体外培养中CSA对缺氧复氧所致MSCs凋亡的影响和机制初探

发布时间：2018-01-15 10:45

本文关键词：体外培养中CSA对缺氧复氧所致MSCs凋亡的影响和机制初探　出处：《浙江大学》2007年硕士论文　论文类型：学位论文

【摘要】： 骨髓间充质干细胞(MSCs)是组织工程研究中普遍采用的种子细胞。于1867年德国病理学家Cohnheim首次提出骨髓中存在MSCs，随研究进一步深入，人们发现这种细胞能分化为多种间充质细胞并形成骨、软骨、肌腱、脂肪等组织，甚至可被诱导形成心肌细胞，，加之其易于获取、具有免疫耐受性、能长期存活、易于转染并能长期表达外源基因等特点，干细胞应用研究日益引起科学家们的关注。目前MSCs在临床上已开始实验性用于心脏修复，但由于局部微环境促凋亡因素的存在使其在移植后不可避免出现凋亡。而已有许多基础实验和临床研究证实环胞霉素A(CSA)可以减少多个细胞系发生的凋亡，CSA对MSCs是否也具有相近的作用目前尚不得而知。本实验采取不同浓度CSA处理大鼠MSCs后，在体外环境中给予缺氧再复氧后应用多个方法检测其凋亡的情况并初步测定部分有关的凋亡通路上关键因子初步解释其可能的抗凋亡机制。方法：采用密度梯度离心法分离获取SD大鼠的MSCs，再以贴壁筛选法纯化MSCs。取传代后第三代细胞进行流式细胞学鉴定，进入实验。先采取不同浓度(0.5-5μmol/L)CSA对MSCs进行预处理，实验组根据CSA浓度分为(0uM CSA组，0.5uM CSA组，1uM CSA组，2.5uM CSA组，5uM CSA组)以及常规培养组。然后实验组在体外环境中给予缺氧再复氧(H/R)后和常规培养组应用多个方法检测其凋亡的情况，并检测细胞色素C的释放和caspase-3的活性，线粒体膜电位(△ψm)的变化，以及AIF的转位等。结果：在缺氧复氧处理后的MSCs出现了CSA(0.5-5μmol/L)剂量依赖下的凋亡减少。其可能的一个途径就是通过直接或间接对抑制线粒体细胞色素C的释放和AIF的转位从而稳定△ψm，控制胞浆caspase-3的活性来达到抗凋亡的作用。结论：CSA在体外培养下可能通过直接或间接抑制线粒体细胞色素C的释放和AIF的转位从而稳定△ψm并进一步影响胞浆caspase-3的活性来减少对缺氧再复氧培养所诱发MSCs的凋亡。
[Abstract]:Bone marrow mesenchymal stem cells (MSCs) is widely used as seed cells in the tissue engineering. In 1867 the German pathologist Cohnheim MSCs first proposed the existence of the bone marrow, with further research, people found that the cells can differentiate into multiple mesenchymal cells and the formation of bone, cartilage, tendon, fat, even by inducing the formation of myocardial cells, and it is easy to get, with immune tolerance, which can survive for a long time, easy transfection and long-term expression of exogenous gene characteristics of stem cells research has attracted the attention of scientists. The MSCs in clinical experiments have been started for cardiac repair, but due to the existence of local micro environment and promote apoptosis the factors in the inevitable emergence of apoptosis after transplantation. But there are many basic experiments and clinical studies confirmed that cyclosporin A (CSA) can reduce apoptosis in multiple cell lines occurred, CSA to MSCs Whether it has the same effect. This experiment is still can make nothing of it take different concentrations of CSA after MSCs in rats in vitro to hypoxia reoxygenation after application of multiple methods to detect the apoptosis and preliminary determination of key factor related apoptosis pathway on the part of preliminary interpretation of the possible mechanism of anti apoptosis.
Methods: using density gradient centrifugation to obtain SD rats MSCs, with adherence screening method for purification of MSCs. from cultured cells of the third generation of flow cytometric identification into the experiment. First take different concentration (0.5-5 mol/L) CSA pretreatment on MSCs, the experimental group was divided into 0uM (according to CSA CSA 0.5uM group, CSA group, 1uM CSA group, 2.5uM CSA group, 5uM CSA group) and the conventional culture group. Then the experimental group in vitro to hypoxia reoxygenation (H/R) and conventional culture group application of multiple methods to detect the apoptosis, and the detection of cytochrome C release and caspase-3 the activity of mitochondrial membrane potential (Delta Psi m) changes, and the translocation of AIF.
Results: in hypoxia treated MSCs CSA (0.5-5 mol/L) the dose dependent decrease of apoptosis. A possible way is through direct or indirect inhibition of mitochondrial cytochrome C release and AIF translocation to stable Delta Psi m, active control of cytoplasmic caspase-3 to resist the role of apoptosis.
Conclusion: CSA can directly or indirectly inhibit the release of cytochrome C and the translocation of AIF and m and further affect the stability of delta psi cytosolic caspase-3 activity to reduce the apoptosis of hypoxia reoxygenation induced by MSCs cultured in vitro.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

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