我们感谢 Reata 制药公司提供 CDDO DFPA。我们也承认, 简和李塞德曼主席在儿科癌症创新 (约翰 Letterio) 的支持。这项工作得到了国防部的支持 [W81XWH-12-1-0452];安吉福勒青少年和青年癌症研究倡议在案例综合癌症中心;琼斯卡拉汉基金会的卡拉汉研究生奖学金。

所有的研究都是按照西方储备大学医学院的机构动物护理和使用委员会批准的程序进行的。
1. 制备骨髓源性树突状细胞 (BMDCs)
<ol><li>通过热处理消毒所有手术器械, 并在第二类生物安全柜中进行实验, 并进行安全程序。</li>
	<li>弄死 C57BL/6 小鼠 8-10 周的年龄通过使用 CO2室。将鼠标放在解剖板上, 用70% 乙醇冲洗。使用外科剪刀将胫骨-腓骨和股骨骨放在10厘米的培养皿中, 将70% 乙醇放在其中。</li>
	<li>使用手术刀片和镊子解剖组织尽可能多地在10厘米培养皿的盖子上, 隔离胫骨和股骨在6厘米培养皿中放置70% 乙醇。</li>
	<li>使用手术刀片修剪两端的胫骨和股骨。使用3毫升 PBS 在3毫升注射器与23G 针, 以冲洗的内容骨髓从一个端骨到一个圆锥管包含12毫升 PBS。对每个骨骼的末端重复此步骤3x。</li>
	<li>离心机细胞悬浮在 300 x g 为5分钟。</li>
	<li>取出上清液, 用1毫升 ACK 株溶藻缓冲器并用重悬细胞颗粒, 清除红细胞。</li>
	<li>添加9毫升 PBS 稀释 ACK 株溶藻缓冲和离心机在 300 x g 5 分钟。</li>
	<li>去除上清和并用重悬10毫升培养基 (RPMI-1640 加 l-谷氨酰胺, 10% 血清, 1% 非必需氨基酸 (100x), 10 毫米 HEPES, 50 nM β巯基乙醇, 5% 青霉素/链霉素)。 注: 血清内毒素水平必须少于0.1 欧盟/毫升。</li>
	<li>通过40微米细胞过滤器的细胞悬浮, 并采取20µl 的细胞悬浮和混合80µl 的台盼蓝, 以计数的活细胞的数量, 通过使用。</li>
	<li>将单元格数调整为 1 x 106单元格/ml, 其 15 ng/毫升为 GM-CSF 和 10 IL-4/毫升。</li>
	<li>板3毫升 1 x 106细胞/毫升在每井6井板和孵化细胞在 37°c, 5% CO2和95% 湿气在 CO2孵化器。 注: 一只老鼠的骨髓细胞约为 3-5 x 107细胞, 表明它可以从2到 3 6-井板中放置。</li>
	<li>在3天, 从每个井中移除所有3毫升培养基, 在每个井中加2毫升新鲜的 PBS, 然后轻轻地旋转盘子以确保去除所有非黏附的细胞。</li>
	<li>将3毫升新鲜培养基与 15 IL-4 和 10 ng/毫升在每个井中更换。在 co2孵化器中孵化37°c、5% co2和95% 湿度的单元格。</li>
	<li>在5天, 直接添加另外3毫升新鲜培养基与 15 ng/毫升基因脑脊液和 10 ng/毫升 IL-4 在每个井。孵育细胞在 37°c, 5% co2,和95% 湿度 co2孵化器。 注: 每个井的总容积现在是6毫升。</li>
	<li>7天, 把整个盘子放在冰上10分钟。轻轻地将每个井中的培养基注入, 将松散附着的 BMDCs 作为 iDCs 进入悬浮。 注: 粘附巨噬细胞仍然附着在盘子上。低温温和的程序是避免 DC 活化影响进一步实验的关键。</li>
	<li>离心细胞悬浮在 300 x g 为5分钟和并用重悬以新鲜培养基为进一步实验。 注: BMDCs 可以通过流式细胞仪识别荧光标记的 CD11c 抗体, 我们在图 1中展示了 BMDCs 的浇口策略以进行进一步的实验。</li>
</ol>2. TolDC 基因和蛋白质剖面特征
<ol><li>板2毫升 1 x 106 BMDCs/毫升每井在6毫微米 CDDO-DFPA 的存在或不存在 100-400 nM 的存在或不存在, 为孵化 1 h 在 37°c, 5% co2和95% 湿气在 CO2孵化器。 注: CDDO-DFPA, 合成萜, 是一个核因子 (红衍生 2) 样-2 因子 (Nrf2) 诱导剂和核因子 nf-κb (NF nf-κb) 抑制剂。应用其他制剂诱导 TolDCs 从 iDCs, 如 IL-10, 维生素 D3, 地塞米松或海湾 11-7085, 并修改这一步骤, 以最佳条件为每个代理。</li>
	<li>添加10或 100 ng/毫升的 LPS 孵化4-24 小时诱导 mDCs (孵化时间是不同的, 由于 mRNA 或蛋白质测量)。</li>
	<li>用1毫升的吸管轻轻地将培养基在每一个井中, 并收获细胞悬浮。离心机以 300 x g 为5分钟, 分别收集细胞和上清液。</li>
	<li>用流式细胞仪分析细胞颗粒的表面配体, 如刺激配体: CD40、CD80、CD86、MHC II、OX40L、ICOSL 或抑制配体: PD-L1、PD-L2、ILT3、ILT4。</li>
	<li>用定量实时 pcr (qRT pcr) 和 ELISA 法分别从细胞颗粒中分离 rna 和清液样品, 并对细胞因子、基因和蛋白质水平进行分析。 注: 例如, 炎症细胞因子: TNF α、IFNγ、EDN-1、IL-6、IL-12 和 IL-23, 或抗炎性细胞因子: IL-4、IL-10、IL-15、TGF-β1和 HO-1。</li>
</ol>3. 评估 TolDCs体外和体内的功能
<ol><li>
T 细胞自体增殖试验
	<ol>
<li>通过热处理消毒所有手术器械, 并在第二类生物安全柜中进行实验, 并进行安全程序。</li>
