【摘要】：目的：用含有骨保護素(OPG)的慢病毒載體(PGC-FU-OPG-RFP)轉染大鼠骨髓間充質(zhì)干細胞(BMSCs),觀察其對細胞活性的影響及表達情況。 方法：選取清潔級4周齡SD大鼠,雌雄不限,無菌條件下取脛骨和股骨,進行骨髓間充質(zhì)干細胞的分離和培養(yǎng)。培養(yǎng)到第三代時進行轉染,設立未轉染對照組、空慢病毒載體組、含OPG慢病毒載體組。未轉染對照組未經(jīng)特殊處理,空慢病毒載體組轉染PGC-FU-RFP,含OPG慢病毒載體組轉染PGC-FU-OPG-RFP.轉染后48h,免疫熒光顯微鏡觀察熒光表達；流式細胞儀檢測慢病毒的轉染效率；用M T T法評價慢病毒轉染對BMSCs增殖的影響；用Western Blot檢測轉染后基因的表達情況。 結果： 1.成功培養(yǎng)出第三代SD大鼠BMSCs,貼壁的BMSCs散在分布于瓶底,呈長梭形、多角形,生長良好。 2.按10、50、100的感染復數(shù)(MOI)轉染第3代骨髓間充質(zhì)干細胞,48小時后免疫熒光顯微鏡觀察可見紅色熒光蛋白表達,末轉染組未見表達。流式細胞儀檢測慢病毒的轉染效率分別為12.0%、41.8%、70.0%。 3.MTT檢測發(fā)現(xiàn)：轉染后細胞增殖狀態(tài)良好,細胞增殖能力無明顯改變,3組比較無顯著差異(P0.05) 4. Western Blot結果顯示：轉染PGC-FU-OPG-RFP組72kDa處有條特征帶,其大小等于OPG-RFP融合蛋白(-44kDa+28kDa=72kDa),基本判斷OPG農(nóng)達。 結論： 1.慢病毒載體成功轉染BMSCs, MOI為100時轉染率可達70.0%。 2.經(jīng)慢病毒轉染后的BMSCs, MTT檢測細胞增殖活力與末轉染組無明顯差異,Western Blot檢測目的基因可表達。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346

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