骨保护素基因转染大鼠骨髓基质干细胞的表达及对其生物学行为的影响

发布时间：2018-10-11 15:16

【摘要】：目的探讨含有骨保护素(osteoprotegerin,OPG)的真核表达载体转染大鼠骨髓基质干细胞(BMSCs)表达情况并分析OPG基因转染对BMSCs生物学行为的影响。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,进行骨髓基质干细胞的分离和培养;设立质粒载体组、空载体组、未转染组。质粒载体组转染plRES2-EGFP-OPG,空载体组转染plRES2-EGFP,未转染组未经特殊处理。转染后48小时,在激光扫描共聚焦显微镜下检测EGFP表达;RT-PCR检测各组骨髓基质干细胞中OPGmRNA的表达;免疫细胞化学检测OPG蛋白的表达;MTT法检测细胞增殖能力;细胞碱性磷酸酶染色法检测细胞向成骨细胞分化的能力。结果1.骨髓基质细胞分离培养形态观察:初始分离的骨髓细胞呈圆形;大小不一;24 h后有少量细胞贴壁; 96 h后贴壁生长的细胞主要为梭形的成纤维样细胞;分瓶培养后细胞形成克隆;传代后细胞贴壁生长,分裂相增多,并不断增殖分化形成均一的梭形细胞。 2.激光扫描共聚焦显微镜观察结果:质粒载体组转染后可见绿色荧光蛋白表达,未转染组未见表达。 3. RT-PCR及免疫细胞化学检测结果:反转录聚合酶链反应观察到质粒载体组在1200bp有明显条带,空载体组及未转染组未见表达;免疫细胞化学检测质粒载体组OPG蛋白表达阳性,空载体组及未转染组为阴性表达。 4. MTT检测细胞增殖结果:转染后细胞在其表面及孔内平铺、伸展、生长及增殖状态良好,细胞增殖能力无明显改变,3组比较无显著差异(P 0.05)。 5.细胞碱性磷酸酶(ALP)染色结果:plRES2-EGFP-OPG转染的BMSCs合成ALP的能力显著提高,胞浆呈阳性蓝染颗粒,与空载体组及未转染组相比存在显著差异(P 0.05)。结论:1.成功分离培养大鼠BMSCs,证明了BMSCs可作为基因强化骨组织工程中理想的种子细胞。 2.转染OPG基因的BMSCs后可可稳定、高效的表达OPG,建立了OPG基因修饰的骨髓基质干细胞瞬时基因表达体系。 3.经plRES2-EGFP-OPG转染后的BMSCs增殖能力未受到影响,并具有一定的生物学功能,能促进其向成骨细胞分化。
[Abstract]:Objective to investigate the expression of (BMSCs) in rat bone marrow stromal stem cells transfected with eukaryotic expression vector containing osteoprotegerin (osteoprotegerin,OPG) and analyze the effect of OPG gene transfection on the biological behavior of BMSCs. Methods Bone marrow stromal cells (BMSCs) were isolated and cultured from the tibia and femur of 4-week-old SD rats of clean grade under sterile conditions, and the plasmid vector group, empty vector group and untransfected group were set up. Plasmid group transfected plRES2-EGFP-OPG, empty vector group transfected plRES2-EGFP, without special treatment. 48 hours after transfection, the expression of EGFP was detected by laser scanning confocal microscope, the expression of OPGmRNA in bone marrow stromal cells was detected by RT-PCR, the expression of OPG protein was detected by immunocytochemistry, the proliferative ability of cells was detected by MTT method. The ability of cells to differentiate into osteoblasts was detected by alkaline phosphatase staining. Result 1. Morphological observation of bone marrow stromal cells: the primary separated bone marrow cells were round and varied in size, a small number of cells adhered to the wall after 24 hours, and the cells that adherent to the bone marrow cells after 96 hours were mainly fusiform fibroblasts. Cell clone was formed after flask culture, cell adherent growth, mitotic phase increased after passage, and the cells proliferated and differentiated into uniform fusiform cells. 2. The results of laser scanning confocal microscopy showed that the expression of green fluorescent protein was observed in the plasmid vector group after transfection, but not in the untransfected group. Results of RT-PCR and immunocytochemistry: reverse transcriptase polymerase chain reaction showed that there were obvious bands in 1200bp in plasmid vector group, but no expression in empty vector group and untransfected group, OPG protein expression in plasmid vector group was positive by immunocytochemistry. Negative expression was found in empty vector group and untransfected group. 4. The results of MTT: after transfection, the cells were flattened, stretched, grown and proliferated well, and the ability of cell proliferation did not change obviously, but there was no significant difference among the three groups (P 0.05). The results of alkaline phosphatase (ALP) staining showed that the ability of BMSCs transfected with plRES2-EGFP-OPG to synthesize ALP was significantly increased, and the cytoplasm was blue stained, which was significantly different from that of empty vector group and untransfected group (P 0.05). Conclusion: 1. The successful isolation and culture of rat BMSCs, proved that BMSCs could be used as an ideal seed cell in gene strengthening bone tissue engineering. 2. The BMSCs transfected with OPG gene was stable and highly expressed OPG,. The transient gene expression system of bone marrow stromal cells modified with OPG gene was established. 3. 3. The proliferation of BMSCs transfected with plRES2-EGFP-OPG was not affected and had certain biological function, which could promote the differentiation of BMSCs into osteoblasts.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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