【作者】 杨思远；

【导师】 石应康；

【作者基本信息】 四川大学 ， 外科学， 2007， 博士

【摘要】 目的：通过研究流动切应力因素作用于大鼠骨髓间充质干细胞(mesenchymal stem cells, MSCs)后，对其内皮诱导所得细胞抗血栓形成、血管活性物质生成及粘附因子表达等生物学性质的变化，探讨流动切应力加载与MSCs内皮分化过程相结合后，能否在体外构建出满足缺血性心脏病(ischemic heart disease, IHD)治疗及心血管组织工程需要的种子细胞。方法：1．采用Percoll分离液密度梯度分离法，体外分离及纯化SD大鼠MSCs，经过形态学特征、表面标志物表达及多向分化潜能等多方面鉴定所得细胞。将纯化后细胞置入含VEGF及bFGF的内皮诱导液中定向诱导2周后，倒置相差显微镜观察细胞形态变化，免疫荧光染色观察细胞CD31表达情况，免疫组织化学方法检测Ⅷ因子表达情况，透射电子显微镜观察细胞内超微结构。2．将分离纯化所得的大鼠MSCs按照加载切应力大小分为静态对照组、5 dyn／cm~2切应力加载组和10dyn／cm~2切应力加载组，切应力加载组完成3小时切应力加载后，三组均于静态下定向内皮诱导14天，分别以荧光实时定量逆转录-聚合酶链反应(real-time fluorescent quantitation reverse transcription-polymerase chain reaction,real-time RT-PCR)及流式细胞仪(flow cytometry)观察所得细胞的t-PA、eNOS、ET-1、ICAM-1、VCAM-1等因子mRNA及蛋白表达水平的变化情况。流动切应力加载组所得细胞培养3天后传代，再次检测传代后细胞上述因子的表达情况。3．同时也观察所得细胞的NO合成量、总抗氧化能力(T-AOC)、及NADPH氧化酶gp91phox亚单位mRNA的变化情况。结果：1．内皮诱导2周后，培养细胞获得了内皮特异性细胞形态、排列方式及超微结构，并表达内皮特异性抗原CD31及Ⅷ因子相关抗原。2．对MSCs施加生理范围内大小的层流切应力3h后，其内皮诱导所得P1代细胞的t-PA、eNOS、ET-1、ICAM-1、VCAM-1等因子mRNA表达水平均明显升高，且与切应力强度呈依赖关系。传代后P2代细胞上述因子中，除ICAM-1表达继续升高外，其余因子表达下降，其中eNOS和ET-1表达下降明显，而t-PA和VCAM-1表达下降不明显。3．所得P1代细胞的eNOS、ET-1、ICAM-1、VCAM-1等因子蛋白表达水平变化趋势不同。5 dyn／cm~2加载后，eNOS表达下降，而10 dyn／cm~2加载后eNOS表达增加。其余因子无论切应力强度大小，均表达增加。传代后P2代细胞中，5 dyn／cm~2组eNOS表达仍低于对照水平，而10dyn／cm~2组水平降低至对照水平。同时其余因子蛋白表达下降至对照水平。4．对MSCs施加生理范围内大小的层流切应力3h后，其内皮诱导所得细胞的NO产量及T-AOC增加，gp91phox亚单位mRNA表达下降，均与切应力强度呈依赖关系。结论：1．成年SD大鼠骨髓MSCs能在体外定向诱导为内皮细胞，有望成为心血管疾病的细胞治疗及相关心血管组织工程理想的种子细胞来源。2．生理范围内流动切应力能对由MSCs分化而来的内皮细胞抗血栓能力、抗氧化能力、血管活性物质生成及粘附分子表达等多方面进行精细的调控。其调控效果及强度因细胞因子的不同而不同。这可能是由于不同的细胞因子是通过各自特异的分子机制来对同一类型切应力作出反应的结果。3．向MSCs阶段施加生理范围大小的层流切应力，可作为一种有效的种子细胞预处理方法及手段，能使种子细胞具有更强的增殖分化能力，更优良的抗血栓形成、抗炎症及抗动脉粥样硬化性质，有助于IHD细胞治疗及心血管组织工程的发展。更多还原

