和 HF 从暴露的平板上的排气。我们发现, 暴露48板到 1 x LD50的高放并没有导致任何可探测的 HF 脱气 (数据没有显示)。对于 cp 曝光, 我们发现有足够的 cp 离气从48个板块暴露到 1 x LD50 , 至少提出一些安全问题 (未显示的数据)。公开/refeed 和公开仅对高放暴露的方法进行了评估。SV40-HCEC 细胞暴露于高放, 经 mtt 法测定24小时后的生存能力。剂量反应迭代在不同的天执行。与公开/refeed 方法相比 (图 3a), 我们发现, 仅公开方法产生了更一致的细胞生存能力测定结果 (图 3b)。因此, 选择此方法作为最终曝光方法的一部分。对于 CP, 两种不同的暴露方法之间的细胞生存率测定结果的一致性的任何差异都无法评估, 因为安全考虑排除了从化学油烟机中去除 CP 暴露的板, 以24小时孵化的标准细胞培养孵化器。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57372/57372fig3v2.jpg"> 图 3: 曝光一致性与公开/refeed 和仅公开方法.数据表示为生存率的平均值 n=6。(a) 高放在培养基中稀释, 室温下孵化10分钟, 然后用于通过暴露/refeed 方法暴露细胞。(b) 高放在培养基中稀释, 室温下孵化10分钟, 然后用于通过公开的方法公开 SV40-HCEC。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
单元格模型细化
中型成分
我们坚持由始发调查员为通过和维持这些细胞系而建立的文化条件;然而, 我们在实验中对细胞进行了修改。SV40-HCEC 细胞的起源调查员所规定的细胞培养培养基不包括氢化可的松, 但在暴露研究中, 我们利用了低剂量的松 (0.5 µg/毫升) 来抑制细胞因子产生的间歇性峰值。在幼稚的 SV40-HCEC (未显示的数据) 中看到, 在细化研究中, 从一个迭代到下一次的数据分析会产生负面影响。
表型稳定性
在我们的细胞模型细化研究中, 我们发现, SV40-HCEC 开始减少细胞因子的产生, 以应对毒物暴露从60号通道开始 (没有显示数据)。因此, 我们维持 cryostocks 低通道细胞, 执行所有研究使用60号通道的细胞, 并监测细胞因子的生产在筛选过程中。
板材布局
在高通量研究中, 实验变异性的一个重要来源是多井板块的边缘效应。我们发现边缘井细胞细胞因子的反应与高放和 CP 研究中的内水井不一致, 或等同于内部井 (未显示数据)。数据的中值波兰语 (用于将数据集的边缘效果最小化的统计练习) 没有充分解决问题 (未显示数据)。因此, 不使用细胞板边缘井进行实验或控制 (图 4)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57372/57372fig4.jpg"> 图 4: 图书馆和细胞板块布局.库板布局中的灰色阴影区域表示包含 siRNA 的井。白色的水井是空的。在 "顶板集" 中使用了库板的行 A D 中的40个目标, 在 "底板集" 中使用了库板的行 E H 中的40个目标。井 B2-B6 用于负池 siRNA 控制。水井 C2-C6 未转染。井 B7-B11 用于小豆蔻明或 SKF 86002 正控药物如所述, 井 C7-C11 用于亚砜汽车控制。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
高温超导体外模型验证
用 Z 因子分析法确定了 SV40-HCEC 在高温超导研究中的适用性, 因为这项统计试验用于测定高温超导24的测定质量。对于 HF Z 的因子分析, 细胞被播种在 1.25 x 103细胞/好, 转染了负池 siRNA (如上所述) 24 小时播种后, 并暴露48小时后向0.0036% 高放 (1 x LD50), n=3。对于 CP Z 的因子分析, 细胞被播种和转染如上所述, 并暴露48小时后染成 0.0008% CP (1 x LD50), n=5。由于这种暴露模式的更大的变异性, 在 CP 研究中使用了更大的 n。Z ' 因子是计算 IL-8 表达数据从未暴露的与外露的细胞和负池 siRNA 转染未曝光的与暴露细胞的 CP 和高放暴露。我们还分析了非转染细胞, 以确定是否有影响的检测质量, 由于 siRNA 染。验证研究的许多迭代都是在精炼曝光和培养条件的同时进行的, 目的是最大限度地提高 Z 因子的结果。SV40-HCEC 通过了验证, 大多数迭代对 HF 曝光率 (表 1) 和 CP 曝光 (表 2) 的 Z 因子值大于0.50。这些表中的每个复制都来自于不同天的电镀细胞。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">曝光组</td>