		<li>用0.5% 牛血清白蛋白 (BSA) 和2毫米 EDTA 在500毫升 PBS 中制备 mac 缓冲液。通过过滤0.2 µm 过滤器来杀菌缓冲。 注意: 在下面的实验中, 把缓冲器放在冰上。</li>
		<li>使用 CO2室获得 CD4+ t 细胞, 弄死 t 细胞受体 (TCR) 转基因小鼠8-10 周龄。将鼠标放在解剖板上, 用70% 乙醇冲洗。用手术剪刀和镊子将脾脏从腹部左侧分离出来。</li>
		<li>将脾脏与2毫升 PBS 放在6厘米的培养皿中, 使用3毫升注射器的后推, 通过40微米细胞过滤器将脾脏剁碎。</li>
		<li>收集细胞悬浮液和离心机在 300 x g 5 分钟。</li>
		<li>并用重悬在 400 ul 的 mac 缓冲细胞颗粒。</li>
		<li>添加 100 ul CD4+ T 细胞生物素抗体鸡尾酒在 4°c 5 分钟。 注: 抗体鸡尾酒结合其他细胞类型, 除了 CD4+ T 细胞, 如 CD8a, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b (DX5), CD105, 抗 MHC 类 II, Ter-119 和 TCRγ/δ。</li>
		<li>添加 300 ul 的 mac 缓冲器和 200 ul 的抗生物素珠 (材料表) 在 4°c 10 分钟。</li>
		<li>将铁磁球 (材料表) 和预分离过滤器组成的柱体放在磁场中, 并用3毫升的 mac 缓冲器冲洗。</li>
		<li>在细胞中添加9毫升的 mac 缓冲液, 离心机在 300 x g 处, 5 分钟。</li>
		<li>并用重悬3毫升的 mac 缓冲器中的细胞颗粒, 并应用到该柱上。收集包含 CD4+ T 单元格的流。</li>
		<li>用另一3毫升的 mac 缓冲器洗涤柱, 同时收集流经的流量。</li>
		<li>利用 CO2室, 弄死 C57BL/6 小鼠8-10 周龄, 获得脾泛 DCs。将鼠标放在解剖板上, 用70% 乙醇冲洗。用手术剪刀和镊子将脾脏从腹部左侧分离出来。将脾脏放在含有2毫升胶原酶 d 溶液的6cm 培养皿中 (2 毫克/毫升胶原酶 d 溶于含钙、镁的 HBSS)。</li>
		<li>用1毫升注射器和25G 针注射1毫升的胶原酶溶液到脾脏两次。用小剪刀把脾脏切成小块。</li>
		<li>在室温下摇动和孵化25分钟。</li>
		<li>在室温下添加 500 ul 0.5 米 EDTA, 5 分钟。 注: 3.1. 13-3. 1.14 的步骤对于提高 DCs 的产量至关重要。</li>
		<li>现在使用3毫升注射器的背部, 通过一个40微米细胞过滤器, 并收集细胞悬浮在 300 x g 离心5分钟, 以剁碎脾浆。</li>
		<li>并用重悬在 350 ul 的 mac 缓冲器, 50 ul 的 FcR 阻断试剂, 100 ul 的 Pan 树突状细胞生物素抗体鸡尾酒在4°c 为10分钟。 注: 抗体鸡尾酒对抗原不是由 dc 表达。</li>
		<li>通过添加9毫升的 mac 缓冲器和离心机在 300 x g 5 分钟内冲洗细胞。</li>
		<li>并用重悬在 800 ul 的 mac 缓冲细胞颗粒, 并添加 200 ul 的抗生物素珠在 4°c 10 分钟。</li>
		<li>重复步骤从 3.1. 9-3. 1.11 收集 DCs。</li>
		<li>再用3毫升的 mac 缓冲器清洗一列, 同时收集流量。</li>
		<li>将 2 x 105 DCs/ml 处理为1小时, 在37°c 存在或不存在 100-400 nM CDDO DFPA. 分别, 将 CD4+ T 单元格 (1 x 107/毫升) 标记为15分钟, 在37°c 使用1微米 CFSE, 用 PBS 清洗, 并重新调整音量以获得 2 x 106 T 细胞/毫升的最终浓度。</li>
		<li>接下来, 在一个96井板块, 共培养 100 ul 的处理树突状细胞具有相同体积的 CFSE 标记 CD4+ T 细胞, 两者都收集从上述步骤获得1:10 的比率。现在添加 100 ng/毫升卵清蛋白 (卵) 肽323-329 每井, 并通过流式细胞术在2-3 天的孵化后测量 T 细胞的 CFSE 强度。 注意: 从我们以前的工作37中优化了 DCs 和 T 单元格的数目和比率。</li>
	</ol>
</li>
	<li>
髓鞘少突胶质糖蛋白 (MOG) 注射 BMDCs 脉冲诱导无源 EAE (35-55)
	<ol>
<li>板2毫升 1 x 106 BMDCs/毫升每井在6个有文化媒介的 CDDO 的存在或没有100-400 毫微米 DFPA 为孵化1小时。</li>
		<li>加入10毫升的 LPS, 孵化24小时。</li>
		<li>加入100微克/毫升的 MOG (35-55) 孵化4小时。</li>
		<li>在 300 x g 的5分钟内收获细胞悬浮液和离心机。</li>
		<li>并用重悬细胞颗粒与 PBS, 计数细胞。</li>
		<li>皮下注射 200 ul 2 x 106细胞到8-10 周老女性 C57BL/6 小鼠 (100 ul 到每条后腿)。</li>
		<li>在 BMDC 注射日和48小时。然后, 在每只老鼠身上注射200的百日咳毒素 (PTX)。</li>
		<li>每周重复步骤3.2、6-3、2.7, 连续4周。</li>
		<li>采用标准标准37 (0.5 跛尾、1跛尾、2中下肢无力、3-严重后肢无力、4-完全后肢麻痹、5-四肢或垂死状态、6-死亡) 评价 EAE 的临床症状。</li>
	</ol>
</li>
</ol>

