这项工作得到了国家心脏, 肺和血液研究所的资助 R01088554, T32HL007284 和美国心脏协会 predoctoral 奖学金14PRE20420024。我们特别感谢 St. 裘德细胞成像共享的研究核心, 包括兰德尔和霍纳的电子显微镜图像, 亚当 Straub 为协助与代表性的免疫荧光图像, 和 Pooneh 巴盖尔·阿萨迪为她对原稿协议有价值的反馈。

1. 用于 VCCC 的电镀单元 <ol> <li> 区域性大 225 cm 2 瓶 EC 和 SMC 在不育条件下在37和 #176; C, 直到70-90% 汇合。确保使用的 EC 比 SMC 多;如果使用初级人类 ec 和 smc, 通常3大烧瓶 ec 到1大烧瓶 smc. 
	<ol> <li> 使用较低通道的主单元格, 并且不使用过去的段落10。选择基于单元格的区域性媒体共。对于原发性人冠状动脉 ec, 使用 EBM-2 ec 基底介质与 EGM2 MV 生长因子。对于原发性人冠状动脉 smc, 使用 SmBM smc 基底介质与 SmGM-2 生长因子。在使用培养细胞之前, 将生长因子添加到介质中. </li>
	</ol> </li> <li> VCCC 1 天的构造: 用消毒剂 (如 、Cavicide) 喷洒大 (150 mm) 培养皿和盖子, 然后用纸巾或无绒抹布擦拭。然后用70% 乙醇 (乙醇) 喷雾培养皿, 将它们放在引擎盖上晾干. </li>
	<li> 在无菌条件下打开过滤器插入板 (参见 材料表 ), 并将插入物的 SMC 侧 (过滤器的底部) 涂上纤维连接蛋白溶液。保持插入在提供的6井板, 吸管纤维连接蛋白解决方案通过侧缝到底部的板, 确保底部一半的过滤器覆盖 (不超过1毫升). </li>
	<li> 经过30分钟的处理, 纤维连接蛋白溶液在室温下在细胞培养罩, 采取插入出的板, 真空任何过剩的解决方案, 并倒置, 放置到底部一半的清洁培育皿。预留。在吸痰过剩溶液时, 一定不要干扰过滤器插入膜;这可以避免通过轻轻地倾斜板, 并清除解决方案边缘的插入. </li>
	<li> 处理 225 cm 2 烧瓶与3毫升的 5ml 2X 胰蛋白酶-EDTA 溶液, 一旦细胞解除了板块, 添加9毫升 smc 介质, 以中和胰蛋白酶 (3:1, 媒体: 胰蛋白酶)。转移到锥形管, 混合好, 和吸管10和 #181; L 到例上以确定单元格号. 
	<ol> <li> 计算 5 x 5 框中的单元格数 ( 即 , 18), 将其乘以1万以获取 1 mL (18万) 中的单元格数。然后乘以12毫升 (细胞悬浮总体积, 216万 SMC)。确保有足够的 SMC, 以满足所需的油井数量. 
		注: 例如, 1 井应该有 7.5万 smc, 因此18口井将有135万总 smc。除以每1毫升18万细胞所需的135万细胞, 这导致7.5 毫升的细胞悬浮, 将需要电镀。然后计算所需的最终体积 (18 井 x 750 和 #181; L = 13.5 毫升)。因此, 采取7.5 毫升的细胞悬浮, 使体积高达13.5 毫升与 5ml SMC 介质, 并混合良好. </li>
	</ol> </li> <li> 板750和 #181; L 单元悬浮物包含大约 7.5万 SMC 到每个过滤器的底部, 注意不要泄漏任何介质和电池解决方案。将盖子放在顶部, 并在37和 #176 中小心地将培养皿与插入器一起转移到恒温箱中. 
	注: 其他细胞类型的最佳细胞密度将需要根据经验确定. </li>
	<li> 在2天, 用2毫升新鲜的 5ml SMC 介质填充干净的6个菜肴。从孵化器中取出盘子。一次取出一个插入物, 然后通过抽吸将介质取出, 然后放入 SMC 介质中的6井盘中. </li>
	<li> 将1毫升的0.5% 牛凝胶溶液涂在过滤器 (EC 侧) 的另一侧, 在37和 #176 上至少30分钟; C. </li>
	<li> 处理 225 cm 2 烧瓶, 3 毫升的 5ml 2X 胰蛋白酶-EDTA (10x 稀释在无菌 Dulbecco 和 #39; s PBS) 溶液中, 轻轻敲击烧瓶, 以解除板上的细胞. 
