人胰岛素样生长因子-1和RNA干扰多聚蛋白聚糖酶-1基因对人骨髓干细胞骨架的影响研究

发布时间：2018-09-19 18:45

【摘要】：背景:目前慢病毒介导aggrecanase-1sh RNA及h IGF-1双向作用于类风湿患者骨髓间充质干细胞的研究还处在初级阶段。目的:观察在将aggrecanase-1sh RNA、h IGF-1基因同时转染到人骨髓间充质干细胞后,目的基因在细胞内的表达及细胞分化情况。方法:体外培养人骨髓间充质干细胞,并应用携带aggrecanase-1sh RNA、h IGF-1基因和绿色荧光蛋白的慢病毒载体转染第3代人骨髓间充质干细胞,同时以空载体转染人骨髓间充质干细胞作为阴性对照组。待病毒转染率到最高时分别提取各组细胞的蛋白检测从而得出目的基因表达量。并应用激光共聚焦显微镜观察转染成功后细胞的骨架及细胞形态。结果与结论:成功于体外培养出人骨髓间充质干细胞,而且以慢病毒为载体成功的将目的基因转染到细胞内。荧光显微镜下可观察到细胞发出绿色荧光,各组的平均转染效率可达到88%。Western blot检测结果及激光共聚焦显微镜下细胞形态显示,目的基因在人骨髓间充质干细胞内可高效持续的表达出相应蛋白,且细胞形态变化也可间接证明aggrecanase-1sh RNA、h IGF-1基因可共同促进人骨髓干细胞向软骨的定向分化。
[Abstract]:BACKGROUND: At present, the research of lentivirus-mediated aggrecanase-1sh RNA and H IGF-1 acting on bone marrow mesenchymal stem cells of rheumatoid patients is still in its infancy. Objective: To observe the expression and cell differentiation of the target gene after transfecting aggrecanase-1sh RNA and H IGF-1 gene into bone marrow mesenchymal stem cells. METHODS: Human bone marrow mesenchymal stem cells were cultured in vitro and transfected into the third generation of human bone marrow mesenchymal stem cells with lentiviral vector carrying aggrecanase-1sh RNA, h IGF-1 gene and green fluorescent protein, and the human bone marrow mesenchymal stem cells were transfected with empty vector as negative control group. RESULTS AND CONCLUSION: Human bone marrow mesenchymal stem cells were successfully cultured in vitro, and the target gene was successfully transfected into cells by lentivirus vector. The results were observed under fluorescence microscope. The results of Western blot and confocal laser microscopy showed that the target gene could efficiently and sustainably express the corresponding protein in human bone marrow mesenchymal stem cells, and the changes of cell morphology could indirectly prove aggrecanase-1sh RNA and H IGF-1 gene. It can promote the directional differentiation of human bone marrow stem cells into cartilage.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R684

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