本文描述了一种有效的协议, 可用于重现性生成 iDCs, 并随后将其分化为 TolDCs, 我们建议, 这可以用于评估新分子靶剂的能力, 以诱导 TolDC表。如本报告所述, 我们遵循了一个序列, 我们首先分析了流式细胞术对表面配体的 TolDC 表达, 然后通过 qRT PCR 和 ELISA 测定对 DC 细胞因子剖面的评价。最后, 利用 MS 的临床前小鼠 EAE 模型, 证明了 TolDCs在体外抑制 T 细胞增殖的能力, 以及在体内抑癌的功效, 证实了其免疫调节功能。
在我们的协议中, iDCs 是由小鼠骨髓前体产生和分化而成的, 并结合了 GM-脑脊液和 IL-4。其他协议在培养基中使用类似于形式的酪氨酸激酶3配体 (Flt3L) 来生成 iDCs44。虽然使用 Flt3L 增加了 iDCs 的产量, 这些 iDCs 通常需要2天 (9 天) 的收获, 比 GM-CSF/IL-4 加法 (7 天)44。更重要的是, 从 Flt3L 生成的 iDC 通常区分为招揽 (CD8 和 CD8 +) 和 pDCs45 , 这些功能和表型等同于体内的稳定状态 DCs。然而, GM-CSF/IL-4 总是诱导 iDC 向招揽 (CD8-) 的分化, 只有44和成熟期间, 它们显示了类似的特点, 炎症 DCs46。值得注意的是, GM-CSF/IL-4 经常被用于临床试验和基础研究, 这是因为区分 DCs 与单核细胞或 CD34+祖,44的优势。结果表明, 从这两种方法产生的 iDCs 产生形态学上不同的细胞, 它们也具有不同的细胞表面标记物, 并在其活化时表现出明显的细胞因子。此外, 这些方法诱发的 TolDCs 在反应趋化因子和诱导抗原特异性 T 细胞响应44、45时的迁移能力各不相同。我们了解到, 由于 CD8+ dc 的唯一容量为交叉公差47, 因此在 TolDC 归纳中可能会表现出更多的潜力。但是, 需要从 Flt3L 派生的 BMDCs 进一步进行单元格排序, 以调查特定的 CD8+ DC 子集。此外, 由于 GM-CSF/IL-4 诱发 BMDCs 在 T 细胞刺激和炎症介质的生产后, LPS 治疗44,45, 我们发现这一策略给出了更多可重现的结果, 在我们的实验。
BMDCs 从 GM-CSF/IL-4 开始在4天前后表达 CD11c, 并丰富了6天后的表达式48。这种培养过程可以持续到10-12 天, 在 8-1049日, 骨髓细胞的电镀密度较低, 而 GMC SF 的剂量也较低。在该协议中, CDDO-DFPA (合成萜) 和 LPS 作为代理加入, 分别诱导直流耐受和成熟。我们最近报告说, 利用这种 BMDC 生成方法从文化中收获的 iDCs 在用 CDDO DFPA37预处理时表现出功能性 TolDC 表型。值得注意的是, CDDO-DFPA, 这只是添加到文化后, idc 收获 (7 天)37, 与像维生素 D3 的代理, 必须在 idc 分化期间多次添加到文化 (2 天, 4, 6)50。此外, 这种诱导 TolDCs 维生素 D3 可能取决于不同的机制, 必须启动在和之后, iDC 分化51。我们的数据表明, 在 iDC 分化过程中添加 CDDO DFPA 对 CD11c+ DCs 的纯度没有影响, 但剂量依赖性降低了7天 iDCs 的产量 (未显示数据)。我们的分析还表明, 在这些实验中使用的 CDDO DFPA 浓度较高可能对骨髓祖细胞有毒性, 尽管 iDC 显示 CDDO-DFPA37浓度的正常生存能力。预计 TolDC 诱导可以通过仔细分析的时间和长度的影响, 如 CDDO-DFPA 的代理商, 优化。
重要的是要考虑的替代方法和机制, 通过它可以诱导 iDCs mDCs 其他人比 LPS, 如 CD40L, TNF α, 和干扰素-γ52。由脂多糖的 DC 成熟通过收费样受体 4 (TLR4) 导致激活的几个转录因子, 包括核因子 nf-κb (NF-nf-κb), p38 丝裂原活化蛋白激酶 (p38 MAPK), c 君 n-末端激酶 (JNK) 和胞外信号调节蛋白激酶 (ERK1/2)。CD40L 和 tnf-α通过 CD40 和 tnf 受体分别诱导 DCs 的成熟, 诱发了 NF-nf-κb 通路。与此相反, 干扰素-γ刺激了不同的途径, 包括活化的 JAK 激酶, 酪氨酸激酶 (TYK), 和信号传感器和转录蛋白 (统计) 活化剂, 这些下游效应也辅以活化NF-nf-κb 路径52。此外, 虽然 CD40L/TNF-α-based dc 成熟基因表达谱表明, 这些 DCs 分化 T 细胞向 Th2 细胞反应53, DC 成熟的干扰素-γ是强烈偏向于 Th1 细胞反应54。因此, 这些诱导的 DC 成熟与分化为特定的 T 细胞子集没有使用的协议。
有几份报告显示, 体外分化的 DCs 能够诱导小鼠55的 EAE 临床症状和病理的全谱。采用体外活化 DCs 对被动 EAE 诱导的方法进行了不同实验室的各种修改. MOG 引 BMDCs 术前7天, MOG 和完全弗氏佐剂 (CFA) 的免疫被动和主动 EAE 诱导的结合导致临床症状评分较高 (峰值临床评分: 3.5) 相比被动 EAE诱导由管理 MOG 引物 BMDCs 单独 (峰值临床评分: 2)56。结果采用 MOG 引物 BMDCs 与 MOG 免疫相结合的方法表明, 免疫后9天发生疾病发作, MOG 和 CFA56。然而, 由于众所周知, CFA 在管理57站点引起了明显的炎症, 我们在协议中仅利用 DCs 进行被动 EAE 诱导, 以便有效地分析 TolDCs 在 EAE 中的作用。为了进一步确定 TolDCs 的功能表型, 还应在抑制活性 EAE 小鼠的临床症状方面进行试验。DCs 的管理路径对于诱导 EAE 症状58也是至关重要的。正如我们的实验室和其他人所报告的, 皮下注射 BMDCs 到 C57BL/6 小鼠的侧面区域一周一次, 总共3-4 个注射, 导致 EAE 的峰值临床评分约 2-2. 日 11-1237可重现性疾病发病 5,58。我们提供了一个简短的剪辑, 以演示不同的临床症状之间的主动和被动 EAE 感应 (视频 1)。
我们的议定书所承认的局限性包括所使用的方法所需的时间和对 BMDC 分化的非生理相关微环境的依赖性。然而, 虽然从小鼠脾脏中分离出富含抗体的分选方法来收集 DCs 和 T 细胞, 但其所需的 DC 种群的产量却很低。因此, 与全脾抗体柱分离相比, 骨髓源性 dc 发育方法的优越性是 dc 种群的测井率较高。如上所述, DCs 通过此方法生成的体外要求高浓度的细胞因子补充, 这与 DCs体内微环境59没有生理相关, 也不能保证长期DC 的可持续性。在我们的协议中使用的 GM CSF 也不是正常 DC 分化在体内60中的基本细胞因子。然而, 建议的 BMDC 生成协议重现性产生的功能 TolDCs 的一个很高的分数, 应该被视为一个高度可靠和可重复的方法来生成功能 DCs 与致耐受性属性。
总之, 从这里所描述的协议产生的 TolDCs 可以有效地表征其基因和蛋白质表达谱, 通过确定其调节 T 细胞依赖性免疫应答的能力在体外, 并通过评估其作为领养细胞治疗的功能, 以抑制 EAE 诱导在体内。我们的议定书提供了一个框架, 以评估任何新的药物认为有能力诱发 TolDCs, 并进一步加快治疗发展过程中 TolDCs 从板凳到临床。

