小鼠骨髓间质干细胞和脂肪间质干细胞保护心肌作用及机制研究

发布时间：2018-05-13 02:37

本文选题：脂肪间质干细胞 + 骨髓间质干细胞　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:骨髓来源的间质干细胞(Bone marrow mesenchymal stem cells,BM-MSCs)和脂肪来源的间质干细胞(Adipose derived mesenchymal stem cells,AD-MSCs)均在心肌损伤修复中发挥重要作用,并且这种作用主要通过分泌exosomes来实现。但是,AD-MSCs和BM-MSCs修复作用的优劣尚不明确。本实验将探究C57BL/6小鼠来源的AD-MSCs和BM-MSCs在细胞特性、exosomes释放量和对缺血缺氧心肌的保护作用的区别及机制。方法:6-10周雄性C57BL/6小鼠用于分离培养AD-MSCs和BM-MSCs。MTS实验和细胞计数检测AD-MSCs和BM-MSCs的增殖能力和倍增时间。流式细胞术检测AD-MSCs和BM-MSCs的表面标志(CD29,Sca-1和CD34)和autophagy的表达。使用无血清培养基培养48h分别收集第四代AD-MSCs和BM-MSCs上清,利用超滤离心和Exo Quick-TC试剂盒从上清中分离纯化出AD-MSCs来源的exosomes(Exo-AD)和BM-MSCs来源的exosomes(Exo-BM),通过Nanosight仪器分析鉴定exosomes的大小和分布。DC蛋白定量检测exosomes的浓度及其他蛋白浓度。Western blot检测exosomes的表面标志CD63和细胞自噬相关分子Beclin-1。根据心肌细胞提取试剂盒的操作方法体外分离培养新生大鼠心肌细胞,根据MTS和LDH实验的检测指标进行体外构建心肌缺血缺氧模型。使用带红色荧光的PKH26标记Exo-AD和Exo-BM后与心肌细胞孵育12小时,荧光显微镜下检测exosomes的被摄取情况。MTS实验检测Exo-AD和Exo-BM处理心肌细胞缺血缺氧72小时后细胞的存活情况,JC-1实验检测Exo-AD和Exo-BM处理后心肌细胞线粒体膜电位(ΔΨm)的变化。miRNAs芯片分析检测Exo-AD和Exo-BM内各miRNAs的含量。结果:实验结果表明AD-MSCs较BM-MSCs的倍增时间短,增殖能力更强,并且分泌exosomes的能力更强。相反地,BM-MSCs内自噬体的表达量则更高。本实验成功构建了体外心肌缺血缺氧模型,Exo-AD和Exo-BM均能被新生大鼠心肌细胞摄取。与对照组相比,两者均具有心肌细胞缺血缺氧过程的保护作用,但是当同量exosomes处理时,Exo-BM对心肌细胞的保护作用则更强。miRNAs芯片分析检测,Exo-AD和Exo-BM内miRNAs的含量不尽相同,有243种miRNAs的表达在Exo-AD和Exo-BM中有差异,其中包括与心肌保护作用相关的miR-21、miR-30、miR-221和miR-223。结论:本研究发现AD-MSCs和BM-MSCs在细胞特性、exosomes释放量及对缺血缺氧的保护作用均有区别,其主要原因可能与Exo-AD和Exo-BM中miRNAs含量的差异有关。这些为临床MSCs的选择提供了理论依据。
[Abstract]:Objective: bone marrow mesenchymal stem cells (BM-MSCs) and adipose derived mesenchymal stem cells (AD-MSCs) play an important role in the repair of myocardial injury, and this effect is mainly achieved by secreting exosomes. However, the effect of AD-MSCs and BM-MSCs repair is not clear. The purpose of this study was to investigate the difference and mechanism of AD-MSCs and BM-MSCs derived from C57BL/6 mice in the release of exosomes and their protective effects on ischemic and hypoxic myocardium. Methods male C57BL/6 mice were isolated and cultured for 6 to 10 weeks. The proliferation and doubling time of AD-MSCs and BM-MSCs were measured by cell count and AD-MSCs assay. The expression of CD29 Sca-1 and CD34) and autophagy on the surface of AD-MSCs and BM-MSCs were detected by flow cytometry. The supernatants of the fourth generation AD-MSCs and BM-MSCs were collected in serum-free medium for 48 h. Exosomesus exo-ADfrom AD-MSCs and Exo-BMN from BM-MSCs were isolated and purified from supernatant by ultrafiltration centrifugation and Exo Quick-TC kit. The size and distribution of exosomes were identified by Nanosight instrument. The concentration of exosomes and other protein concentrations were detected by Nanosight. The surface marker of exosomes is CD63 and the cell autophagy related molecule Beclin-1. According to the method of cardiomyocyte extraction kit, neonatal rat cardiomyocytes were isolated and cultured in vitro. Myocardial ischemia and hypoxia model was established in vitro according to MTS and LDH test. Exo-AD and Exo-BM were labeled with red fluorescent PKH26 and incubated with cardiomyocytes for 12 hours. Detection of uptake of exosomes under fluorescence microscope. Detection of survival of cardiomyocytes treated with Exo-AD and Exo-BM for 72 hours after ischemia and hypoxia. JC-1 experiment to detect changes of mitochondrial membrane potential (螖蠄 m) of cardiomyocytes treated with Exo-AD and Exo-BM. The contents of miRNAs in Exo-AD and Exo-BM were detected by chip analysis. Results: the results showed that the multiplication time of AD-MSCs was shorter than that of BM-MSCs, and the ability of exosomes secretion was stronger. On the contrary, the expression of autophagy in BM-MSCs was higher than that in BM-MSCs. Both Exo-AD and Exo-BM can be absorbed by neonatal rat cardiomyocytes successfully. Compared with the control group, both of them had protective effects on myocardial cells during ischemia and hypoxia, but the protective effect of Exo-BM on cardiomyocytes was stronger when treated with the same amount of exosomes. The contents of miRNAs in Exo-AD and Exo-BM were different by microarray analysis. There were differences in the expression of miRNAs between Exo-AD and Exo-BM, including miR-21 miR-30 miR-221 and miR-223 related to myocardial protection. Conclusion: in this study, we found that there were differences between AD-MSCs and BM-MSCs in the release of exosomes and their protective effects on ischemia and hypoxia. The main reasons may be related to the difference of miRNAs content in Exo-AD and Exo-BM. These provide a theoretical basis for the selection of clinical MSCs.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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