肝细胞生长因子基因转染缺氧培养骨髓间充质干细胞凋亡相关蛋白的影响

发布时间：2019-04-21 11:08

【摘要】：背景:研究已证实肝细胞生长因子基因转染可提高骨髓间充质干细胞移植效果,但其具体机制尚未完全明确。目的:探讨肝细胞生长因子基因转染对缺氧、无血清培养骨髓间充质干细胞c-Met、Bax、Bcl-2、Caspase-3表达及细胞迁移的影响。方法:1贴壁法体外分离、扩增培养骨髓间充质干细胞。用x-gal染色法检测含肝细胞生长因子基因的重组腺病毒对骨髓间充质干细胞的感染率。2在缺氧、无血清条件下培养0,3,6,9,12 h,采用RT-PCR及Western blot测定骨髓间充质干细胞Bax、Bcl-2、Caspase-3的表达。3骨髓间充质干细胞缺氧、无血清培养6 h,采用RT-PCR及Western blot测定HGF、c-Met、Bax、Bcl-2、Caspase-3的表达。4细胞迁移划痕法观察缺氧、无血清培养6 h后肝细胞生长因子转染对骨髓间充质干细胞迁移的影响。结果与结论:1重组腺病毒对骨髓间充质干细胞的转染率与病毒感染复数具有量效关系,病毒感染复数为150时细胞的感染率达96.4%。2随着缺氧时间的延长骨髓间充质干细胞Bax、Bcl-2表达逐渐升高(P0.05),缺氧6 h时Bax/Bcl-2比值、Caspase-3蛋白达到最小(P0.05)。3缺氧及无血清培养6 h后,与对照组及空载体组相比,Ad-HGF组HGF、c-Met、Bcl-2表达增高,而Bax、Caspase-3表达下降(P0.05),对照组与空载体组差异无显著性意义(P0.05)。4Ad-HGF组骨髓间充质干细胞在缺氧培养6 h后,迁移率比对照组及空载体组高(P0.05)。结果表明肝细胞生长因子基因转染上调缺氧、无血清培养骨髓间充质干细胞c-Met、Bcl-2表达,下调Bax、Caspase-3表达,增强骨髓间充质干细胞迁移能力。
[Abstract]:Background: it has been proved that hepatocyte growth factor gene transfection can improve the effect of bone marrow mesenchymal stem cell transplantation, but the specific mechanism is not completely clear. Aim: to investigate the effects of hepatocyte growth factor gene transfection on expression and cell migration of bone marrow mesenchymal stem cells (MSCs) in hypoxia and serum-free culture. Methods: 1 Bone marrow mesenchymal stem cells were isolated and cultured by adherent method in vitro. X-gal staining was used to detect the infection rate of recombinant adenovirus containing hepatocyte growth factor gene on bone marrow mesenchymal stem cells (BMSCs). 2 cultured for 0, 3, 6, 9, 12 h under hypoxia and serum-free conditions. The expression of Bax,Bcl-2,Caspase-3 in bone marrow mesenchymal stem cells (BMSCs) was determined by RT-PCR and Western blot. 3BMSCs were exposed to hypoxia and cultured for 6 hours without serum. HGF,c-Met,Bax,Bcl-2, was measured by RT-PCR and Western blot. The effect of hepatocyte growth factor transfection on the migration of bone marrow mesenchymal stem cells was observed after 6 hours of serum-free culture. The expression of Caspase-3 was observed by 4 cell migration scratch method. Results and conclusion: 1 the transfection rate of recombinant adenovirus to bone marrow mesenchymal stem cells was dose-dependent with the number of virus infection. When the number of virus infection was 150, the cell infection rate reached 96.4%. With the prolongation of hypoxia time, the expression of Bax,Bcl-2 in bone marrow mesenchymal stem cells increased gradually (P0.05), and the ratio of Bax/Bcl-2 was increased at 6 h after hypoxia. Caspase-3 protein reached the minimum (P0.05) .3After hypoxia and serum-free culture for 6 hours, the expression of HGF,c-Met,Bcl-2 in Ad-HGF group was higher than that in control group and empty vector group, while the expression of Bax,Caspase-3 was decreased (P0.05). The migration rate of bone marrow mesenchymal stem cells in Ad-HGF group was higher than that in control group and empty vector group after 6 hours of hypoxia culture (P0.05). 4 the migration rate of bone marrow mesenchymal stem cells in Ad-MSCs group was higher than that in control group and empty vector group (P0.05). The results showed that hepatocyte growth factor gene transfection could upregulate hypoxia, increase the migration ability of bone marrow mesenchymal stem cells (BMSCs) by up-regulating the expression of c, Bcl-2 and down-regulating the expression of Bax,Caspase-3 in serum-free cultured bone marrow mesenchymal stem cells (BMSCs).
【作者单位】：福建医科大学附属第一医院心内科高血压研究所;福建省立医院北院福建省老年医院心内科;
【基金】：福建省卫生厅青年基金(2011-1-32,2011-1-15) 福建省自然科学基金(2014J01433)~~
【分类号】：R329.2

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