	注: 可能需要将烧瓶放回37和 #176 的恒温箱中; C 为1-3 分钟。
	<ol> <li> 一旦细胞从板上脱落, 加入9毫升 EC 培养基来中和胰蛋白酶 (3:1)。转移到锥形管, 池一起, 混合好, 和吸管10和 #181; L 到例上以确定单元格数。
		<ol> <li> 计算 5 x 5 框中的单元格数 ( 即 , 60), 将其乘以1万以获取 1 mL (60万) 中的单元格数。然后乘以12毫升 (细胞悬浮总体积, 720万 EC)。确保有足够的 EC, 以满足所需的油井数量. 
			注: 每个井需要大约 36万 EC。例如, 18 口井需要 648万 EC。除以每1毫升 60万 EC 所需的648万细胞, 这结果在10.8 毫升的细胞悬浮, 将需要电镀。然后计算所需的最终体积 (18 口井 x 1 毫升 = 18 毫升)。因此, 采取10.8 毫升的细胞悬浮, 使体积高达18毫升与 5ml EC 介质, 并轻轻重. </li>
		</ol> </li> </ol> </li> <li> 板 1 mL 36万 EC 到每个插入过滤器的顶部。让细胞在37和 #176 不受干扰地孵育; C 为 24 h. 4 天更换培养基。5天收获. </li>
</ol> 2。从 VCCC <ol> 中获取分数 <li> 在4和 #176 中执行隔离, 并将所有仪器放在冰上. </li>
	<li> 请记住单独插入的数量, 并将裂解缓冲器添加到标记为梅杰的50毫升管中 (用于隔离过滤器)。然后贴上两个 10 mm 的盘子;一个用于 EC, 一个用于 SMC. </li>
	<li> 根据裂解物的浓度需要调整裂解缓冲液的体积; 对于15插入, 500 和 #181; 梅杰管的裂解缓冲液和250和 #181; 推荐使用每培养皿. </li>
	<li> 一次使用一个 6-好的插入板。吸入细胞培养罩中的培养基, 然后将其输送到冰上的冷室. 
	注意: 以下说明用于1插入隔离. </li>
	<li> 吸管10和 #181; L 1x PBS 到插入的 SMC 一侧. </li>
	<li> 使用细胞提升器将细胞刮到插入物的一端, 并将电池从升降器转移到具有裂解缓冲液的 SMC 培养皿中. </li>
	<li> 当电池升降器触到盘中的裂解缓冲液后, 不要让它接触到插入式滤光片, 直到它被完全擦干并在纸巾上晾干。重复对 smc 过滤器的刮擦至少一到两次, 以完全去除其余的 smc 细胞碎片. </li>
	<li> 将 "插入" 和 "吸管 10" #181; 将 1x PBS 转到插入过滤器的上部/EC 一侧。重复步骤2.6 和2.7 与细胞刮刀和 EC 培养皿. </li>
	<li> 用吸管从过滤器的 ec 一侧收集剩余的电池和 PBS 浆料, 并将其转移到 ec 培养皿中进行裂解缓冲。要非常彻底地刮除过滤器两侧的细胞, 以使梅杰分数最小的 EC/SMC 污染. </li>
	<li> 小心使用手术刀从塑料插入物中切出过滤器。做到这一点, 削减70-80% 它远离塑料和使用镊子拉过滤器完全关闭塑料插入结构。将过滤器放在50毫升锥形管中, 用裂解缓冲液标记梅杰, 确保它位于裂解缓冲液中, 并保持湿润. </li>
	<li> 按6组重复执行隔离步骤, 直到处理完所有插入操作. </li>
	<li> 确保不同的治疗组保持在单独的管和菜中. </li>
	<li> 涡旋50毫升梅杰锥形管在全强度十五年代或直到混合好。将过滤器从梅杰的锥形带钳上取下, 将它们沿管子的侧壁拖动, 以使试管中的蛋白质和液体达到最大值。在此点之后, 丢弃筛选器. </li>
	<li> 一旦所有的过滤器已经从梅杰锥管中删除, 做一个快速旋转 (10-15 s 在最大速度〜 1.6万 x g) 在离心机, 把所有的液体和蛋白质的底部的管。将梅杰管和 EC 和 SMC 培养皿的内容转移到分离的小型离心管中. </li>
	<li> 可选: 几种梅杰/SMC/EC 管的内容在设置4三十年代脉冲在冰或质轻轻地用手. </li>