【Abstract】 Objective: Through investigating the effects on the abilities of anti-thrombus, synthesis of vasoactive substances and expression of adhesion molecules of the endothelial cells induced from rat bone marrow mesenchymal stem cells (MSCs) after flow loading. And approaching possibility to construct seeded cells which can well satisfied with the needs for ischemic heart disease treatment and cardiovascular tissue engineering by connecting flow shear stress and the process of endothelial differentiation of MSCs.Methods: (1). S-D rats bone marrow MSCs could be separated and purified in vitro by the Percoll density gradient separation,. Morphology characteristics, cell surface markers and the potency of multi-directional differentiation were applied to identify the cells. The purified cells were induced to endothelial linage in special media contain VEGF and bFGF for 2 weeks, the changes of cell morphology were observed under an inverted phase contrast microscope, the expression of CD31 was measured by immunofluorescence strain(IF), immunohisto- chemistry (IH) strain were used to investigate the factorⅧrelated antigen, a transmission electron microscope (TEM) was applied to observe the cells ultramicro-structure.. (2) the purified MSCs were divided into control group, 5 dyn/cm~2 group and 5 dyn/cm~2 group according the intensity of shear stress loading. As soon as the 3 hours stress loading finished , all groups were induced to endothelial linage for 14 days, the mRNA and protein expression levels of t-PA, eNOS, ET-1, ICAM-1, VCAM-1 were measured by real-time RT-PCR and flow cytometry. The shear loading groups cells were cultured for more 3 days and then passaged to next generation, and all mentioned factors were measured again. (3). At the same time, the amount of NO synthesis, total anti-oxidation competence(T-AOC), and the mNRA expression level of gp91phox, a subunit of NADPH oxidase, were all detected.Results: (1). After induced for 2 weeks ,the cells acquired endothelial cell morphology, arrangement and ultramicro structure, and expressed the endothelial special antigen-CD31 andⅧfactor. (2). after exposing to physiological shear stress, the mRNA expression levels of t-PA, eNOS, ET-1, ICAM-1, VCAM-1 of P1 group had a obviously increase in an intensity depend manner. When the cells were passaged ,the mRNA expression levels of ICAM-1 kept on increasing,and that of the rest decreased .among them , mRNA expression of eNOS and ET-1 decreased significantly. (3). after exposing to physiological shear stress, the change of the protein expression levels of eNOS、ET-1、ICAM-1、VCAM-1 of P1 group differed with shear stress intensity, the expression levels of eNOS decreased in 5 dyn/cm~2 and increased in 10 dyn/cm~2. the other factors level increased regardless of shear intensity. When cells passaged to next generation, eNOS remained lower in 5 dyn/cm~2 and decreased to control level in 10dyn/cm~2. (4). after exposing to physiological shear stress for 3 hours, the yield of NO and T-AOC of endothelial cells induced from MSCs increased, and the mRNA level of gp91phox decreased in an intensity-dependent manner.Conclusion: (1). Adult SD rat bone marrow MSCs can be induced to endothelial cells in vitro,it would be an ideal seeded cell source for cardiovascular disease treatment and cardiovascular tissue engineering. (2). Physiological fluid shear stress could refined regulate the anti-thrombus ability, the anti-oxidation ability, the formation of vasoactive substances and the expression levels of cell adhesion molecules. The effects and intensity of regulation differed with different factors, which may due to different factors reacted to shear stress in respectively special molecule mechanism. (3). It would be a efficient method for preconditioning the seeded cells by exposing physiological fluid shear stress to MSCs, it could make the cells acquire strong proliferation and differentiation ability, superordinary anti-thrombokinesis, anti-inflammation and anti atherosclerotic character, and it would helpful to the development of IHD treatment and cardiovascular tissue engineering.更多还原