马尼斯戴夫是由国防部补助金 PR141774 和克罗恩和结肠炎基金会美国职业发展奖的支持。Cominelli 的实验室由 NIH 赠款 DK042191 (俱乐部)、DK055812 (俱乐部)、DK091222 (俱乐部) 和 DK07948 (俱乐部) 支持。阿诺德卡普兰的实验室部分由 l. 大卫和 e. 弗吉尼亚鲍德温基金会支持。供资机构在研究分析或手稿的撰写方面没有任何作用。它的内容完全是作者的责任。

该研究中的所有小鼠实验和程序均通过案例西储大学和 #39 的机构动物保育和使用委员会批准。这些程序是在实验室动物保育 (AAALAC) 认可设施的评估和认可协会上进行的。BM 是根据大学医院机构审查委员会批准的协议在干细胞核心设施的情况下, 西储备大学在知情同意后. 
 1. 人骨髓间充质干细胞的培养和转导 (hMSCs) 注意: 在以前的出版物中详细介绍了 hMSCs 的隔离情况 15 , 20 . 
<ol> <li> 在 Dulbecco 和 #39 中分离的 hMSCs 中的培养鹰中低血糖 (DMEM-LG) 培养基, 辅以10% 胎牛血清 (FBS)。培养37和 #176 的细胞; C, 95% 湿度, 5% CO 2 在 T175 烧瓶中, 25 毫升的生长培养基。每 3-4 天更换介质。14天, 执行 hMSCs 的主要通道。
	<ol> <li> 当单元格达到80% 汇流时, 完全移除旧介质, 并用5毫升无菌磷酸盐缓冲盐水 (PBS; 用于75厘米的组织培养烧瓶) 轻轻冲洗细胞. </li>
		<li> 将 PBS 从烧瓶中取出, 并加入4毫升的0.25% 胰蛋白酶-乙酸酸 (EDTA)。将烧瓶放回37和 #176 的恒温箱中; C 为 5-10 分钟. 保持曝光时间尽可能简短. </li>
		<li> 当大多数细胞变得全面或脱离培养皿时, 通过加入一卷小牛血清来停止反应, 相当于胰蛋白酶的一半. </li>
		<li> 将电池悬浮液转移到大小离心管。离心 400 x g 的细胞5分钟, 然后小心地除去上清. </li>
		<li> 重5毫升培养基中的细胞 (DMEM-LG + 10% FBS), 并用例计数细胞. 
		注意: 在这一点上, 细胞可以继或转与三重记者. </li>
	</ol> </li> <li> 使用三重报告器执行 hMSCs 的转导。
	<ol> <li> 种子 1.5 x 10 6 单元格在一个 T175 烧瓶中。培养25毫升生长培养基中的细胞在37和 #176; C、95%、湿度和 5% CO 2 。等待24小时才能附加单元格. </li>
		<li> 将病毒鸡尾酒放入15毫升的试管中, 如参考 18 所述。
		<ol> <li> 在37和 #176 处解冻慢, 在水浴中进行 C。添加160和 #181; 5 毫克/毫升 proteamine 硫酸盐溶液在 Dulbecco 和 #39; s 改性鹰培养基 (DMEM) 到8毫升培养基 (DMEM-LG + 10% FBS), 最终浓度为100和 #181; g/毫升。漩涡为 3 s. 添加病毒感染多样性 (莫伊) 的 5 (第一次解冻) . </li>
		</ol> </li> <li> 从单元格培养瓶 (步骤 1.2.1) 中删除介质, 并将病毒鸡尾酒添加到单元格中。孵育10小时, 在37和 #176; C, 95% 湿度, 5% CO 2 . </li>
	</ol> </li> <li> 添加 4 ml 100 和 #181; 在4毫升的 DMEM-LG + 10% FBS 和孵化额外的 14 h proteamine 硫酸盐
	<li> 用25毫升培养基 (DMEM-LG + 10% FBS) 替换 24 h 后停止转导. </li>
	<li> 通过流式细胞仪评价 mRFP 表达, 确认转导效率 18 . 
	注: 在这里, 使用超过65% 效率的 hMSCs 转. </li>
	<li> 用25毫升的新鲜培养基 (DMEM-LG + 10% FBS) 每周更换两次, 以扩展细胞。将单元格培养为37和 #176; C、95%、湿度和 5% CO 2 。通道细胞后, 他们达到80% 汇合. </li>
	<li> 达到所需的单元格数后, 每步执行 trypsinization 1.1.2-1.1.4. </li>
	<li> 计算单元格并将其悬浮在无菌 PBS (1x) 中, 最终浓度为 1 x 10 6 cells/75 和 #181; l 保持细胞在冰上的运输到超声波机。为实验, 使用 hMSCs 从段落数字 2-5. </li>
</ol> 2。超声引导心内注射 hMSCs 入左心室 注意: 使用 SAMP1/YitFc 与已建立的小肠炎症和年龄和性别匹配的 AKR/J 小鼠进行实验。保持桑普和 AKR 小鼠在特定的无病原体条件下, 喂养他们标准的实验室周, 并保持他们在12小时的光/暗循环。在这里, 超声引导心脏注射进行了专门的无病原体心脏超声室位于 CWRU 动物研究设施, 被消毒消毒剂和70% 酒精冲洗。实验室研究人员在心内注射时必须佩戴个人防护设备, 包括长袍、面罩和无菌手套。在过程的持续时间内, 必须保持鼠标的体温. 
<ol> <li> 在麻醉鼠标之前设置超声成像系统。对于本协议, 使用为鼠标超声心动图设计的超声波机。
	<ol> <li> 打开电源并初始化传感器 (30 MHz) 以设置新的研究. </li>