	<li> 离心机在最大转速 (〜 1.6万 x g) 15 分钟在4和 #176; c. 用二辛可宁测定法吸管出上清液和测定蛋白质含量的裂解物. </li>
</ol> 3。在 VCCC 孵化的5天, 为 En 免疫荧光 <ol> <li> 收割 VCCC, 从孵化箱中取出插入物。在细胞培养罩中工作, 从插入式过滤器的下井中抽离 SMC 介质, 用 1x PBS 冲洗两次。在 EC 方面重复. </li>
	<li> 保持插入完好, 并在板中, 添加4% 甲醛 (粉煤灰) 10-20 分钟在一个步入式的4和 #176; C 室在一个温和的振动筛。将插入过滤器的两侧用 1x PBS 清洗5分钟, 重复两次. 
	注意: 粉煤灰是有毒的, 必须小心处理. </li>
	<li> 在板中执行阻断、主、二级抗体孵化, 确保过滤器的两侧都保存在抗体 + 阻断液中 (9 毫升 PBS、25和 #181; l X100、50毫克牛血清白蛋白、500和 #181; l 正常血清) </li>
	<li> 要在幻灯片上安装滤镜, 请将过滤器从塑料插入物中取出, 并将手术刀安装在手柄上, 并放置在带有安装介质的显微镜幻灯片上. </li>
</ol> 4。采集 VCCC 的横向免疫荧光 <ol> <li> 在 VCCC 孵化的5天, 从孵化箱中取出插入物。吸出介质从两边的插入过滤器在细胞培养罩和冲洗每一侧的过滤器两次与 1x PBS. </li>
	<li> 保持插入完好, 并在6井板, 增加4% 粉煤灰和孵化过夜在一个步入式的4和 #176; C 室在一个温和的振动筛. 
	注意: 粉煤灰是有毒的, 必须小心处理. </li>
</ol> 5。嵌入 VCCC 为横向免疫荧光 <ol> <li> 在过滤器插入物被固定了大约 24 h 在4% 煤灰之后, 转移到70% 乙醇为至少 24 h, 但至多几星期. </li>
	<li> 在自动组织处理器上长时间 (通宵) 运行过滤器。使用程序如下: 70% 乙醇30分钟, 85% 乙醇30分, 95% 乙醇30分, 100% 乙醇30分, 100% 乙醇45分, 100% 乙醇45分, 二甲苯30分, 二甲苯30分, 二甲苯45分, 60 和 #176; c 石蜡 30 min, 60 和 #176; c 石蜡 30 min, 60, #176; C 石蜡 45 min. </li>
	<li> 经过处理后, 将采集到的过滤器转移到液体石蜡中, 将过滤器保存在卡带中. </li>
	<li> 从卡带上取下过滤器, 小心地用镊子把它放在边缘。用剪刀把过滤器切成两半, 形成半圆状半。放置在埋置站的冷板部分的2一半, 只要足够长的时间, 以填补嵌入模具与液体石蜡. </li>
	<li> 一旦充满液体石蜡, 很简单地将嵌入模具接触到冷板, 然后将每半部分的过滤器嵌入模具 (切边); 这确保在剖切过程中, 一个将从过滤器的中心向边缘切割.</li>
	<li> 在嵌入过程中, 使用镊子将过滤器放置到位, 以防止两个部分相互掉落, 直到石蜡冷却到足以让它们单独站立为止。将嵌入模具移至冷板, 直到完全凝固. 
	注: 冰盘上的冷却块有助于防止皱纹的部分. </li>
</ol> 6。VCCC 横向免疫荧光的切片和染色 <ol> <li> 用于石蜡切片和滑动安装: 切割5和 #181; m 节, 将剖面放置到42和 #176 中; C 水浴, 并拿起的部分使用正电荷幻灯片.然后在42和 #176 中一夜之间保持幻灯片; C 烘箱晾干. </li>
	<li> 在开始补液步骤之前, 在60和 #176 烘烤幻灯片; C 将石蜡融化, 并帮助将 VCCC 部分粘附到幻灯片上。冷却简单, 然后水化的幻灯片通过两个变化的二甲苯, 100% 乙醇, 95% 乙醇, 一个变化的70% 乙醇, 和两个变化的蒸馏水. </li>
	<li> 避免抗原检索方法, 涉及微波或加热的部分可能脱落的幻灯片. </li>
	<li> 在室温下执行1小时的标准阻塞, 然后在4和 #176 中使用主抗体进行过夜孵育, 其次是室温下的洗涤步骤和1小时的二次抗体。安装介质和片. </li>
</ol>