			<td colspan="3">Z 因子分析</td>
		</tr>
<tr>
<td>代表 #1</td>
			<td>代表 #2</td>
			<td>代表 #3</td>
		</tr>
<tr>
<td>高频暴露负控制与未曝光负控制</td>
			<td>0.68</td>
			<td>0。5</td>
			<td>0.56</td>
		</tr>
<tr>
<td>HF 暴露与天真</td>
			<td>0。4</td>
			<td>0.68</td>
			<td>0.56</td>
		</tr>
</tbody></table>表 1: SV40-HCEC 高放暴露 IL-8 的 Z 因子分析
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="2">曝光组</td>
			<td colspan="3">Z 因子分析</td>
		</tr>
<tr>
<td>代表 #1</td>
			<td>代表 #2</td>
			<td>代表 #3</td>
		</tr>
<tr>
<td>CP 暴露的负控制与未曝光负控制</td>
			<td>0。6</td>
			<td>0.18</td>
			<td>0.46</td>
		</tr>
<tr>
<td>CP 暴露与天真</td>
			<td>0.42</td>
			<td>0.29</td>
			<td>0.42</td>
		</tr>
</tbody></table>表 2: IL-8 的 SV40-HCEC CP 暴露 Z 因子分析
高放损伤细胞的初步 siRNA 筛查结果
在筛选前进行转染优化研究, 并将 siRNA 靶向 cyclophilin b 用于这些研究。采用原位RNA 检测法测定 cyclophilin b mRNA 水平, 采用高含量分析仪对结果进行量化。我们取得了近90% 击倒 cyclophilin b 和一般不超过5-10% 细胞损失与 4 pmol siRNA/井和0.3 µL/良好转染试剂 (数据没有显示)。更高的 siRNA 实际上导致了更穷的 cyclophilin b 击倒 (数据没有显示)。同样水平的击倒是达到了与 1.2 pmol/井的 siRNA 和0.09 µL/井转染试剂, 但我们选择使用 4 pmol 每井, 以解决任何目标, 可能需要更多的 siRNA, 以实现有效的击倒。同时对靶向击出动力学进行了评价, 观察到靶向击倒在转染后两天最大值 (未显示数据)。还验证了整个板块的目标击倒一致性 (未显示数据)。抗炎药物小豆蔻明和 SKF 86002 被用来作为抑制细胞因子生产高放和 CP 暴露的积极控制。这些都是通过测试在暴露的细胞中已知的抗炎性质的各种化合物来选择的。通过剂量反应优化研究, 选择了在筛查研究中使用的剂量。
我们采用的高通量 siRNA 筛选策略是行业标准, 涉及三主要步骤: 1) 初级筛选, 2) 库反褶积, 3) 目标验证。在初级筛查中, 用不同 siRNA 序列的混合物作为单个试剂来评估每个靶的表型。然后将目标向下选择到库卷积。在这个步骤中, 每一个不同的 siRNA 序列, 汇集到目标的每个基因在初级筛查单独测试。目标在目标验证中进行另一种向下选择, 其中目标表型经药物、表型恢复和/或 siRNA 来自另一供应商。对于我们的主要屏幕, 我们选择执行一个聚焦的亚基因组屏幕, 而不是一个基因组宽屏幕的经济原因。从 HF 和 CP 损伤小鼠角膜的微阵列数据和巧妙途径分析 (IPA) 被用来集中我们的目标图书馆的主要屏幕 (手稿准备)。简单地说, 小鼠角膜暴露在毒物蒸汽使用与早先描述的方法25相似的蒸汽杯子。暴露的角膜按钮和控制被收获在不同的时间点后暴露。rna 被分离, 并且 rna 的质量和数量被监视。所有的微阵列实验都是根据制造商的26号协议进行的。利用主分量分析 (PCA) 对原始信号强度进行归一化分析, 确定数据的重要变化源。基因按统计学意义排序。数据被挖掘, 并与预先配置的 siRNA 库的基因列表进行了比较。从这些基因列表中, 我们选择了3120目标进行筛选。siRNA 库在 SV40-HCEC 单元格中进行了筛选 (参见图 5中的主要屏幕的高温超导协议流程图)。