	</ol> </li> <li> 麻醉在感应腔中使用 3-4% 异氟醚在100% 的氧气中以 0.2-0.5 升/分的速度进行操作. 用2% 异氟醚在100% 氧气中通过鼻锥维持动物的整个过程。通过捏脚趾、滚动鼠标和观察没有运动的情况, 在执行成像之前确认正确的麻醉. 
	注意: 眼部软膏应应用于眼后的麻醉诱导, 以防止角膜干燥。将麻醉小鼠转移到成像机 ( 即, 生物发光成像系统和超声波), 在那里他们将得到一个麻醉异氟醚的维持剂量。确定麻醉开始时间和麻醉深度, 观察到在整个麻醉期间, 每10分钟的尾巴和脚垫上都缺乏对捏的反应. </li>
	<li> 将成像台和超声凝胶的温度设置为37和 #176; C. </li>
	<li> 用脱毛膏完全清除麻醉鼠标胸部区域的皮毛. </li>
	<li> 将鼠标放在仰卧位的成像台上, 用胶带固定上、下肢体, 避免在手术过程中身体运动。在所有小鼠的过程中使用心电图监测器. </li>
	<li> 用10% 的聚维酮/碘棉签清洁胸部区域的皮肤, 然后用70% 的乙醇拭子擦拭。将一层厚的凝胶涂敷在老鼠的胸腔内. </li>
	<li> 在支架上安装传感器并调整其位置, 直到左心室在视野中清晰可见. </li>
	<li> 加载150和 #181; l 转 hMSC 细胞悬架 (2 x 10 6 hMSCs 150 和 #181; l 无菌 PBS) 成1毫升注射器与28克针和安全注射器的适当持有人。为了使脉动血液形象化, 在注射器里保持一个小的空气柱。为了避免结块, 在注射前暂停细胞. </li>
	<li> 向鼠标胸部推进注射器, 用超声引导调整针的轨迹, 进入左心室. </li>
	<li> 在超声成像指导下, 通过肋间间隙穿透注射器针并进入鼠标左心室. 
	注: 在超声图像的左心室, 注射器针尖应清晰可见。适当的安置应该进一步证实由新鲜的动脉血液流出入注射器。这些都是成功插入的标志. </li>
	<li> hMSC 细胞悬浮液非常缓慢地注入2分钟以上, 应用轻柔前压力. </li>
	<li> 在注射悬浮细胞后, 轻轻地取出针头, 清除超声波凝胶, 并从胶带上松开鼠标. </li>
	<li> 将动物放在一个带有预热垫的新的干净的笼子里. 
	注意: 在手术完成后, 应为小鼠提供一个温暖的环境, 直至恢复。应避免过度加热, 因为外围血管舒张会损害恢复。小鼠应密切监测, 并与其他小鼠保持隔离, 直到其完全恢复, 表明他们的能力, 以保持胸骨卧床. </li>
	<li> 监测动物, 直到他们达到完全恢复从麻醉和每天直到实验结束. </li>
</ol> 3。生物发光 (BLI) 成像 hMSCs <ol> <li> 在生物荧光成像系统中的心内注射后, 将小鼠24小时图像化。启动 在体内 成像软件。初始化成像系统并打开一项新的研究。
	<ol> <li> 在控制面板中, 通过单击和 #34 来设置图像参数; 序列设置. #34; 在成像模式下, 选择和 #34; 发光和 #34; #34;p hotograph; #34; 将曝光时间从0.5 秒设置为10分钟, 将分设置为/#34; 中和 #34; 和 F/停止 #34; 1. #34; 设置励磁过滤器, #34; 块和 #34; 发射过滤器, #34; 打开. #34; 为两只老鼠设置视野, #34; #34。通过单击 "购置控制面板", 将序列设置添加到图像向导中. </li>
	</ol> </li> <li> 开关在氧气泵上, 并将异氟烷设置在感应腔和机器内的鼻锥2.5% 上. </li>
	<li> 将鼠标 ip 注入300和 #181; D 型素 (12.5 毫克/毫升). </li>
	<li> 将鼠标置于麻醉感应室。
	<ol> <li> 在 2-3 分钟后, 将鼠标移入 体内 成像室, 将其头部置于麻醉歧管的鼻锥上。应用光学软膏保护眼睛在成像。要对多个小鼠进行图像处理, 请使用黑色光挡板, 以防止光线反射到相邻的小鼠身上. </li>
	</ol> </li> <li> 单击 #34; 获取和 #34; 按钮以在素注入后10分钟开始图像获取. </li>
	<li> 重复步骤 3.3-3.5 以映像更多的鼠标. </li>
	<li> 后 在体内 图像采集, 安乐小鼠经 CO 2 吸入后再经颈椎脱位, 确认死亡。执行 ex 活体 分析。
	<ol> <li> 如果在20分钟后执行 ex 体内 分析, 则在牺牲鼠标之前重复素注入 (步骤 3.3); BLI 信号在素注入后可能会减少20分钟. </li>
	</ol> </li> <li> 解剖小鼠以清除整个胃肠道 ( 即从胃到直肠)、肠系膜淋巴结 (MLN)、肺、脾脏和肝脏。将它们放入培养皿中使用镊子和剪刀 2 , 4 。
	<li> 将包含说明器官的培养皿放置在成像室中, 并每步骤 3.1-3.6 获取 BLI 图像. </li>
	<li> 图像采集后, 将说明器官转移到含有最佳切削温度 (OCT) 化合物的试样模具中。Cryofreeze-80 和 #176 的模具; C 使用干冰。在冰冻切片上使用 anti-luciferase 抗体进行免疫组化, 以确认是否存在 hMSCs 16 . </li>
	<li> 对于光强的量化, 请使用工具调色板中的 "感兴趣区域 (ROI)" 工具 16 , 19 。选择 ROI 框架并将其移动到图像上的感兴趣区域。单击和 #34; ROI 度量和 #34; 并以 SEQ 文件格式保存数据。将数据导出为 .txt 文件, 并使用统计软件执行量化 16 的基本统计测试. </li>
</ol>