<ol>
	<li>Isakson, B. E., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterocellular+contact+at+the+myoendothelial+junction+influences+gap+junction+organization.">Heterocellular contact at the myoendothelial junction influences gap junction organization.</a> Circ Res. 97 (1), 44-51 (2005).</li><li>Little, T. L., Xia, J., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+tracers+define+differential+endothelial+and+smooth+muscle+coupling+patterns+within+the+arteriolar+wall.">Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.</a> Circ Res. 76 (3), 498-504 (1995).</li><li>Heberlein, K. R., Straub, A. C., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myoendothelial+junction:+breaking+through+the+matrix?.">The myoendothelial junction: breaking through the matrix?.</a> Microcirculation. 16 (4), 307-322 (2009).</li><li>Straub, A. C., Zeigler, A. C., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myoendothelial+junction:+connections+that+deliver+the+message.">The myoendothelial junction: connections that deliver the message.</a> Physiology (Bethesda). 29 (4), 242-249 (2014).</li><li>Isakson, B. E., Ramos, S. I., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++and+inositol+1,4,5-trisphosphate-mediated+signaling+across+the+myoendothelial+junction.">Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction.</a> Circ Res. 100 (2), 246-254 (2007).</li><li>Dora, K. A., Doyle, M. P., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+calcium+in+smooth+muscle+causes+endothelial+cell+generation+of+NO+in+arterioles.">Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.</a> Proc Natl Acad Sci U S A. 94 (12), 6529-6534 (1997).</li><li>Sonkusare, S. K., 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=Elementary+Ca2++signals+through+endothelial+TRPV4+channels+regulate+vascular+function.">Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function.</a> Science. 336 (6081), 597-601 (2012).</li><li>Ledoux, 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=Functional+architecture+of+inositol+1,4,5-trisphosphate+signaling+in+restricted+spaces+of+myoendothelial+projections.">Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.</a> Proc Natl Acad Sci U S A. 105 (28), 9627-9632 (2008).</li><li>Boerman, E. M., Everhart, J. E., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+age+decreases+local+calcium+signaling+in+endothelium+of+mouse+mesenteric+arteries+in+vivo.">Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo.</a> Am J Physiol Heart Circ Physiol. 310 (9), H1091-H1096 (2016).</li><li>Toussaint, F., Charbel, C., Blanchette, A., Ledoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+regulates+intracellular+Ca(2)(+)+dynamics+in+native+endothelial+cells.">CaMKII regulates intracellular Ca(2)(+) dynamics in native endothelial cells.</a> Cell Calcium. 58 (3), 275-285 (2015).</li><li>Truskey, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Cell+Vascular+Smooth+Muscle+Cell+Co-Culture+Assay+For+High+Throughput+Screening+Assays+For+Discovery+of+Anti-Angiogenesis+Agents+and+Other+Therapeutic+Molecules.">Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules.</a> Int J High Throughput Screen. 2010 (1), 171-181 (2010).</li><li>Biwer, L. 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=Two+functionally+distinct+pools+of+eNOS+in+endothelium+are+facilitated+by+myoendothelial+junction+lipid+composition.">Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.</a> Biochim Biophys Acta. 1861 (7), 671-679 (2016).</li><li>Straub, A. C., 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=Endothelial+cell+expression+of+haemoglobin+alpha+regulates+nitric+oxide+signalling.">Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling.</a> Nature. 491 (7424), 473-477 (2012).