终点评估包括在细胞培养培养基中的 IL-8 水平的无洗涤珠基检测和细胞活力的 mtt 法。这些细胞暴露于 0.0036%HFA, 如前所述, 大约 1 x LD50。用 MTT 法测定24小时后细胞活力。通过严格标准化平均差 (SSMD)27, 分析了各 siRNA 池对细胞生存能力的影响, 以统计意义相对于每个板块暴露的负池平均值。图 6显示了 SSMD 和细胞活力褶皱变化的双手电筒图。选择的命中选择阈值的目标, 加剧细胞死亡或改善细胞活力是 SSMD ≤-1.28 和 SSMD ≥ 1.28, 分别。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57372/57372fig5v2.jpg"> 图 5: 主屏幕高温超导协议流程图.此流程图显示了高温超导协议每天执行的基本步骤。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57372/57372fig6.jpg"> 图 6: siRNA 对高放暴露 SV40-HCEC 细胞生存能力的双手电图.每个目标显示的结果平均为六个复制 (n=6)。细胞活力数据表示为相对于暴露的负池控制 siRNA 的折变, 并通过 SSMD 分析统计学意义。SSMD ≤-1.28 或≥1.28 的目标被选为命中。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
分析了各 siRNA 池对高放诱导 IL-8 生产的影响, 并对各板块暴露的负池平均值的统计意义进行了比较。IL-8 在细胞培养上清液中的含量在暴露后24小时进行评估。图 7显示了 SSMD 和 IL-8 折变的双手电筒图。为降低 IL-8 产量的目标选择的命中选择阈值减少了 40%, SSMD ≤1.0。为提高 IL-8 产量的目标选择的命中选择阈值为五倍, SSMD > 1.28。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57372/57372fig7.jpg"> 图 7: 双手电筒剧情 siRNA 对 IL-8 生产的影响高放暴露 SV40-HCEC 细胞.结果显示为六复制 (n=6) 的平均值。IL-8 表达水平数据表示为相对于暴露的负池 siRNA 的折变, 并通过 SSMD 分析统计学意义。命中被定义为目标有40% 减少 IL-8 水平和 SSMD ≤-1.0, 或者五倍增加 IL-8 水平和 SSMD > 1.28。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57372/57372fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

高通量小抑制 RNA 筛选是一种重要的工具, 有助于更迅速地阐明化学角膜上皮损伤的分子机制。本文对氟化氢-氯化苦诱导的角膜上皮损伤进行高通量筛选的暴露模型和方法进行了开发和验证。

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

high-throughput-sirna-screening-for-chloropicrin-hydrogen-fluoride

Nanyang Technological University

Research

JoVE Journal

Biology

5.8K 次观看.  US Army Medical Research Institute of Chemical Defense. 高通量小抑制 RNA 筛选是一种重要的工具, 有助于更迅速地阐明化学角膜上皮损伤的分子机制。本文对氟化氢-氯化苦诱导的角膜上皮损伤进行高通量筛选的暴露模型和方法进行了开发和验证。

视频: High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

科研

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

内向整流钾通道的小分子调节剂的高通量筛选

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

高通量 SiRNA 筛选氯化苦和氟化氢引起的角膜上皮细胞损伤

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