<ol>
	<li>Duijvestein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+with+interferon-gamma+enhances+the+therapeutic+activity+of+mesenchymal+stromal+cells+in+animal+models+of+colitis.">Pretreatment with interferon-gamma enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.</a> Stem Cells. 29 (10), 1549-1558 (2011).</li><li>Sala, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cells+Reduce+Colitis+in+Mice+via+Release+of+TSG6,+Independently+of+Their+Localization+to+the+Intestine.">Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine.</a> Gastroenterology. 149 (1), 163-176 (2015).</li><li>Gonzalez, M. A., Gonzalez-Rey, E., Rico, L., Buscher, D., Delgado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+mesenchymal+stem+cells+alleviate+experimental+colitis+by+inhibiting+inflammatory+and+autoimmune+responses.">Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses.</a> Gastroenterology. 136 (3), 978-989 (2009).</li><li>Dave, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+murine+interstitial+cells+of+cajal+suppress+cellular+immunity+and+colitis+via+prostaglandin+e2+secretion.">Stem cells for murine interstitial cells of cajal suppress cellular immunity and colitis via prostaglandin e2 secretion.</a> Gastroenterology. 148 (5), 978-990 (2015).</li><li>Manieri, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosally+transplanted+mesenchymal+stem+cells+stimulate+intestinal+healing+by+promoting+angiogenesis.">Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis.</a> J. Clin. Invest. 125 (9), 3606-3618 (2015).</li><li>Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Cominelli, F., Kollias, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+on/off+regulation+of+TNF+biosynthesis+in+mice+lacking+TNF+AU-rich+elements:+implications+for+joint+and+gut-associated+immunopathologies.">Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies.</a> Immunity. 10 (3), 387-398 (1999).</li><li>Abraham, C., Cho, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease.">Inflammatory bowel disease.</a> N. Engl. J. Med. 361 (21), 2066-2078 (2009).</li><li>Dave, M., Papadakis, K. A., Faubion, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+inflammatory+bowel+disease+and+molecular+targets+for+biologics.">Immunology of inflammatory bowel disease and molecular targets for biologics.</a> Gastroenterol. Clin. North Am. 43 (3), 405-424 (2014).</li><li>Dave, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cell+Therapy+for+Inflammatory+Bowel+Disease:+A+Systematic+Review+and+Meta-analysis.">Mesenchymal Stem Cell Therapy for Inflammatory Bowel Disease: A Systematic Review and Meta-analysis.</a> Inflamm. Bowel Dis. 21 (11), 2696-2707 (2015).</li><li>Panes, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+allogeneic+adipose-derived+mesenchymal+stem+cells+(Cx601)+for+complex+perianal+fistulas+in+Crohn's+disease:+a+phase+3+randomised,+double-blind+controlled+trial.">Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial.</a> Lancet. 388 (10051), (2016).</li><li>Lee, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+hMSCs+improve+myocardial+infarction+in+mice+because+cells+embolized+in+lung+are+activated+to+secrete+the+anti-inflammatory+protein+TSG-6.">Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6.</a> Cell Stem Cell. 5 (1), 54-63 (2009).</li><li>Gao, J., Dennis, J. E., Muzic, R. F., Lundberg, M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+in+vivo+distribution+of+bone+marrow-derived+mesenchymal+stem+cells+after+infusion.">The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.</a> Cells Tissues Organs. 169 (1), 12-20 (2001).