【作者】 王珊；

【导师】 吴耀炯；

【作者基本信息】 清华大学 ， 生物学， 2016， 博士

【摘要】 间充质干细胞(mesenchymal stem cells,MSCs)具有多分化潜能、强大的旁分泌作用和低免疫原性等优点。动物试验研究显示,MSCs对多种组织损伤具有修复作用,表现出治疗多种重大疾病的应用前景,目前全球有多个MSCs治疗心肌梗塞和脑中风的临床试验在进行。对于MSCs的移植,静脉注射具有安全性高和容易操作等优点,是目前干细胞移植最常用的途径;但研究表明,体外培养的MSCs在静脉注射后到达靶器官的数量极少,绝大多数细胞不能通过肺循环而滞留在肺脏中,并很快死亡,严重影响其对靶器官的修复效果。然而造成MSCs肺滞留的原因和机制并不清楚。对MSCs肺滞留分子机制的研究将有助于研发减少MSCs肺阻塞提高其到达损伤器官数量的方法,对MSCs应用于临床治疗具有重大意义。在本论文中,流式细胞仪和蛋白质免疫印迹分析显示经体外连续贴壁培养的MSCs表面的很多整合素如integrinβ1,α5和αVβ3的表达水平都显著升高,而免疫荧光染色和流式细胞仪分析整合素的配体纤维粘连蛋白和玻连蛋白均在正常小鼠肺血管内皮细胞的表面表达。使用功能性阻断抗体阻断MSCs表面的integrinβ1,α5和αVβ3,使MSCs在静脉注射后在小鼠肺脏中的滞留量显著降低,多种检测方法如荧光定量PCR和组织学分析均验证了此结果;MSCs肺滞留量显著降低的同时伴随着在血液循环中的水平增加,并有更多的MSCs迁徙到小鼠心肌梗塞的心脏和耳朵发炎区域。本论文研究还发现,将连续传代并贴壁培养的MSCs经过短时间的三维立体培养,很多整合素的表达水平显著下降,静脉注射后在肺脏中的滞留量大幅度降低。我们的研究结果表明,整合素的过多表达和活化是MSCs肺滞留的一个重要因素。综上所述,本研究证明了传统贴壁培养的MSCs表面整合素过多表达和活化,产生过度的粘附能力,导致其静脉注射后粘附在肺血管壁上。使用特异性功能阻断抗体阻断整合素的功能或者将MSCs经过三维立体培养降低整合素的表达可以大幅度降低MSCs在肺中的滞留量,并增加MSCs在血液循环和到达缺血心肌和炎症组织的数量。因此本研究提示,通过降低MSCs表面整合素的功能是降低其肺阻塞,增加其损伤组织到达量的有效方法,对MSCs临床治疗具有重大的科学意义。更多还原

【Abstract】 Mesenchymal stem cells(MSCs)have shown a profound therapeutic potential in tissue repair and regeneration,and numerous clinical trials for a variety of diseases such as myocardial infarction and stroke are underway.Moreover,intravenous infusion has been a popular transplantation route of MSCs for its convenience and safety.However,recent studies indicate that MSCs are largely entrapped in the lungs after intravenous delivery and die shortly.The underlying mechanisms have been poorly understood.In this study,flow cytometry and western blot analysis showed that the expression levels of many integrins such as β1,α5,and αVβ3 in MSCs increased markedly upon cultured expansion in 2D monolayers,whose ligands fibronectin and vitronectin were detected on the surface of vascular endothelial cells in the lungs by immunostaining and flow cytometry.Blockade of integrin β1,integrin α5,or integrins αVβ3 with functional blocking antibodies significantly decreased the amount of MSCs entrapped in the lungs following intravenous infusion as determined by real-time PCR and histological analysis;meanwhile,corresponding increases in the levels of circulating MSCs in the blood and MSCs homed to the ischemic myocardium and inflamed ear were found.Intriguingly,a short period of 3D spheroid culture of MSCs,which had been expanded for several passages in monolayers,substantially reduced the expression levels of many integrins and the number of MSCs entrapped in the lungs.Our results indicate that excess expression and activation of integrins is a significant cause of lung entrapment of MSCs.In conclusion,in this study we provided forceful evidence to show that MSCs in conventional monolayer culture developed excess adhesive ability due to increased expression and activation of certain integrins and were capable of binding to the normal endothelium in the lungs.Blockade of integrins with functional blocking antibodies or downregulation of integrin expression through 3D spheroid culture resulted in substantially reduced lung entrapment of MSCs,increased levels of circulating MSCs in the blood,and enhanced homing and trafficking of MSCs to the ischemic myocardium and inflamed ear.Thus our study suggests a novel approach to increase MSC homing and engraftment to sites of tissue injuries by reducing the ECM binding activity of surface integrins.更多还原