胃癌细胞来源exosome重塑微环境细胞作用及机制

发布时间：2018-03-30 20:21

本文选题：exosome　切入点：miR-4669　出处：《江苏大学》2017年硕士论文

【摘要】：目的:探讨胃癌细胞旁分泌外泌体(exosome)促进骨髓间质干细胞(bone marrow mesenchymal stem cell,BMMSC)转分化的重要作用和途径,阐明其诱导BMMSC转分化的分子机制,为胃癌微环境细胞重塑提供新的实验依据。方法:采用Nanosight检测分析exosome直径大小、数量并验证Rab27a-siRNA干扰组肿瘤细胞培养上清中exosome的量;透射电镜检测exosome形态和直径大小;Western blot检测exosome特异性标记CD81、CD9蛋白的表达。免疫荧光检测诱导前后BMMSC中α-SMA的表达,Transwell迁移实验检测胃癌细胞SGC-7901、HGC-27迁移能力的改变,定量PCR检测比较诱导前后BMMSC中相关因子的表达和mi R-4669表达的差异。采用Rab27a-siRNA抑制exosome分泌,免疫荧光和Transwell迁移实验检测Rab27a干扰的胃癌细胞上清诱导后BMMSC表型和功能的改变,评估exosome的诱导作用。运用mi R-4669 micmis在胃癌细胞中上调mi R-4669的表达,分析转染mi R-4669 micmis的SGC-7901和HGC-27上清及exosome分别作用BMMSC后其表型、功能的变化,确定mi R-4669对BMMSC向胃癌间质干细胞(gastric cancer tissue-derived MSC,GC-MSC)转分化的调控作用。体外血管生成试验评价miR-4669过表达组BMMSC对血管形成能力的影响。定量PCR检测转染mi R-4669mimics的胃癌细胞上清或exosome诱导后BMMSC相关基因表达变化,分析胃癌细胞来源的exosome通过miR-4669重塑BMMSC向GC-MSC转化的分子机制。结果:免疫荧光和Transwell迁移试验结果显示,胃癌细胞来源的exosome可再现胃癌细胞上清的作用,诱导BMMSC使其获得GC-MSC样表型和功能,α-SMA表达显著增强,且诱导后BMMSC具有更强的促进胃癌细胞迁移的能力。通过转染Rab27a-siRNA抑制胃癌细胞exosome的分泌,与对照组相比,干扰组诱导后BMMSC的基质细胞活化表型和促进胃癌细胞迁移的能力均显著减弱。qRT-PCR结果显示,胃癌细胞来源exosome作用BMMSC后miR-4669表达水平较对照组显著上调。与对照组相比,转染mi R-4669 mimics的胃癌细胞上清和exosome诱导后的BMMSC显示出增强的基质细胞活化表型和促进胃癌细胞迁移的能力,且转染mi R-4669 mimics的胃癌细胞上清诱导后的BMMSC具有更强的促进血管生成能力。转染mi R-4669mimics的胃癌细胞上清或exosome诱导后BMMSC相关基因检测结果显示,miR-4669的靶基因RXRA以及pri-4669、pre-4669的表达并无显著变化,而成熟的miR-4669表达上调,且相关炎性因子CCL2、IL-6、IL-8表达上调。结论:胃癌细胞分泌的exosome是胃癌细胞培养上清中诱导BMMSC向GC-MSC转化的重要组分,其富含的mi R-4669可被直接递送至BMMSC中,进而促使其获得GC-MSC样表型和功能,是介导胃癌细胞旁分泌作用BMMSC的关键信号分子。为MSC在胃癌微环境中的改造与重塑提供了新的机制。
[Abstract]:Objective: to investigate the important role and pathway of paracrine exosome-exosome (exosome) in promoting the transdifferentiation of bone marrow mesenchymal stem cells (BMSCs), bone marrow mesenchymal stem cell (BMMSCC), and to elucidate its molecular mechanism of inducing BMMSC transdifferentiation. Methods: the diameter and quantity of exosome were analyzed by Nanosight and the amount of exosome in the supernatant of tumor cell culture in Rab27a-siRNA interference group was verified. Transmission electron microscopy (TEM) was used to detect the morphology and diameter of exosome. Western blot was used to detect the expression of CD81G + CD9 protein specifically labeled by exosome. The expression of 伪 -SMA in BMMSC before and after induction was detected and the transwell migration assay was used to detect the migration ability of gastric cancer cell line SGC-7901HGC-27. Quantitative PCR was used to detect the expression of related factors and the expression of miR-4669 in BMMSC before and after induction. Rab27a-siRNA was used to inhibit the secretion of exosome, immunofluorescence and Transwell migration assay were used to detect the changes of BMMSC phenotype and function in the supernatant of gastric cancer cells induced by Rab27a interference. To evaluate the induction effect of exosome, the expression of mi R-4669 was up-regulated by mi R-4669 micmis in gastric cancer cells. The phenotypic and functional changes of the SGC-7901 and HGC-27 supernatants transfected with mi R-4669 micmis and the BMMSC treated with exosome were analyzed. To determine the regulatory effect of mi R-4669 on the transdifferentiation of BMMSC into gastric cancer cancer tissue-derived MSC. In vitro angiogenesis test was used to evaluate the effect of BMMSC on angiogenesis in miR-4669 overexpression group. Quantitative PCR was used to detect the supernatant of gastric cancer cells transfected with mi R-4669mimics. Or exosome induced changes in the expression of BMMSC related genes, To analyze the molecular mechanism of the transformation of exosome from gastric cancer cells to GC-MSC through miR-4669 remodeling of BMMSC. Results: the results of immunofluorescence and Transwell migration test showed that exosome derived from gastric cancer cells could reproduce the role of supernatant of gastric cancer cells. BMMSC was induced to obtain GC-MSC like phenotype and function, 伪 -SMA expression was significantly increased, and the induced BMMSC had a stronger ability to promote the migration of gastric cancer cells. Transfection of Rab27a-siRNA inhibited the secretion of exosome in gastric cancer cells, compared with the control group. The activation phenotype of stromal cells and the ability to promote the migration of gastric cancer cells in BMMSC induced by interference group were significantly decreased. The results of qRT-PCR showed that the expression of miR-4669 in gastric cancer cells derived from exosome was significantly up-regulated than that in the control group, compared with the control group. The supernatant of gastric cancer cells transfected with mi R-4669 mimics and BMMSC induced by exosome showed enhanced phenotype of stromal cell activation and enhanced migration of gastric cancer cells. The BMMSC induced by the supernatant of the gastric cancer cells transfected with mi R-4669 mimics has stronger ability of promoting angiogenesis. The detection of BMMSC related genes in the supernatant of gastric cancer cells transfected with mi R-4669mimics or induced by exosome showed that the target gene RXRA of miR-4669 and the surface of pri-4669 gene pre-4669 were detected. Da has not changed significantly. The expression of mature miR-4669 was up-regulated, and the related inflammatory factor CCL2, IL-6, IL-8 was up-regulated. Conclusion: exosome secreted by gastric cancer cells is an important component in inducing the transformation of BMMSC to GC-MSC in the supernatant of gastric cancer cell culture, and its rich mi R-4669 can be directly delivered to BMMSC. It is a key signal molecule that mediates paracrine action of gastric cancer cells to BMMSC and provides a new mechanism for the transformation and remodeling of MSC in gastric cancer microenvironment.
【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】：R735.2

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