</li><li>English, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mesenchymal+stromal+cell+immunomodulation.">Mechanisms of mesenchymal stromal cell immunomodulation.</a> Immunol Cell Biol. 91 (1), 19-26 (2013).</li><li>Pizarro, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAMP1/YitFc+mouse+strain:+a+spontaneous+model+of+Crohn's+disease-like+ileitis.">SAMP1/YitFc mouse strain: a spontaneous model of Crohn's disease-like ileitis.</a> Inflamm. Bowel Dis. 17 (12), 2566-2584 (2011).</li><li>Lennon, D. P., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+marrow-derived+mesenchymal+stem+cells.">Isolation of human marrow-derived mesenchymal stem cells.</a> Exp. Hematol. 34 (11), 1604-1605 (2006).</li><li>Lin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+transplantation+and+long-term+engraftment+of+intra-arterially+delivered+clonally+derived+mesenchymal+stem+cells+to+injured+bone+marrow.">Serial transplantation and long-term engraftment of intra-arterially delivered clonally derived mesenchymal stem cells to injured bone marrow.</a> Mol. Ther. 22 (1), 160-168 (2014).</li><li>Dennis, J. E., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+ceramic+vehicles+for+rat-marrow-derived+(Rattus+norvegicus)+osteogenic+cell+delivery:+effects+of+pre-treatment+with+fibronectin+or+laminin.">Porous ceramic vehicles for rat-marrow-derived (Rattus norvegicus) osteogenic cell delivery: effects of pre-treatment with fibronectin or laminin.</a> J. Oral Implantol. 19 (2), 106-115 (1993).</li><li>Lin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+lentiviral+transduction+of+human+mesenchymal+stem+cells+that+preserves+proliferation+and+differentiation+capabilities.">Efficient lentiviral transduction of human mesenchymal stem cells that preserves proliferation and differentiation capabilities.</a> Stem Cells Transl. Med. 1 (12), 886-897 (2012).</li><li>Love, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+mesenchymal+stem+cell+transplant+by+bioluminescence+and+PET.">Imaging of mesenchymal stem cell transplant by bioluminescence and PET.</a> PET. J. Nucl. Med. 48 (12), 2011-2020 (2007).</li><li>Haynesworth, S. E., Goshima, J., Goldberg, V. M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cells+with+osteogenic+potential+from+human+marrow.">Characterization of cells with osteogenic potential from human marrow.</a> Bone. 13 (1), 81-88 (1992).</li><li>Ishizaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+cell+transplantation+provides+timing-dependent+cell+distribution+and+functional+recovery+after+stroke.">Intra-arterial cell transplantation provides timing-dependent cell distribution and functional recovery after stroke.</a> Stroke. 44 (3), 720-726 (2013).</li><li>Schenk, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+chemotactic+protein-3+is+a+myocardial+mesenchymal+stem+cell+homing+factor.">Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor.</a> Stem Cells. 25 (1), 245-251 (2007).</li><li>Hayashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+implantation+of+mesenchymal+stem+cells+has+beneficial+effects+on+healing+of+experimental+colitis+in+rats.">Topical implantation of mesenchymal stem cells has beneficial effects on healing of experimental colitis in rats.</a> J. Pharmacol. Exp. Ther. 326 (2), 523-531 (2008).</li><li>Kodani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+colonoscopy+in+mice+to+evaluate+the+severity+of+colitis+and+colorectal+tumors+using+a+validated+endoscopic+scoring+system.">Flexible colonoscopy in mice to evaluate the severity of colitis and colorectal tumors using a validated endoscopic scoring system.</a> J. Vis. Exp. (80), e50843 (2013).</li></ol>

