人脐带间充质干细胞来源微囊泡对化疗损伤大鼠卵巢颗粒细胞的保护作用

发布时间：2018-06-28 17:10

本文选题：微囊泡 + 颗粒细胞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：第一部分人脐带间充质干细胞来源微囊泡的分离纯化目的:建立一个高效人脐带间充质干细胞来源微囊泡的分离纯化方法。方法:收集第3代至第8代间充质干细胞上清液,分别采用差速离心法,改良超速离心法,蔗糖梯度纯化法提取MVs,Bradford法蛋白定量,共聚焦显微镜及透射电镜观察MVs的形态学特征。结果:改良超速离心方法MVs的蛋白含量显著高于传统差速离心方法和蔗糖梯度纯化法,每1×106个细胞大约释放162μg左右的MVs。电镜下MVs直径介于30-200nm之间,呈单层膜、类圆形囊泡结构,中间为低电子致密物质。结论:改良超速离心法能获得高蛋白浓度的微囊泡。第二部分微囊泡对化疗损伤卵巢颗粒细胞的保护作用目的:探讨MSCs来源MVs对化疗损伤颗粒细胞的保护作用。方法:1.机械分离法分离3周SD雌性大鼠卵巢颗粒细胞,原代无血清培养,FSHR免疫荧光鉴定颗粒细胞的纯度;将PKH26标记的MVs添加入正常颗粒细胞以及顺铂损伤颗粒细胞中,6h后荧光显微镜观察MVs在颗粒细胞的定位;将不同浓度MVs与颗粒细胞共培养,确定最佳MVs作用浓度。2.实验分组:实验分为正常颗粒细胞组(GCs)、顺铂组(CTx)和顺铂+微囊泡组(MVs+CTx);MTT法检测各组细胞存活率;化学发光法检测各组培养液上清雌二醇和孕酮浓度;Annexin V-FITC/PI双染流式细胞仪检测各组颗粒细胞凋亡率;实时荧光定量PCR检测各组颗粒细胞凋亡相关基因(Bax、Bcl-2、Caspase3)以及类固醇急性调节蛋白基因(St AR)的表达水平。结果:1.无血清培养的大鼠卵巢颗粒细胞生长良好,免疫荧光染色证实本研究所获颗粒细胞纯度达95%以上;MVs在共培养6h后,损伤颗粒细胞中可见红色荧光标记的MVs。2.MVs与化疗损伤颗粒细胞共培养后,颗粒细胞凋亡率明显减少,雌激素水平明显升高,颗粒细胞凋亡相关基因Caspase3的表达水平显著降低,Bcl-2与Bax比值及St AR基因表达水平显著升高,且MVs对化疗损伤颗粒细胞的保护作用呈浓度依赖性。结论:MVs能进入化疗损伤的颗粒细胞中,在体外能够减轻颗粒细胞的损伤,促进损伤颗粒细胞增殖和分泌功能的恢复。
[Abstract]:The first part: isolation and purification of microcapsules derived from human umbilical cord mesenchymal stem cells objective: to establish an efficient method for isolation and purification of human umbilical cord mesenchymal stem cells derived from microcapsules. Methods: the supernatants of mesenchymal stem cells from the third to the eighth generation were collected. The MVs were extracted by differential centrifugation, modified ultracentrifugation and sucrose gradient purification. The morphologic characteristics of MVs were observed by confocal microscope and transmission electron microscope. Results: the protein content of MVs by modified ultracentrifugation was significantly higher than that by conventional differential centrifugation and sucrose gradient purification, and about 162 渭 g MVsper 1 脳 106 cells were released. Under electron microscope, the diameter of MVs is between 30-200nm, monolayer membrane, circular vesicle structure and low electron dense material in the middle. Conclusion: the modified ultracentrifugation method can obtain microvesicles with high protein concentration. The second part was the protective effect of microvesicles on ovarian granulosa cells induced by chemotherapy. Objective: to investigate the protective effect of MSCs derived from MVs on granulosa cells induced by chemotherapy. Method 1: 1. The ovarian granulosa cells of SD female rats were isolated by mechanical separation for 3 weeks, and the purity of granulosa cells was identified by FSHR immunofluorescence in primary serum-free culture. After adding PKH26 labeled MVs into normal granulosa cells and cisplatin damaged granulosa cells for 6 h, the localization of MVs in granulosa cells was observed by fluorescence microscope, and the optimal concentration of MVs was determined by co-culture of different concentrations of MVs with granulosa cells. The experiment was divided into normal granulosa cell group (GCs), cisplatin group (CTX) and cisplatin microvesicular group (MVs CTX). The concentration of estradiol and progesterone in supernatant of each group was detected by chemiluminescence assay. The apoptosis rate of granulosa cells in each group was detected by flow cytometry with Annexin V-FITC / Pi double staining. The expression of apoptosis-related gene (Bax-Bcl-2Caspase3) and steroid acute regulatory protein gene (St AR) in granulosa cells were detected by real-time fluorescent quantitative PCR. The result is 1: 1. The granulosa cells of rat ovary cultured in serum-free medium grew well. Immunofluorescence staining confirmed that the purity of granulosa cells in this study was over 95%, and MVs was cocultured for 6 h. After co-culture of red fluorescent labeled MVs.2.MVs and chemotherapy-injured granulosa cells, the apoptotic rate of granulosa cells was significantly decreased, and the level of estrogen was significantly increased. The expression of apoptosis-related gene Caspase3 significantly decreased the ratio of Bcl-2 to Bax and the expression of St AR gene, and the protective effect of MVs on granulosa cells was concentration-dependent. ConclusionMVs can enter the granulosa cells injured by chemotherapy, which can attenuate the injury of granulosa cells in vitro and promote the recovery of proliferation and secretory function of injured granulosa cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75

【相似文献】