树突状细胞 (DCs) 是先天免疫系统不可分割的一部分, 并首次发现和特征的拉尔夫斯坦曼和 Zanvil 科恩在1973年作为主要的专业抗原呈现细胞1。DCs 在免疫活化方面发挥了重要作用, 通过在次级淋巴器官中通过主要的组织相容性复合物 (MHC) 向 T 细胞和 B 细胞呈现经过处理的抗原, 将先天免疫系统和自适应的免疫力结合在一起2。在哺乳动物免疫系统中, 至少有两类 dcs 被描述为髓系 dcs 和浆 dcs (pDCs)3。髓细胞 dcs, 也称为常规 dcs (招揽), 其特点是 CD11c 的表达, 并可区分为未成熟的 dcs (iDCs)体外从骨髓前体细胞或外周血单核单位使用粒细胞-巨噬细胞集落刺激因子 (GM-CSF) 和 IL-4 的小鼠或人类物种, 分别为4。
激活 ' 危险 ' 信号, 如病原体相关的分子模式 (玉兰) 或损伤相关的分子模式 (潮湿), 将推动 iDCs 成熟免疫 dcs 作为成熟的 dcs (mDCs) 通过捆绑各种模式识别受体对DC 曲面5。免疫 DCs 进一步通过上调 MHCII2、刺激配体 (CD80、CD86 和 CD40)6、细胞因子和其他可溶性调解人7, 进一步促进天真的 T 细胞增殖和分化。免疫 DCs 对细胞因子介导的 T 细胞分化至关重要。例如, 干扰素和 IL-12 都是 Th1 分化所必需的8和 IL-1、IL-6 和 IL-23 对于天真的 T 细胞极化对 Th17 细胞9是至关重要的。虽然成熟的 DCs 对外来抗原有反应, 但自抗原控制的 dc 活化可能导致耐受性消融, 并通过生成自身反应性 T 细胞来促进自身免疫性疾病的发展, 其活化导致组织破坏10.
最近的报告提供了直流可塑性的明确证据, 其例证是它们能够在组织微环境中与不同的线索相互作用, 并区分为不同的效应/抑制 dc 子集。抗炎介质 (如 IL-1011、TGF β12和 HO-113 ) 在诱导致耐受性 DCs (TolDCs) 的免疫抑制中发挥了重要作用。这些 TolDCs 获取调节功能并抑制 T 细胞增殖14。此外, DCs 缺乏协同刺激和 TolDCs 的抗炎介质的产生, 都有助于诱导调节 T 细胞 (Tregs), 同时也有效抑制 Th1 和 Th17 分化和扩张15。在过去的两年中, TolDCs 的治疗潜力已经被几个调查员报告。在这些研究中, 体外产生的 TolDCs 不仅改善了自身免疫性疾病的不同临床前模型的病理症状 16 , 而且还导致了患者免疫耐受的发展17 ,18。有趣的是, 今天 TolDCs 疗法已被认为是一种替代或辅助方法的自身免疫性疾病的几个临床试验, 包括1型糖尿病19, 类风湿关节炎20, 21, 多发性硬化症 (MS)22,23,24, 克罗恩病25。
有各种各样的协议被使用了开发 TolDCs 和几个实验室报告了方法为 TolDCs 的世代和表型特征。这些方法可用于从造血祖重现性生成 TolDCs体外, 并稳定地维护它们在致耐受性状态在体内26,27,28,29。iDCs 可以通过接触各种免疫调节药理剂或抗炎性细胞因子转化为 TolDCs。例如, 维生素 D3 是已知的一种已知的药理剂, 可以增加 IL-10 的生产和抑制 IL-12 分泌物, 从而增强其免疫抑制功能30。此外, 当 DCs 暴露于强烈的炎症刺激, 如脂多糖 (LPS), 一些药理剂, 如地塞米松 31, 雷帕霉素 32, 皮质类固醇33已显示, 以诱导TolDC 表型通过减少 CD40、CD80、CD86 和 MHCII34的 DC 表面表达式。IL-10 和 TGF β是最受研究的抗炎性细胞因子, 以诱导直流耐受35和伴随暴露于这两种细胞因子已被证明诱导致耐受性表型在dc36。
由于致耐受性 DC 是由功能特征而不是表型标记来定义的, 所以需要对 TolDCs 的细胞和功能特性进行一致的方法。此外, 必须建立一个严格和一致的协议, 以便对致耐受性 DC 表型进行一致的评价和表征, 如果我们要有效和重现性比较新的药物诱导 TolDC 表型的能力实验室。在这里, 我们提供了一个详细的协议, 分步方法分离 iDCs 与造血祖细胞的小鼠, 并随后分析的功效, 评估新的药物的能力, 以转换 iDCs 为 TolDCs, 提供了一个强大的TolDCs 的功能和表型特性体外和体内。这个描述包括一个精心的方法来表征 TolDCs 的表面配体, 细胞因子剖面和免疫抑制功能在体内。我们还提供了一个方法的例子, 探讨这些 TolDCs 的潜在治疗应用在 MS, 实验性自身免疫脑脊髓炎 (EAE) 的临床前模型。这项既定的议定书将帮助调查人员评估新的药物促进诱导 TolDCs 的能力, 并将促进扩大 TolDC 治疗发展范围的努力。