作者没有什么可透露的。

本研究介绍了一种新的超声引导心内注射 hMSCs 小鼠实验 CD 模型的方法。这项技术有很高的生存率和成功, 由于针的轨迹可以调整基于 real-time, 高分辨率图像的鼠标左心室, 提供的超声。传递到左心室的好处是, hMSCs 然后分布 intra-arterially 和旁路静脉循环, 从而避免了肺部细胞的聚集。以前, 颈动脉插管用于动脉分娩, 用于注入 MSCs16,21。颈动脉插管法是指在动脉内放置导管的手术方法。导管向主动脉弓方向推进, 用于细胞的输血。导管随后被切除, 颈动脉结扎。这种新的技术用于超声引导下的骨髓间充质干细胞在左心室, 与颈动脉插管相比, 具有更少的侵入性, 速度快得多 (约10分钟/小鼠与60分钟/小鼠), 并有较少的发病率和死亡率 (和 #60;10% vs 30%)。小鼠超声引导心内注射 hMSCs 对人临床试验的临床翻译将通过肠系膜上动脉插管 (目前为治疗慢性肠系膜缺血而进行难以消化的胃肠道出血) 将 hMSCs 送到患病的小肠。
对超声引导心内注射 hMSCs 在小鼠左心室的改变和诊断有多种考虑。该过程的一个关键方面是掌握使用超声引导注射针进入左心室。与任何程序一样, 适当的培训、练习和专家反馈是成功执行此过程的重要组成部分。不良的技术可以导致心包积液和死亡的心脏压塞。为了减少细胞结块和栓塞到大脑的风险 (即,中风), 必须在每次注射前重 hMSCs。在实施这一程序之前, 重要的是要咨询负责当地动物研究机构的兽医接受专家的意见。确保遵循安全和有效使用动物的准则, 以尽量减少对小鼠的无意伤害。
为了检测小鼠注射后 hMSCs 的分布, hMSCs 成功地转了三位记者, 效率高。鉴别试验表明, 转 hMSCs 分化成软骨细胞、脂肪脂和骨18的能力。在成骨功能测定中, 将羟基磷灰石/磷酸磷酸盐基质的陶瓷立方体植入免疫缺陷的小鼠皮下, 并显示出 hMSCs 形成异位供体骨的能力17,18.这些分化和功能性的化验表明, 转 hMSCs 保留其干细胞的性质。本研究的结果表明, 转 hMSCs 用于心内注射桑普小鼠定位到小肠, 这是炎症部位的桑普小鼠。在肠道无炎症的 AKR 小鼠, 只有少数的 hMSCs 局部化到肠道24小时后注射, 暗示, 在没有炎症, hMSCs 不留在肠道。这一数据与多项其他研究一致, 它们显示了 MSCs 在损伤部位迁移和嫁接的能力, 如急性心肌梗塞22的心脏和受辐射损伤的腿部16中受伤的 BM。与其他疾病一样, 骨髓间充质干细胞在结肠损伤模型中的应用在改善炎症23方面表现出了有效性。在 24, 6-trinitrobonzene 磺酸 (TNBS) 模型的大鼠结肠炎, 林et al.注入 BM 衍生的 MSCs 进入大鼠结肠黏膜下层, 周围的区域暴露给 TNBS, 并显示加速愈合的结肠炎23.
Manieri et al.最近的一项研究使用了一种新的内窥镜辅助注射结肠间充质干细胞的方法, 在结肠损伤部位, 在前列腺素缺乏小鼠中, 并显示出它们在防止形成穿透性溃疡5。桑普是一种小肠炎症模型, 这些小鼠不会在结肠内产生自发炎症。我们的实验室已经开发并验证了一种内镜方法来评估和定量的结肠炎症和肿瘤的发展小鼠。但是, 内窥镜设备无法在活鼠24中到达小肠。该评分系统仅可作为桑普小鼠安乐死后炎症反应的终点评价方法。因此, 内镜方法不适用于桑普模型中 MSCs 的局部应用。采用这种新的注射技术, 可以实现对小肠间充质干细胞的增强定位。是否增加局部化到发炎的小肠将转化为提高疗效是未知的, 将成为未来研究的焦点。除了桑普模型外, 超声引导下的骨髓间充质干细胞是一种有效的增强 hMSCs 在其他小肠炎症模型中的定位的方法, 如 TNFΔRE6小鼠实验性 CD 模型。
BLI 是一种灵敏和方便的技术, 可以连续地用于跟踪 hMSCs在体内, 但它不允许精确量化 hMSC 归巢。使用三重记者转 hMSCs 在小鼠的优势是, 在三重记者的酶 ttk 允许更多定量正电子发射断层扫描 (PET) 成像19。结合高定量 PET 成像 (与示探针) 与计算机断层扫描 (CT) (提供本地化) 允许定量估计的数量嫁接 hMSCs 在患病的站点, 因为伽玛计数是成正比的ttk 基因标记的可行 hMSCs 的数量。精确定量的 hMSC 定位可以帮助确定 hMSCs 归巢和治疗效果的百分比, 将成为未来研究的重点。
这种技术尽管有许多优点, 但仍有一些局限性: i) 对小鼠进行昂贵的心脏超声成像系统的需求, ii) 与心腔注射有关的死亡率和发病率, iii) 无法用于慢输液 (即,数小时) 的细胞和其他分子。尽管有这些限制, 它有许多好处在目前的技术颈动脉插管的动脉注射 hMSCs, 如上所述。
最后, 本研究的结果表明, 超声引导注射技术的有效性和方便性, 以改善 hMSCs 的定位, 在桑普模型的实验 CD 的炎症部位。未来的研究将确定是否增加 hMSCs 的动脉内传递的归巢可以导致在小肠炎症模型的治疗效果增加。

