间充质干细胞内皮分化前流动加载对所得细胞表型及生物学行为的影响

发布时间：2018-06-03 04:28

本文选题：流动切应力 + 间充质干细胞　；参考：《四川大学》2007年博士论文

【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。
[Abstract]:Objective: To investigate the changes in the biological properties of antithrombotic, vasoactive substances and adhesion factor expression induced by endothelial induced cells after the effect of flow shear stress factors on rat bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs), and explore the combination of flow shear stress loading and MSCs endothelium differentiation. Can seed cells be constructed in vitro to meet the needs of ischemic heart disease (IHD) treatment and cardiovascular tissue engineering.
Methods: 1. the density gradient separation method of Percoll separation liquid was used to separate and purify the SD rat MSCs in vitro. The cells were identified by morphological characteristics, expression of surface markers and multidirectional differentiation potential. After the purified cells were placed in the endothelial induction solution containing VEGF and bFGF for 2 weeks, the cells were inverted with phase contrast microscope to observe the cells. Morphological changes, immunofluorescence staining were used to observe the expression of CD31 in cells. Immunohistochemical method was used to detect the expression of factor VIII factor. The ultrastructure of.2. was observed by transmission electron microscope. The isolated rat MSCs was divided into static control group according to the loading stress size, 5 dyn / cm~2 shear stress loading group and 10dyn / cm~2 shear stress group. In the force loading group, after the shear stress loading group completed the 3 hour shear stress loading, the three groups were induced by the static direction endothelium for 14 days. The real-time quantitative reverse transcription polymerase chain reaction (real-time fluorescent quantitation reverse transcription-polymerase chain reaction, real-time RT-PCR) and the flow cytometry (flow cytometry) were used respectively. The changes of t-PA, eNOS, ET-1, ICAM-1, VCAM-1 and other factors mRNA and protein expression levels were observed. The cell culture of the flow shear stress loading group was subcultured 3 days later, and the expression of the above factors after the passage was detected again..3. also observed the NO synthesis of the cells, the total antioxidant capacity (T-AOC), and NADPH oxidase. Changes in the gp91phox subunit mRNA.
Results: 1. after 2 weeks of endothelium induction, the cultured cells obtained the endothelial specific cell morphology, arrangement and ultrastructure, and expressed the endothelial specific antigen CD31 and factor VIII associated antigen.2. to apply the physiological range of laminar shear stress 3H to MSCs, and the endothelial cells induced the t-PA, eNOS, ET-1, ICAM-1, VCAM-1, etc. of the P1 generation cells. The expression level of factor mRNA increased obviously, and was dependent on the shear stress intensity. Among the above factors, the expression of eNOS and ET-1 decreased obviously, except that the expression of eNOS and ET-1 decreased, but the decrease of t-PA and VCAM-1 expression was not obvious in the eNOS, ET-1, ICAM-1, and other factors of the t-PA and VCAM-1 expressions. After the loading of.5 dyn / cm~2, the expression of eNOS decreased, and the expression of eNOS increased after 10 dyn / cm~2 loading. The expression of the remaining factors increased in both the shear stress intensity and the magnitude of the shear stress intensity. The eNOS expression of the 5 dyn / cm~2 group was still lower than the control level in P2 generation cells after the passage, and the level of 10dyn per cm~2 group decreased to the control level. After the expression of other factor proteins decreased to the control level.4., the NO production and T-AOC increased and the mRNA expression of gp91phox subunit decreased, and all of them were dependent on the shear stress intensity after applying the laminar shear stress 3H in the physiological range of MSCs.
Conclusion: 1. the bone marrow MSCs of adult SD rats can be induced to be endothelial cells in vitro, and it is expected to become a cell therapy for cardiovascular diseases and the ideal seed cell source of cardiovascular tissue engineering. The flow shear stress in the.2. physiological range of MSCs can be used for the anti blood thrombus ability, antioxidant capacity and vasoactive activity of endothelial cells derived from MSCs The effect and intensity of the regulatory effect and intensity vary with the cell factor. This may be because different cytokines are the result of the response to the same type of shear stress by the specific molecular mechanism,.3. exerts a physiological range of laminar cutting to the MSCs stage. Stress, as an effective seed cell preconditioning method and means, can make the seed cells more capable of proliferation and differentiation, better antithrombotic formation, anti-inflammatory and anti atherosclerotic properties, and can help IHD cell therapy and the development of cardiovascular tissue engineering.
【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R329

【参考文献】