<table>
	<tbody>
		<tr>
			<td>CDDO-DFPA (RTA-408)</td>
			<td>Reata Pharmaceuticals</td>
			<td>in house synthesis</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Mouse GM-CSF</td>
			<td>Peprotech Inc.</td>
			<td>315-03</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>Mouse IL-4</td>
			<td>Peprotech Inc.</td>
			<td>214-14</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS)</td>
			<td>Sigma Aldrich Inc.</td>
			<td>L2880</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich Inc.</td>
			<td>516732</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pertussis toxin (PTX)</td>
			<td>R&#38;D systems</td>
			<td>3097</td>
			<td>EAE induction</td>
		</tr>
		<tr>
			<td>MOG (35&#8211;55) peptide</td>
			<td>21stCentury Biochemicals</td>
			<td>in house synthesis</td>
			<td>EAE induction</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco, Life Technologies</td>
			<td>15250-061</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>RPMI-1640 plus L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>11875-093</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Non-essential amino acid (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td>Cell isolation</td>
		</tr>
		<tr>
			<td>PE-conjugated CD80</td>
			<td>BD Biosciences</td>
			<td>557227</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD86</td>
			<td>BD Biosciences</td>
			<td>555665</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated PD-L1</td>
			<td>BioLegend</td>
			<td>124307</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated MHCII</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-112-388</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated CD11c</td>
			<td>BD Biosciences</td>
			<td>340544</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched PE</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-091-835</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched APC</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-091-836</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>BioLegend</td>
			<td>423801</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pan dendritic cell isolation kit</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pan Dendritic Cell Biotin-Antibody Cocktail</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation kit</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>CD4+ T cell Biotin-Antibody Cocktail</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>ACK lysing buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>A1049201</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD Biosciences</td>
			<td>309626</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>3 ml syringe</td>
			<td>BD Biosciences</td>
			<td>309588</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD Biosciences</td>
			<td>309626</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td>BD Biosciences</td>
			<td>309588</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich Inc.</td>
			<td>A2058</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575020</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-042-401</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pre-Separation Filter</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-095-823</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>collagenase D</td>
			<td>Sigma Aldrich Inc.</td>
			<td>11088858001</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025076</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>ovalbumin (OVA) peptide 323&#8211;329</td>
			<td>Sigma Aldrich Inc.</td>
			<td>O1641</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01168134_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-12a TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00434165</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-12 p70 DuoSet ELISA</td>
			<td>R&#38;D systems</td>
			<td>DY419-05</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>Mouse EDN-1 ELISA</td>
			<td>RayBiotech</td>
			<td>ELM-EDN1-1</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>TNF-&#945; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00443258</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>MTA00B</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-6 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00446190</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-6 Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>M6000B</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-23a TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01160011</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-23 DuoSet ELISA</td>
			<td>R&#38;D systems</td>
			<td>DY1887-05</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-4 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm99999154_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>IL-10 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01288386_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>TGF-&#946; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01178820_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Anti-Heme Oxygenase 1 antibody</td>
			<td>Abcam</td>
			<td>ab13248</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin antibody</td>
			<td>Abcam</td>
			<td>ab8226</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad Inc.</td>
			<td></td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>BD FACSCalibur Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry</td>
		</tr>
	</tbody>
</table>