克罗恩病 (CD) 是一种常见的慢性炎症性疾病的小和大的肠道和被认为是由不适当的反应宿主免疫系统的肠道微生物7,8。最近的研究表明, 小鼠和人骨髓间充质干细胞 (msc) 可以抑制炎症的老鼠模型的肠道炎症1,3,4,5。有多个正在进行的临床试验, 使用从骨髓或脂肪组织获得的人骨髓间充质干细胞治疗炎症性肠病 (IBD) 患者, 其中包括 CD9。在这些临床试验中使用了两种 msc 治疗方法: 一种是对腔 IBD (包括 CD) 的骨髓间充质干细胞进行全身输注 (即静脉注射), 另一条涉及在瘘管中应用/注射骨髓细胞。肛周患者的道。在最近一次 meta 分析中, MSC 治疗 IBD, 系统性 (即,静脉) msc 治疗腔 IBD (包括 CD) 是有效的多达 40% (95% ci: 7-79%) 的患者, 而疗效更高, 在 61% (95% ci:36-85%), 当 MSCs局部注射到患病 CD 瘘9。最近的 III 期多中心随机安慰剂对照试验, 直接注入 CD 患者肛周瘘的同种异体脂肪干细胞, 有统计学意义的临床和放射学证据表明肛周瘘愈合,确证 meta 分析的结果10。腔镉的静脉化疗疗效低下的原因未得到充分调查, 但原因之一可能是 MSCs 对炎症部位的归巢不足。在结肠炎症模型中的小鼠研究表明, 只有一小部分的 mscs (1-5%) 静脉注射到发炎的结肠; 其余的 mscs 由肺部过滤 (第一次通过效果)1,2 ,5,11,12。因此, 多项小鼠研究已使用腹腔路径 (ip) 的 MSC 管理的动物模型结肠炎4。然而, 他们也表明, 只有一小部分细胞到达和嫁接的结肠, 并与疗效相关的可溶性分泌因子的分泌, 如肿瘤坏死因子诱导基因6蛋白 (TSG-6)2。免疫抑制和愈合的 MSC 机制涉及下的方法, 涉及分泌;细胞接近度-独立因素, 如 TSG-6;和细胞接近相关因素, 如程序性死亡-配体 1 (PD-L1);或锯齿状1。因此, MSC 局部化到炎症部位可能会导致效果提高9,13。事实上, 最近的一项研究表明, 在结肠损伤部位直接植入的骨髓间充质干细胞通过分泌血管内皮生长因子 (VEGF) 促进了愈合。另一方面, 静脉注射后注意到了最小的愈合效果5。为了增加其对炎症部位的定位 (即,桑普小鼠的小肠), 该超声引导下的心内注射技术在左心室的 MSC 管理中得到了发展。图像引导注射确保了准确的注射, 从而导致更高的成功率和降低发病率和死亡率。此外, 向左心室注射骨髓间充质干细胞可使它们进入动脉循环, 在那里它们可以到达发炎的小肠, 然后被困在肺部。
在本研究中, 人骨髓间充质干细胞 (hMSCs) 用于注射 SAMP-1/YitFc (桑普) 小鼠模型的 CD14。桑普是一种良好自发的小鼠慢性炎症模型, 它能发展出近100% 显14的小肠炎症。在没有任何化学、免疫学或基因操作的情况下, 炎症发生在对微生物群的反应中, 与人类 CD11相似。本研究采用性别和年龄匹配的无炎症 AKR/J (AKR) 小鼠, 即桑普的父母对照小鼠。
hMSCs 在实验室中从正常的、不明的捐赠者获得的骨髓样本中分离和扩展, 使用经过验证和以前发布的协议15,16。在分离和扩展后, 在实验室中通过多项试验对成骨、脂和软骨分化的 MSC 能力进行了评价15。通过在免疫缺陷 CB17-Prkdc 小鼠体内植入含有 hMSCs 皮的羟基磷灰石/磷酸磷酸盐基质的陶瓷立方体, 进行成骨功能测定17。立方体检测显示成骨和软骨电位, 被认为是评估个别 MSC 准备工作的终极测试17。为了形象化 hMSCs在体内注射后, 慢被用来传感器 hMSCs 与三重报告基因结构, 包括萤火虫荧光 (佛罗里达州), 单体红色荧光蛋白 (mRFP), 和单纯疱疹病毒胸苷激酶 (ttk), 由改良的骨髓肉瘤病毒 (国防部) 启动子18驱动。萤火虫荧光在三重记者是一种酶, 羽注入素氧化在 hMSCs 和产生光子/白光。这是检测的敏感电荷耦合器件 (CCD) 相机 (生物荧光) 在一个体内光学成像系统, 使可视化活 hMSCs 在小鼠。生物发光成像 (BLI) 是一种敏感的技术, 可以连续用于跟踪细胞和ex 体内分析。使用一个强大的国防部启动器驱动的三重融合记者基因结构的连续表达, 并允许成像的注入 hMSCs 超过16周19。hMSCs 不易传感器, 且转导效率低。利用优化的协议, 提高了 hMSCs 转导效率, 提高了转基因表达的18。利用 mRFP 表达 (三重报告基因之一) 对流式细胞术, 传感器 hMSCs 的能力, 高效率高达83% 的证明。鉴别分析和在体内立方体分析证明了转 hMSCs 分化成软骨细胞、脂肪和骨17的能力。

