慢病毒介导的人碱性纤维生长因子基因的构建及在鼠骨髓间充质干细胞中表达的实验研究

发布时间：2018-06-07 22:23

本文选题：大鼠骨髓间充质干细胞 + 成碱性纤维细胞因子　；参考：《福建医科大学》2010年硕士论文

【摘要】：目的:探讨构建携带人碱性成纤维生长因子(bFGF)的重组慢病毒载体方法并观察碱性成纤维细胞生长因子基因转染大鼠骨髓间充质干细胞后bFGF目的基因的表达情况。方法:采用RT—PCR扩增的方法获取人源性的bFGF与FGF4基因,将目的基因与载体pGC-FU连接[含绿色荧光蛋白(GFP)]。产生pGC FU- FGF4-bFGF慢病毒载体。PCR筛选阳性克隆,测序鉴定。通过lipofectamine2000的介导把pGC FU- FGF4-bFGF载体,pHelper 1.0载体和pHelper 2.0载体三质粒共转染至包装细胞293T.应用Real time-PCR法鉴定和测定滴度。而后转染大鼠骨髓间充质干细胞并检测其表达。MTT法检测到间充质干细胞经过基因修饰以后和未经过基因修饰的干细胞生长情况结果: 1.流式细胞仪显示体外分离培养的第3代大鼠骨髓间充质干细胞表面抗原表达: CD11b/c阳性细胞表达率为14.2%±0.7%,CD34阳性细胞表达率为1.3%±0.5%,CD44阳性细胞表达率97.8%±0.9%,CD90阳性细胞表达率96.9%±1.5%。 2. PCR鉴定证实重组慢病毒有人FGF4信号肽-人BFGF ,病毒滴度为2.00E+9 TU/ml。 3.转染大鼠骨髓间充质干细胞后,行RT-PCR检测到BFGF基因转入干细胞内,其大小约500bp。 4.在荧光显微镜下,观察到转染的干细胞内GFP的表达;通过Western Blot检测转染的细胞可以观察到36～55KDr之间有明显表达,与FGF4-BFGF -GFP融合蛋白(~21+28KDr=~49KDr)基本吻合。(备注:构建的pGC-FU-FGF4-BFGF基因插入片断大小549bp)综上:基本判断FGF4-BFGF表达正常。ELISA检测结果表明转染bFGF的细胞培养上清液中碱性成纤维细胞生长因子浓度显著高于空白对照。 5.MTT法检测到间充质干细胞经过基因修饰以后和未经过基因修饰的干细胞生长情况对比,基因修饰后的目的细胞明显优于未经修饰的干细胞。结论:成功构建的携带人成碱纤维生长因子的慢病毒载体能在293T细胞扩增获得足够高的病毒滴度,并转染入大鼠骨髓间充质干细胞,经检测可以有效的表达。可以为下一步基因治疗大鼠下肢缺血提供很好的基础。
[Abstract]:Aim: to investigate the construction of recombinant lentivirus vector carrying human basic fibroblast growth factor (bFGF) and observe the expression of bFGF target gene in rat bone marrow mesenchymal stem cells transfected with basic fibroblast growth factor gene. Methods: human bFGF and FGF4 genes were obtained by RT-PCR. The target gene was ligated with vector pGC-FU [GFP containing green fluorescent protein]. The positive clones of lentivirus vector pGC FU- FGF4-bFGF were screened and sequenced. PGC FU-FGF4-bFGF vector pHelper 1.0 and pHelper 2.0 were co-transfected into packaging cell line 293T by lipofectamine2000. Real time PCR method was used to identify and determine the titer. Then transfected rat bone marrow mesenchymal stem cells and detected its expression. MTT assay detected the growth of mesenchymal stem cells after gene modification and without gene modification: 1. Flow cytometry showed that the expression rate of CD11b / c positive cells in the third passage of rat bone marrow mesenchymal stem cells in vitro was 14.2% 卤0.710%. The positive rate of CD34 positive cells was 1.3% 卤0.5%. The expression rate of CD44 positive cells was 97.8% 卤0.9m + CD90 positive cells. The expression rate of CD11b / c positive cells was 96.9% 卤1.5.2%. PCR confirmed the recombinant lentivirus human FGF4 signal peptide human bFGF, the titer of the virus was 2.00E 9 TU / ml. 3. After transfection of rat bone marrow mesenchymal stem cells, the expression of bFGF gene was detected by RT-PCR and its size was about 500bp.4. The expression of GFP in the transfected stem cells was observed under fluorescence microscope, and the expression of GFP in transfected cells was detected by Western Blot, which was consistent with the FGF4-BFGF -GFP fusion protein (21 28 K Dr). (note: the size of pGC-FU-FGF4-BFGF gene insertion fragment was 549bp). The results of Elisa showed that the concentration of basic fibroblast growth factor in the supernatant of bFGF transfected cell culture was significantly higher than that of blank control. 5. MTT assay showed that the expression of FGF4-BFGF was normal. The growth of mesenchymal stem cells after gene modification was compared with that of unmodified mesenchymal stem cells. Conclusion: the successfully constructed lentivirus vector carrying human alkali-derived fibroblast growth factor can obtain high enough virus titer in 293T cells. It was transfected into rat bone marrow mesenchymal stem cells and expressed effectively. It can provide a good basis for gene therapy of lower extremity ischemia in rats.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【参考文献】