development and functional characterization of murine tolerogenic dendritic cells

免疫系统的运作方式是在协调对外来抗原的反应和保持对自抗原的无反应状态以及从共生生物体中提取的抗原之间保持紧密的平衡。这种免疫稳态的破坏会导致慢性炎症和自身免疫的发展。树突状细胞 (DCs) 是一种专业的抗原呈现细胞的先天免疫系统参与激活幼稚 T 细胞启动免疫应答外来抗原。然而, DCs 也可以被区分为 TolDCs, 以维持和促进 T 细胞耐受性和抑制效应细胞, 促进发展的任何一种自身免疫或慢性炎症条件。最近在我们理解 TolDCs 的进展表明, 直流耐受可以通过调节它们的分化条件来实现。这种现象已经导致了巨大的增长, 发展 TolDC 疗法的许多免疫障碍造成的免疫耐受性的突破。对前体免疫性小鼠模型的成功研究进一步证实了 TolDCs 治疗自身免疫性疾病的免疫治疗效用。今天, TolDCs 已成为临床上一个有希望的免疫治疗工具, 以恢复免疫耐受的各种免疫障碍的目标致病性自身免疫反应, 同时保持保护免疫完好。虽然多个实验室提出了一系列的策略来诱导 TolDCs, 但在表征细胞和功能表型方面没有一致性。该协议为骨髓源 DCs 的大量开发提供了一步一步的指导, 一种独特的方法, 用于将其与合成萜 2-氰基-312-dioxooleana-19-奠 28-伊斯兰会议组织区分为 TolDCs。酸-difluoro-丙酰胺 (CDDO-DFPA), 以及用于确认其表型的技术, 包括分析 TolDCs 的基本分子特征。最后, 通过在多发性硬化的临床前模型中测试其免疫抑制反应体外和体内, 我们展示了一种评估 TolDC 功能的方法。