</li><li>Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+expression+of+an+Ins(1,4,5)P3+receptor+at+the+myoendothelial+junction+selectively+regulates+heterocellular+Ca2++communication.">Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication.</a> J Cell Sci. 121 (Pt 21), 3664-3673 (2008).</li><li>Axel, D. I., 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=Induction+of+cell-rich+and+lipid-rich+plaques+in+a+transfilter+coculture+system+with+human+vascular+cells.">Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells.</a> J Vasc Res. 33 (4), 327-339 (1996).</li><li>Hastings, N. E., Simmers, M. B., McDonald, O. G., Wamhoff, B. R., Blackman, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis-prone+hemodynamics+differentially+regulates+endothelial+and+smooth+muscle+cell+phenotypes+and+promotes+pro-inflammatory+priming.">Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming.</a> Am J Physiol Cell Physiol. 293 (6), C1824-C1833 (2007).</li><li>Herzog, D. P., Dohle, E., Bischoff, I., Kirkpatrick, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+communication+in+a+coculture+system+consisting+of+outgrowth+endothelial+cells+and+primary+osteoblasts.">Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts.</a> Biomed Res Int. , 320123 (2014).</li><li>Jacot, J. G., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+injury+induces+vascular+smooth+muscle+cell+proliferation+in+highly+localized+regions+of+a+direct+contact+co-culture+system.">Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.</a> Cell Biochem Biophys. 52 (1), 37-46 (2008).</li><li>Scott-Drechsel, D., 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=A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>van Buul-Wortelboer, M. F., 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=Reconstitution+of+the+vascular+wall+in+vitro.+A+novel+model+to+study+interactions+between+endothelial+and+smooth+muscle+cells.">Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.</a> Exp Cell Res. 162 (1), 151-158 (1986).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).</li><li>Heberlein, K. R., 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=A+novel+mRNA+binding+protein+complex+promotes+localized+plasminogen+activator+inhibitor-1+accumulation+at+the+myoendothelial+junction.">A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.</a> Arterioscler Thromb Vasc Biol. 32 (5), 1271-1279 (2012).</li><li>Straub, A. C., 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=Compartmentalized+connexin+43+s-nitrosylation/denitrosylation+regulates+heterocellular+communication+in+the+vessel+wall.">Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.</a> Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).</li><li>Cucina, 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=Vascular+endothelial+growth+factor+increases+the+migration+and+proliferation+of+smooth+muscle+cells+through+the+mediation+of+growth+factors+released+by+endothelial+cells.">Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells.</a> J Surg Res. 109 (1), 16-23 (2003).</li><li>Heberlein, K. R., 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=Plasminogen+activator+inhibitor-1+regulates+myoendothelial+junction+formation.">Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.</a> Circ Res. 106 (6), 1092-1102 (2010).</li><li>Isakson, B. E., Best, A. K., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+protein+on+actin+bridges+between+endothelium+and+smooth+muscle+in+arterioles+demonstrates+heterogeneous+connexin+expression+and+phosphorylation.">Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).</li><li>Biwer, L. A., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mediated+signalling+in+cellular+microdomains.">Endoplasmic reticulum-mediated signalling in cellular microdomains.</a> Acta Physiol (Oxf). 219 (1), 162-175 (2017).</li></ol>