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DMEM-LG&#160;&#160;</td>
			<td style="width:333px;">Gibco</td>
			<td style="width:188px;">31600-091</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25-200-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteamine Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P4020-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin</td>
			<td>Goldbio</td>
			<td>luck-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bobine Serum</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">175 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">75 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>430720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td>Crysalgen</td>
			<td>23-2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Plus O.C.T. compound</td>
			<td>Fisher HealthCare</td>
			<td>4585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomold Standard</td>
			<td>Tissue-Tek&#160;</td>
			<td>4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lenti-Pac HIV Expression Packaging System</td>
			<td>GeneCopoeia</td>
			<td>HPK-LvTR-20&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Ionidine swabs</td>
			<td>Medline</td>
			<td>MDS093901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hair removal cream</td>
			<td style="width:333px;">Nair</td>
			<td style="width:188px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Isoflurane&#160;</td>
			<td style="width:333px;">Piramal Helathcare&#160;</td>
			<td style="width:188px;">NDC 66794 013 25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Forceps&#160;</td>
			<td style="width:333px;">Fine Science Tools</td>
			<td style="width:188px;">11200-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dissection scissors (Wagner)</td>
			<td style="width:333px;">Fine Science Tools</td>
			<td style="width:188px;">Wagner 14068-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Puralube vet ointment</td>
			<td style="width:333px;">Puralube</td>
			<td style="width:188px;">NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Aquasonic 100 ultrasound transmission gel</td>
			<td style="width:333px;">Parker laboratories</td>
			<td style="width:188px;">01 08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAMP mice</td>
			<td>Cleveland Digestive Disease Research Core Centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKR mice</td>
			<td>Cleveland Digestive Disease Research Core Centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vevo 770 imaging system</td>
			<td>&#160;Visual Sonics, Toronto, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;IVIS spectrum series system&#160;</td>
			<td>PerkinElmer, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living image software&#160;</td>
			<td>CaliperLifeSciences, PerkinElmer, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triple reporter</td>
			<td colspan="2">Kindly provided by Dr. Zheng- Hong Lee, CWRU, (citation 19)</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultrasound-guided intracardiac injection of human mesenchymal stem cells to increase homing to the intestine for use in murine models of experimental inflammatory bowel diseases

克罗恩病 (CD) 是一种常见的小、大肠慢性炎症性疾病。小鼠和人骨髓间充质干细胞具有免疫抑制剂的潜能, 并已被证明可以抑制肠道炎症模型中的炎症, 尽管其管理途径可限制其归巢和有效性1,3,4,5. 骨髓间充质干细胞在结肠损伤模型中的局部应用在改善结肠炎症方面表现出更大的功效。然而, 在实验性克罗恩病的 SAMP-1/YitFc (桑普) 模型中, 关于提高人骨髓间充质干细胞 (hMSCs) 在小肠中的定位的技术数据尚缺乏。本文介绍了一种新的超声引导心内注射 hMSCs 在桑普小鼠, 一个良好自发性模型慢性肠道炎症的新技术。性别和年龄匹配, 无炎症 AKR/J (AKR) 小鼠被用作对照。为了分析分布和本土化, hMSCs 转了一个慢, 其中有三名记者。三重记者包括萤火虫荧光 (fl), 为生物发光成像;单体红色荧光蛋白 (mrfp), 用于细胞分类;并截断单纯疱疹病毒胸苷激酶 (ttk), 用于正电子发射断层扫描 (PET) 成像。本研究结果表明, 24 小时后的心内管理, hMSCs 定位在小肠桑普小鼠, 而不是无炎症 AKR 小鼠。这本小说, 超声引导 hMSCs 在左心室的桑普小鼠保证了高成功率的细胞分娩, 使小鼠快速恢复的发病率和死亡率最低。这一技术可以是一种有效的方法, 增强局部化的 MSCs 在其他模型的小肠炎症, 如 TNFΔRE6。未来的研究将确定 hMSCs 通过动脉内分娩增加的局限性是否会导致治疗效果的提高。