BMDCs 的分化与选择:
骨髓祖细胞在全 RPMI 培养基中培养, 在 GM-CSF 和 IL-4 的存在下, 可分化为 iDCs 7 天 (图 1A)。1天, 细胞体积小, 呈球形形态。在3天更换新鲜培养基之前, 使用 PBS 进行洗涤有助于细胞形成簇群, 也增加了 CD11c+细胞的数量。在4天, BMDCs 扩大了规模, 并开始了集群的形成。黏附的巨噬细胞也被转换了并且观察了在板材的底部以拉长的形状。5天, 形成了 BMDCs 的大型簇群。在6天, 还观察到了大量的半粘附和漂浮 BMDCs。BMDCs 7 天收获, 用流式细胞仪对 CD11c 表达进行分析, 作为小鼠 DCs 的特异标记。如图 1B中的代表性流式细胞术图所示, 此方法获得了约83.6% 的 BMDCs 表达 CD11c。
TolDCs 的诱导和遗传表征:
已知的一些诱导 TolDCs 的制剂, 如维生素 D338和地塞米松39, 都是众所周知的低调节直流表面配体的表达, 包括 MHC II 和刺激分子, CD40, CD80 和 CD86。相比之下, 在 LPS 或 CD40L-induced 成熟的情况下, 钙调磷酸酶抑制剂环孢素 a 和 FK506 对 CD83、CD80、CD86 和 MHC II.33的表达没有影响。流式细胞仪分析表明, CDDO-DFPA 诱导 TolDC 对 LPS 诱导的 DCs 表面配体表达无显著影响, 包括 CD80、CD86、MHC II、PD-L1 和 CD40。(图 2)。
此外, BMDCs 治疗的转录和细胞因子分析的存在或没有 CDDO-DFPA 的存在或没有 LPS 描述调制炎症基因剖面。CDDO-DFPA 治疗显著减少 LPS 诱导的促炎性细胞因子基因, 如干扰素-γ, IL-12, EDN1, TNFα, IL-6 和 IL-23 (图 3A-3F)。这些是已知的细胞因子为 Th1 (干扰素-γ和 IL-12) 和 Th17 (IL-6 和 IL-23) 细胞分化。此外, CDDO DFPA 治疗 BMDCs 显示了抗炎性细胞因子基因的增强表达, 如 IL-4、IL-10、TGF β和 HO-1 (图 4A-4D)。这些抗炎基因是已知的自身免疫调节因子, 促进 Th2 (IL-4) 和 Treg (IL-10 和 TGF β) 细胞分化。40在这里, 重要的是要注意的是, 独特的 IL-12−; IL-10+细胞因子生产36, HO-1 表达式13的诱导, 以及 CDDO DFPA 处理诱导 EDN-141的抑制, 都是已知以验证 DCs 致耐受性功能。
TolDCs 的蜂窝和功能特性体外
DC 介导的 T 细胞增殖是由刺激表面配体的参与以及细胞因子和可溶性介质的分泌所触发的7。然而, TolDCs 通过多种机制抑制 t 细胞反应: 通过诱导 t 细胞无能, 通过积极促进删除自身反应性细胞和促进幼稚 T 细胞极化Tregs42。我们已经报告了 CDDO DFPA 诱导 TolDCs37的功能特性。通过检查表面标记物或蛋白 V/7-AAD 染色, 可以独立分析 t 细胞无能和自身反应性 t 细胞的缺失。然而, 我们通过测量 T 细胞增殖指数间接地证实了这种表型。为此, 我们利用 C57BL/6 OTII 转基因小鼠 T 细胞, 并将其暴露在 C57BL/6 小鼠自体的 DCs 中, 并以或不 CDDO DFPA 治疗。这些 DCs 采用 CFSE 染色的 T 细胞与卵肽进行洗涤和共培养。我们观察到, CDDO-DFPA 预处理的 DCs 显著减少 T 细胞增殖, 表明其致耐受性表型(图 5)。
体内TolDCs 在自身免疫的临床前模型中的功能表征
最后, DFPA 诱导的 TolDCs 的功能可以在许多具有自身免疫或炎症特征的临床前体内模型中进行测试。由于, EAE 是一种广泛接受的临床免疫介导中枢神经系统疾病的前小鼠模型, MS43, 我们在目前的研究中利用了这一点。为了进一步研究 CDDO-DFPA 诱导 TolDCs 的体内功能, 我们在被动 EAE 的前小鼠模型中对它们进行了挑战。为此, BMDCs 在 LPS 和 MOG (35-55) 的存在下, 培养了有或没有 CDDO 的 DFPA。然后, 这些细胞被反复注入, 作为在图 6A中描述的计划协议, 并且经常观察到老鼠的临床体征的表现。如预期, LPS 和 MOG (35-55) 脉冲 BMDCs 显示 EAE 的临床症状, 但;CDDO-DFPA 引物 BMDCs 治疗组显示延迟发病, 明显减少临床症状 (图 6B)。这些结果证实了 CDDO DFPA 治疗 TolDCs 诱导的免疫抑制表型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57637/57637fig1.jpg"> 图 1: BMDCs 的开发和表型特征:(A) 骨髓细胞从 C57BL/6 小鼠的胫骨和股骨骨中冲洗出来, 并进一步分化为 BMDCs。在整个分化期 (鳞片棒、100µm) 中, 通过光镜观察适当的细胞簇形成。(B) BMDCs 分化由 CD11c 表面表达法确认, 如外地资产管制署分析。图描述了展开的 CD11c+单元格填充的百分比。(C) BMDCs 的门控策略首先是封闭的, 以排除碎片 (FSC 与 SSC)。在汗衫细胞 (FL2-W 与 FL2-A) 上采用了双重排斥门。在单线细胞中, 活细胞在 7-反相图 (7-反 FSC) 上被封闭。然后对细胞进行进一步的分析, 并对 CD11c 表达进行封闭。<a href="/files/ftp_upload/57637/57637fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57637/57637fig2.jpg"> 图 2: CDDO-DFPA 不改变 DC 细胞表面配体表达式.细胞被预先治疗的存在或没有 CDDO-DFPA (200 nM) 1 小时之前, 刺激与 LPS (100 ng/毫升) 24 小时。采用流式细胞仪对 CD80、CD86、MHC II、PD-L1、CD40 细胞表面表达进行分析。此数字已从科学报告7 中修改, 文章编号: 9886 (2017),: 10.1038/s41598-017-06907-4。以版权许可的复制和转载。