作者没有什么可透露的。

VCCC 在综述 MEJs 的体外方面有许多优点, 但是在确定是否可以将 VCCC 用于特定的应用程序时, 还有一些讨论点。例如, 如果要与此模型一起使用不同的单元格类型, 则需要对其进行优化。我们已经使用了主要的人类细胞, 但它可以隔离的细胞从牛脉24, 或野生或淘汰赛小鼠, 并使用它们在 VCCC1,5,14。
<p class...

在小直径阻力动脉, 薄内弹性板 (IEL) 分离内皮 (EC) 和平滑肌细胞 (SMC)。细胞可以通过这个弹性矩阵的孔, 并通过缝隙连接通道的直接细胞质连接1,2。这种独特的结构称为 myoendothelial 结 (梅杰)。梅杰是一个小 (大约0.5 µm x 0.5 µm, 取决于血管床), 细胞投射主要组成的内皮细胞, 但可能起源于平滑肌以及3,4。许多调查人员已经证明了在梅杰上有选择地发生的信号网络的巨大复杂性, 使它成为促进内皮和平滑肌之间双向信号传递的一个特别重要的位置5 ,6,7,8,9,10。
然而, 在梅杰的信号通路的机械解剖是很难在一个完整的动脉。由于梅杰是细胞投射, 目前无法将体内的梅杰从血管壁隔离出来。因此, 开发了 VCCC 模型1 。重要的是, VCCC 复制的生理内皮形态学11和信号的极化之间的顶端和梅杰部分的细胞12。正是这种独特的模型, 促进了发现α血红蛋白是在内皮细胞, 极化到梅杰。这与常规培养的内皮细胞不表达α血红蛋白13形成对比。对于对微血管内皮有兴趣的研究人员来说, 使用 VCCC 中培养的内皮细胞可能更合适, 特别是如果目的是解剖在小直径阻力动脉中发生的信号通路。
使用一个坚固的塑料插入物, 含有小孔直径的过滤器 (直径0.4 µm), 以培养两种不同的细胞类型, 防止细胞在层间迁移。它导致细胞之间的10µm 距离, 这是明显长于在体内, 但仍然复制许多的体内特征的 MEJs, 包括蛋白质本地化和第二信使信令1,14. 此外, VCCC 允许通过在特定的蜂窝室中加入一个激动剂或拮抗器来锁定特定的细胞类型的信号。例如, 加载 EC 与 BAPTA, 以螯合钙, 并刺激 SMC 与肾上腺素14。与其他描述共培养模式的15,16,17,18,19,20,21, 这提供关于分离 mRNA 和蛋白质的不同分数的说明, 包括过滤毛孔内的不同的梅杰分数。这项技术添加到 VCCC 允许具体调查的变化 mRNA 本地化或转录22, 蛋白质磷酸化12,23, 和蛋白质活动12。本文将描述 EC 在过滤器和 SMC 底部的电镀, 虽然可以在不同的构象中培养两个单元类型11,18,24。

<table>
	<tbody>
		<tr>
			<td>24mm diameter, 0.4&#181;m Transwell Polyester membrane filter insert</td>
			<td>Corning</td>
			<td>3450</td>
			<td>This is one 6 well plate of inserts</td>
		</tr>
		<tr>
			<td>225cm2 flask</td>
			<td>Corning</td>
			<td>431082</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates (without filter inserts)</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery endothelial cells</td>
			<td>Lonza</td>
			<td>CC-2585</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery smooth muscle cells</td>
			<td>Lonza</td>
			<td>CC-2583</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 EC Basal media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EC growth factors (EGM2 MV Single quots)</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>SmBM SMC basal media</td>
			<td>Lonza</td>
			<td>CC-3181</td>
			<td></td>
		</tr>
		<tr>
			<td>SMC growth factors (SmGM-2 SingleQuot)</td>
			<td>Lonza</td>
			<td>CC-4149</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA 10X</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Dilute to 2X with sterile dPBS</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>large petri dishes for SMC Plating (150mm)</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine gelatin</td>
			<td>Sigma</td>
			<td>G-9382</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td>5&#181;g/ml of fibronectin and store at -20</td>
		</tr>
		<tr>
			<td>10X Hanks&#39; Buffered salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>10X Dulbecco&#39;s Phosphate buffered saline (dPBS)</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>Disposable Curved Scalpel (30mm cutting edge) with handle</td>
			<td>Amazon</td>
			<td>MDS15210</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>17-467-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>BD Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm petri dishes</td>
			<td>Falcon</td>
			<td>35-1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA lysis reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>Make 4% solution with dPBS</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Cavicide disinfectant</td>
			<td>Fisher</td>
			<td>131000</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA protein lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic dismembranator</td>
			<td>Fisher</td>
			<td>Model 50</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS for harvesting EC/SMC and immunocytochemistry</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>does not need to be sterile</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Embedding/Sectioning/Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Fisher</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated tissue processer</td>
			<td>Excelsior</td>
			<td>ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding station + cold plate</td>
			<td>Leica</td>
			<td>HistoCore Arcadia</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek Accu-Edge Microtome blade</td>
			<td>VWR</td>
			<td>25608-961 or 25608-964</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher</td>
			<td>22-230-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Fisher</td>
			<td>12-548-5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other Solutions</td>
			<td></td>
			<td></td>
			<td>**Perform all steps in sterile conditions if using the solution on live cells</td>
		</tr>
		<tr>
			<td>Sterile dPBS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>sterile HBSS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>Fibronectin stock solution</td>
			<td></td>
			<td></td>
			<td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed 
			Final concentration: 100&#181;g/mL</td>
		</tr>
		<tr>
			<td>Fibronectin to coat SMC side of VCCC</td>
			<td></td>
			<td></td>
			<td>thaw one aliquot and add 1mL of fibronectin (100&#181;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&#181;g/mL. 
			Final concentration: 5&#181;g/mL</td>
		</tr>
		<tr>
			<td>2x Trypsin EDTA solution</td>
			<td></td>
			<td></td>
			<td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) . 
			Final concentration: 2X</td>
		</tr>
		<tr>
			<td>Blocking/antibody solution</td>
			<td></td>
			<td></td>
			<td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td>
		</tr>
		<tr>
			<td>Bovine gelatin to coat EC VCCC</td>
			<td></td>
			<td></td>
			<td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions. 
			Final concentration: 0.50%</td>
		</tr>
		<tr>
			<td>1X PBS for harvesting cells</td>
			<td></td>
			<td></td>
			<td>does not need to be sterile</td>
		</tr>
	</tbody>
</table>

