慢病毒载体介导的OPG基因体外转染大鼠骨髓间充质干细胞的研究
发布时间：2018-11-05 12:42
【摘要】：目的：用含有骨保护素(OPG)的慢病毒载体(PGC-FU-OPG-RFP)转染大鼠骨髓间充质干细胞(BMSCs),观察其对细胞活性的影响及表达情况。 方法：选取清洁级4周龄SD大鼠,雌雄不限,无菌条件下取胫骨和股骨,进行骨髓间充质干细胞的分离和培养。培养到第三代时进行转染,设立未转染对照组、空慢病毒载体组、含OPG慢病毒载体组。未转染对照组未经特殊处理,空慢病毒载体组转染PGC-FU-RFP,含OPG慢病毒载体组转染PGC-FU-OPG-RFP.转染后48h,免疫荧光显微镜观察荧光表达；流式细胞仪检测慢病毒的转染效率；用M T T法评价慢病毒转染对BMSCs增殖的影响；用Western Blot检测转染后基因的表达情况。 结果： 1.成功培养出第三代SD大鼠BMSCs,贴壁的BMSCs散在分布于瓶底,呈长梭形、多角形,生长良好。 2.按10、50、100的感染复数(MOI)转染第3代骨髓间充质干细胞,48小时后免疫荧光显微镜观察可见红色荧光蛋白表达,末转染组未见表达。流式细胞仪检测慢病毒的转染效率分别为12.0%、41.8%、70.0%。 3.MTT检测发现：转染后细胞增殖状态良好,细胞增殖能力无明显改变,3组比较无显著差异(P0.05) 4. Western Blot结果显示：转染PGC-FU-OPG-RFP组72kDa处有条特征带,其大小等于OPG-RFP融合蛋白(-44kDa+28kDa=72kDa),基本判断OPG农达。 结论： 1.慢病毒载体成功转染BMSCs, MOI为100时转染率可达70.0%。 2.经慢病毒转染后的BMSCs, MTT检测细胞增殖活力与末转染组无明显差异,Western Blot检测目的基因可表达。
[Abstract]:Aim: to investigate the effect and expression of lentivirus vector (PGC-FU-OPG-RFP) containing osteoprotegerin (OPG) on the activity of rat bone marrow mesenchymal stem cells (BMSCs) transfected with (BMSCs),. Methods: bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured from tibia and femur of 4 weeks old SD rats. After the third generation of culture, untransfected control group, empty lentivirus vector group and OPG lentivirus vector group were established. Untransfected control group without special treatment, empty lentivirus vector group transfected PGC-FU-RFP, containing OPG lentivirus vector group transfected PGC-FU-OPG-RFP. The fluorescent expression was observed by immunofluorescence microscope at 48h after transfection, the transfection efficiency of lentivirus was detected by flow cytometry, the effect of lentivirus transfection on BMSCs proliferation was evaluated by M T T assay, and the expression of genes was detected by Western Blot. Results: 1. The BMSCs of BMSCs, adherent to the third generation of SD rats was successfully cultured and distributed on the bottom of the bottle, with long fusiform, polygonal shape and good growth. 2. The third generation of bone marrow mesenchymal stem cells were transfected with the infected plural (MOI) of 10 ~ 50100. The expression of red fluorescent protein was observed by immunofluorescence microscope 48 hours later, but no expression was found in the final transfection group. The transfection efficiency of lentivirus detected by flow cytometry was 12.0 and 41.8%, respectively. 3.MTT analysis showed that after transfection, the cell proliferation status was good, the cell proliferation ability did not change significantly, there was no significant difference among the three groups (P0.05) 4. Western Blot results showed that there was a characteristic band in the 72kDa of the transfected PGC-FU-OPG-RFP group, whose size was equal to that of the OPG-RFP fusion protein (- 44kDa 28kDa=72kDa). Conclusion: 1. The transfection efficiency of lentivirus vector was 70.0 when BMSCs, MOI was 100. 2. The proliferative activity of lentivirus transfected BMSCs, MTT was not significantly different from that of the final transfection group, and the expression of target gene was detected by, Western Blot.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346
本文编号：2312118
[Abstract]:Aim: to investigate the effect and expression of lentivirus vector (PGC-FU-OPG-RFP) containing osteoprotegerin (OPG) on the activity of rat bone marrow mesenchymal stem cells (BMSCs) transfected with (BMSCs),. Methods: bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured from tibia and femur of 4 weeks old SD rats. After the third generation of culture, untransfected control group, empty lentivirus vector group and OPG lentivirus vector group were established. Untransfected control group without special treatment, empty lentivirus vector group transfected PGC-FU-RFP, containing OPG lentivirus vector group transfected PGC-FU-OPG-RFP. The fluorescent expression was observed by immunofluorescence microscope at 48h after transfection, the transfection efficiency of lentivirus was detected by flow cytometry, the effect of lentivirus transfection on BMSCs proliferation was evaluated by M T T assay, and the expression of genes was detected by Western Blot. Results: 1. The BMSCs of BMSCs, adherent to the third generation of SD rats was successfully cultured and distributed on the bottom of the bottle, with long fusiform, polygonal shape and good growth. 2. The third generation of bone marrow mesenchymal stem cells were transfected with the infected plural (MOI) of 10 ~ 50100. The expression of red fluorescent protein was observed by immunofluorescence microscope 48 hours later, but no expression was found in the final transfection group. The transfection efficiency of lentivirus detected by flow cytometry was 12.0 and 41.8%, respectively. 3.MTT analysis showed that after transfection, the cell proliferation status was good, the cell proliferation ability did not change significantly, there was no significant difference among the three groups (P0.05) 4. Western Blot results showed that there was a characteristic band in the 72kDa of the transfected PGC-FU-OPG-RFP group, whose size was equal to that of the OPG-RFP fusion protein (- 44kDa 28kDa=72kDa). Conclusion: 1. The transfection efficiency of lentivirus vector was 70.0 when BMSCs, MOI was 100. 2. The proliferative activity of lentivirus transfected BMSCs, MTT was not significantly different from that of the final transfection group, and the expression of target gene was detected by, Western Blot.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346
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