<a href="/files/ftp_upload/57637/57637fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57637/57637fig3.jpg"> 图 3: CDDO-DFPA 改变了免疫 DCs 的遗传和蛋白质表型. BMDCs在添加 LPS (100 ng/毫升) 之前1小时对 CDDO (50-400 nM) 的存在或缺失进行预处理, 并为 RNA 提取 (4小时)。或者允许在24小时内培养培养基。在收集细胞因子分析之前。用 qRT PCR 和 ELISA 法测定了 IL-12 (B)、TNFα (D)、IL-6 (E) 和 IL-23 (F) 的水平。而干扰素-γ (A) 的水平由 qRT PCR 和 EDN-1 (C) 单独测定。结果以 S.D. 三实验的平均值表示。* p < 0.05, ** p < 0.01, ** p < 0.001 与 LPS 治疗组比较。未配对的学生 t 测试。此数字已从科学报告7 中修改, 文章编号: 9886 (2017),: 10.1038/s41598-017-06907-4。复制和转载与版权许可。<a href="/files/ftp_upload/57637/57637fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57637/57637fig4.jpg"> 图 4: CDDO-DFPA 诱导的 TolDCs 表型经基因和蛋白表达证实.BMDCs 在 CDDO-DFPA (10-400 nM) 的存在或缺失前接受治疗, 在添加 LPS (100 ng/毫升) 前1小时进行预处理, 并在24小时后采集细胞进行 RNA 提取。用 qRT PCR 法测定 IL-4 (A)、IL-10 (B) 和 TGF β (C) 的含量。(D) 在12小时内收集细胞蛋白裂解物。为分析和 HO-1 的水平, 和β肌动蛋白的表达是由西方印迹。结果以 S.D. 三实验的平均值表示。* p < 0.05, ** p < 0.01, ** p < 0.001 与 LPS 治疗组比较。未配对的学生 t 测试。<a href="/files/ftp_upload/57637/57637fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57637/57637fig5.jpg"> 图 5: CDDO DFPA 暴露的 DCs 抑制 T 细胞增殖.DCs 采用 CDDO-DFPA (100-400 nM) 预处理, 仅1小时, 然后以1:10 的比例与 CFSE 染色的 T 细胞进行洗涤和共培养。(A) 分别从 C57BL/6 OTII 转基因小鼠和 C57BL/6 小鼠中分离出脾脏 T 细胞和 DCs。CDDO-DFPA 预处理 DCs 与 CFSE 染色 T 细胞共培养 (w/) 或没有 (w/o) 卵子添加期间孵化。2天流式细胞术测定 T 细胞增殖。图描述 t 细胞相对于数字 t 细胞分裂的百分比。数据是3独立实验的表示法。此数字已从科学报告7 中修改, 文章编号: 9886 (2017),: 10.1038/s41598-017-06907-4。以版权许可的复制和转载。<a href="/files/ftp_upload/57637/57637fig5large.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/57637/57637fig6.jpg"> 图 6: MOG 引物直流诱导的被动 EAE 被 TolDCs 废止.(A) EAE 诱导的 MOG (35-55)-脉冲 BMDCs. 成熟 BMDCs 在 CDDO-DFPA (400 nM) 的存在或缺失下进行治疗, 随后以 MOG (35-55) 为4小时的脉冲。总共200个 ul 2 x 106细胞是由皮下注射到 C57BL/6 小鼠的侧面区域每周一次, 总共四次注射。每一次, PTX 都是由2天后 (第四周期的0天) 的 ip 注入直接管理。(B) 使用标准标准, 在所有四个注射后记录临床评分。所有的数据都显示为 S.E.M. < 0.05 的平均值。多项 t 检验与圣 Sidak 分析 (n = 7 小鼠在每个小组)。此数字已从科学报告7 中修改, 文章编号: 9886 (2017),: 10.1038/s41598-017-06907-4。以版权许可的复制和转载。<a href="/files/ftp_upload/57637/57637fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Development and Functional Characterization of Murine Tolerogenic Dendritic Cells at JoVE.com

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

在这里, 我们提出了一个协议, 开发和表征致耐受性树突状细胞 (TolDCs), 并评估其免疫治疗效用。

小鼠致耐受性树突状细胞的发育及功能表征

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

development-functional-characterization-murine-tolerogenic-dendritic

Nanyang Technological University

Research

JoVE Journal

Medicine

10.5K 次观看.  Case Western Reserve University. 在这里, 我们提出了一个协议, 开发和表征致耐受性树突状细胞 (TolDCs), 并评估其免疫治疗效用。

视频: Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

利用X射线计算机断层扫描在小鼠肥胖模型的FAT卷的分割和测量

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

科研

医学

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

闭塞性细支气管炎在原位肺移植小鼠模型的发展

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...