图 1显示, hMSCs 可以与三重报告员转, 以高效率地保存其干细胞特性。转 hMSCs 可以通过 BLI (图 1C,图 3) 实时可视化。这本小说, 超声引导注射 hMSCs 进入左心室的桑普小鼠确保了非常高的成功率注射, 允许快速恢复小鼠的发病率和死亡率最低 (图 2)。注射一只小鼠与 hMSCs 的平均时间约10分钟, 而少于 8-9% 的发病率和死亡率用这种技术观察。死亡的主要原因是出血性心包积液的卒中和心脏填塞。在 hMSC 的心内 (动脉) 管理后, 进行了24小时的体外分析, 证实这一管理途径导致 hMSCs 桑普小鼠发炎的小肠被归巢, 而不是无炎症控制鼠标 (图 3C和D)。此外, hMSCs 往往不会留在无炎症 AKR 小鼠的小肠中, 开始积聚或被困在肺部 (图 3B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55367/55367fig1.jpg"> 
图 1. hMSCs 可以在体内可视化和保留干细胞属性的情况下转高效地进行.(A)转 hMSCs 的显微图像。人骨髓间充质干细胞的成功转导, 由流式细胞术 mRFP 表达的检测(B)和 BLI (C)确定。转 hMSCs 保留干细胞的性质, 通过鉴别分析证实转 hMSCs 分化成软骨细胞、脂肪细胞和成骨细胞的能力。在功能性分析中, 将羟基磷灰石/磷酸磷酸盐基质的陶瓷立方体植入到免疫缺陷 CB17-Prkdc 小鼠的皮下, 以证明 hMSCs 形成异位供体骨的能力. (D)。在 A = 500 µm 中缩放条形图 C 表示 BLI (103光子/s/厘米2/度) 的单位。在 D = 200 µm 中缩放条形<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55367/55367fig1large.jpg" target="_blank">图. 请单击此处查看此图形的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55367/55367fig2.jpg"> 
图 2: 超声引导注射仪器.(a)全景的仪器: 心脏超声成像系统 (红场), 传感器安装在其贴切的持有人, 注射器持有人和手术表 nosecone 麻醉 (绿色广场), 吸入麻醉机与感应室 (黄色正方形)。(B)注射器针的超声图像: 针尖 (红色箭头) 在左心室 (黄色箭头) 内清晰可见。(C)新的动脉血流出到注射器确认在 hMSC 管理前成功地将注射器针插入左心室。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55367/55367fig2large.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/55367/55367fig3.jpg"> 
图 3. hMSCs 在桑普小鼠的心内/动脉内部管理后定位到小肠.显示 3-4 小鼠/组的代表性图像, 注射后24小时安乐死和三独立实验。在体内生物发光成像显示在注射后的桑普和 AKR 小鼠的 hMSCs 的存在24小时(A)。在桑普小鼠的前体分析表明, hMSCs 优先定位到小肠 (即,炎症部位) (D), 而它们往往不留在无炎症控制的小肠中 AKR鼠标(C)。事实上, 在第一次动脉通道后, hMSCs 开始在 AKR 小鼠的肺部积聚更多, 而不是桑普老鼠(B)。 在桑普和 AKR 小鼠的脾、肝、肺中也可检出一小部分 hMSCs (B、C、D)。 刻度线表示 BLI (103光子/s/厘米2/度) 的单位。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55367/55367fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflamma

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

小鼠在结肠炎症模型中的研究表明, 一小部分 (1-5%) 的间充质干细胞 (MSC) 静脉注射或腹腔回家到发炎的结肠1,2。本研究表明, 超声引导下的骨髓间充质干细胞注射可增加肠道定位。

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

ultrasound-guided-intracardiac-injection-human-mesenchymal-stem-cells

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K 次观看.  Case Western Reserve University. 小鼠在结肠炎症模型中的研究表明, 一小部分 (1-5%) 的间充质干细胞 (MSC) 静脉注射或腹腔回家到发炎的结肠1,2。本研究表明, 超声引导下的骨髓间充质干细胞注射可增加肠道定位。

视频: Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

科研

医学

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

利用人类纤维原细胞线粒体表型的监测领域的帕金森氏病

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

基因治疗的载体和干细胞视网膜下注射到老鼠的眼睛

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

二硝基苯磺酸/ TNBS结肠炎模型:提供洞察炎症性肠病和膳食脂肪的影响

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同基因小鼠黑色素瘤细胞注射，以确定其在肺转移潜能

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.

分离肺内动脉和平滑肌细胞以研究血管反应

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of ...

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and experimental verification. The effective components and potential targets of Trichosanthis and Fritillaria thunbergii were collected by high-throughput experiment and reference-guided (HERB) database of traditional Chinese medicine and a similarity ensemble approach (SEA) database, and the LUAD-related targets were queried by the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. A drug-component-disease-target network was constructed by Cytoscape software. Protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analyses were conducted to obtain core targets and key pathways. An aqueous extract of Trichosanthes-Fritillaria thunbergii and A549 cells were used for the subsequent experimental validation. Through the HERB database and literature search, 31 effective compounds and 157 potential target genes of Trichosanthes-Fritillaria thunbergii were screened, of which 144 were regulatory targets of Trichosanthes-Fritillaria thunbergii in the treatment of lung adenocarcinoma. The GO functional enrichment analysis showed that the mechanism of action of Trichosanthes-Fritillaria thunbergii against lung adenocarcinoma is mainly protein phosphorylation. The KEGG pathway enrichment analysis suggested that the treatment of lung adenocarcinoma by Trichosanthes-Fritillaria thunbergii mainly involves the PI3K/AKT signaling pathway. The experimental validation showed that an aqueous extract of Trichosanthes-Fritillaria thunbergii could inhibit the proliferation of A549 cells and the phosphorylation of AKT. Through network pharmacology and experimental validation, it was verified that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

This study reveals the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma based on network pharmacology and experimental verification. The study also demonstrates that the PI3K/AKT signaling pathway plays a vital role in the action of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma.

毛滴虫对肺腺癌作用机制的网络药理学预测及实验验证

We aimed to study the mechanism of Trichosanthes-Fritillaria thunbergii in treating lung adenocarcinoma (LUAD) based on network pharmacology and ...

大鼠 DRG 压缩模型的标准化 Tuina 协议

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

作者聚焦： 推拿对压迫背根神经节疼痛的大鼠模型的镇痛作用  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

推拿对大鼠模型的镇痛作用，压迫背根神经节疼痛

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...