a cell culture model of resistance arteries

myoendothelial 结 (梅杰), 一个独特的信号 microdomain 在小口径阻力动脉, 展品定位的特定的蛋白质和信号的过程, 可以控制血管的音调和血压。由于它是一个投射从内皮或平滑肌细胞, 并且由于它的小尺寸 (平均, 面积为 ~ 1 µm2), 梅杰很难孤立地研究。然而, 我们已经开发了一种细胞培养模型, 称为血管细胞共育 (VCCC), 允许体外梅杰形成, 内皮细胞极化, 并解剖的信号蛋白和过程中血管壁的阻力动脉.VCCC 有多种应用, 可以适应不同的细胞类型。该模型由两个细胞类型生长在一个过滤器的相反两侧的0.4 µm 孔, 其中体外MEJs 可以形成。在这里, 我们描述如何创建 VCCC 通过电镀细胞和分离的内皮, 梅杰, 和平滑的肌肉分数, 然后可以用于蛋白质分离或活动化验。具有完整单元格层的过滤器可以固定、嵌入和分段进行荧光分析。重要的是, 这个模型的许多发现都被证实使用了完整的阻力动脉, 凸显了它的生理相关性。

用于 VCCC 的过滤器插入物有小, 0.4 µm 孔。图 1A, VCCC 滤镜嵌入的en 面视图显示了梅杰可以在体外形成的毛孔。该示意图描绘的是光滑的肌肉细胞镀上的过滤器的下部前一天内皮细胞被镀在对面 (图 1B)。一旦细胞被镀, EC, 梅杰, 和 SMC 分数可以隔离三天后。其中的一个示例显示在图 2A中, 其中纤?...

Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

A Cell Culture Model of Resistance Arteries

一个细胞培养模型的阻力动脉描述, 允许解剖的信号通路的内皮, 平滑肌, 或之间的内皮和平滑肌 (myoendothelial 交界处)。选择性应用激动剂或蛋白质隔离, 电子显微镜, 或免疫荧光可以利用这种细胞培养模型。

阻力动脉的细胞培养模型

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

a-cell-culture-model-of-resistance-arteries

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.8K 次观看.  University of Virginia School of Medicine. 一个细胞培养模型的阻力动脉描述, 允许解剖的信号通路的内皮, 平滑肌, 或之间的内皮和平滑肌 (myoendothelial 交界处)。选择性应用激动剂或蛋白质隔离, 电子显微镜, 或免疫荧光可以利用这种细胞培养模型。

视频: A Cell Culture Model of Resistance Arteries

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

使用穆林精密切肺片对肺动脉内下氧肺血管收缩进行视频形态分析

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

的Perigenital敏感性和前列腺肥大细胞活化新生母婴分离的小鼠模型评估

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

分离培养犬从动脉和静脉主血管内皮细胞

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

利用大鼠离体阻力动脉的视频显微镜对血管控制机制的评价

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

肌肉失衡: 运动人群功能性偏心肌筋强度的检测与训练

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...