脂质体介导BMP2和VEGF165双基因修饰小鼠骨髓基质干细胞的表达

发布时间：2018-04-13 18:52

本文选题：组织工程 + 骨髓基质干细胞　；参考：《泸州医学院》2011年硕士论文

【摘要】：目的：通过全骨髓培养法培养、分离、纯化骨髓基质干细胞,构建携带BMP2和VEGF165基因的重组pYr-adshuttle-4质粒,并通过脂质体介导将其转染至骨髓基质干细胞。将转染后的BMSCs进行体外培养,观察生物学特性、向成骨细胞分化的能力,探讨BMP2和VEGF165双基因修饰大鼠骨髓基质干细胞与单基因修饰诱导成骨能力的差异。方法：1.取小鼠双侧股骨骨髓并采用全骨髓培养法培养进行原代培养,通过换液和传代培养分离和纯化骨髓基质干细胞,传至第3代用于后续实验。2.分别构建携带BMP2和VEGF165基因的重组pYr-adshuttle-4质粒。3.按照未转染组、空载体组、BMP2单基因转染组、VEGF165单基因转染组、BMP2和VEGF165双基因共同转染组通过脂质体介导分别转染骨髓基质干细胞。4.转染后行常规细胞形态学观察7天；48h通过RT-PCR检测BMP2和VEGF165的mRNA含量,Western-Blot检测BMP2和VEGF165蛋白表达变化；转染后一周分别检测各组细胞成骨能力—碱性磷酸酶。结果：1.全骨髓细胞培养法成功分离、培养、纯化骨髓基质干细胞,骨髓基质干细胞呈长梭形样贴壁生长。2.成功构建携带骨形态发生蛋白-2、血管内皮生长因子-165的重组pYr-adshuttle-4质粒3.脂质体介导成功将携带BMP2和VEGF165基因的重组pYr-adshuttle-4质粒转染进骨髓基质干细胞,转染率高,无细胞毒性作用,转染后细胞生长活力增强。4.转染后BMSCs:RT-PCR检测显示BMP2+VEGF165双基因共同转染组mRNA水平表达高于骨形态发生蛋白-2组和血管内皮生长因子-165组单基因转染。5.Western-Blot检测显示共同转染组中BMP2和VEGF165基因组在蛋白水平表达量明显大于单基因组。6.双转染组中骨髓基质干细胞的ALP活性明显高于各单转染组(P0.05)结论：1.全骨髓培养法成功的培养、分离并纯化得到骨髓基质干细胞2.利用脂质体介导BMP2和VEGF165基因pYr-adshuttle-4质粒共同转染BMSCs可在体外长期高效稳定共同表达BMP2和VEGF165基因,并诱导向成骨细胞分化。3.BMP-2、VEGF-165双基因修饰骨髓基质干细胞诱导成骨能力明显高于单基因修饰骨髓基质干细胞成骨能力。
[Abstract]:Aim: to isolate and purify bone marrow stromal cells by whole bone marrow culture, construct recombinant pYr-adshuttle-4 plasmid carrying BMP2 and VEGF165 genes, and transfect them into bone marrow stromal cells mediated by liposome.After transfection of BMSCs was cultured in vitro, the biological characteristics and the ability to differentiate into osteoblasts were observed, and the difference of osteogenic ability between BMP2 and VEGF165 double gene modified rat bone marrow stromal stem cells and single gene modified bone marrow stromal cells was discussed.Method 1: 1.Bone marrow stromal cells were isolated and purified from bilateral femur bone marrow of mice and cultured with whole bone marrow culture method. The bone marrow stromal cells were isolated and purified by liquid exchange and passage culture, and then transferred to the third passage for further experiment. 2.The recombinant pYr-adshuttle-4 plasmids containing BMP2 and VEGF165 genes were constructed, respectively.Bone marrow mesenchymal stem cells (BMSCs) were transfected by liposome mediated by liposome in the untransfected group and the empty vector group in the single gene transfection group of VEGF165 and the co-transfected group of BMP2 and VEGF165 genes.After transfection, the expression of BMP2 and VEGF165 protein was detected by RT-PCR and Western-Blot, and the osteogenic ability of each group was detected by alkaline phosphatase one week after transfection.The result is 1: 1.Bone marrow mesenchymal stem cells were isolated, cultured and purified successfully by whole bone marrow cell culture method. Bone marrow stromal cells grew as fusiform adherent cells.The recombinant pYr-adshuttle-4 plasmid containing bone morphogenetic protein-2 and vascular endothelial growth factor-165 was successfully constructed.The recombinant pYr-adshuttle-4 plasmid carrying BMP2 and VEGF165 genes was successfully transfected into bone marrow stromal cells by liposome-mediated transfection. The transfection rate was high and there was no cytotoxicity.After transfection, BMSCs:RT-PCR analysis showed that the expression of mRNA in BMP2 VEGF165 double gene cotransfection group was higher than that in bone morphogenetic protein-2 group and vascular endothelial growth factor -165 group. 5. Western-Blot analysis showed that BMP2 and VEGF165 genomes were in protein in co-transfected group.The level of expression was significantly higher than that of single genome.The ALP activity of bone marrow stromal cells in double transfection group was significantly higher than that in each single transfection group (P 0.05) conclusion: 1.Bone marrow stromal cells 2 were isolated and purified by whole bone marrow culture.Cotransfection of BMP2 and VEGF165 gene pYr-adshuttle-4 plasmid with liposome can effectively and stably co-express BMP2 and VEGF165 genes in vitro.The osteogenic ability of bone marrow mesenchymal stem cells modified with VEGF-165 gene was significantly higher than that with single gene modified bone marrow stromal stem cells.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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