杆状病毒介导肝生长因子在骨髓间充质干细胞中的调控表达

发布时间：2018-01-30 04:27

本文关键词： 骨髓间充质干细胞股骨头坏死杆状病毒供体质粒反应质粒转染肝生长因子强力霉素四环素基因表达调控系统增殖　出处：《南昌大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：建立转染高效可调控的携带肝生长因子(hepatocytegrowthfactor，HGF）为目的基因的重组杆状病毒vAcrtTA2s-Ptight-HGF,并探寻其在转染骨髓间充质干细胞（bonemarrowmesenchymalstemcells,BM-MSCs）后的DOX诱导浓度、转染后细胞形态和增殖速率的变化。方法：采用全骨髓培养法培养兔BM-MSCs,基因工程方法构建重组杆状病毒vAcrtTA2s-Ptight-HGF，随后转染BM-MSCs，用ElISA和WesternBlot检测不同浓度强力霉素（doxycycline，DOX）诱导体外培养的兔BM-MSCs分泌的HGF的浓度，MTT方法检测转染后BM-MSCs的增殖变化。结果：成功获得兔BM-MSCs第5代细胞，成功构建了高效可调控重组杆状病毒载体vAcrtTA2s-Ptight-HGF，并且成功转染兔BM-MSCs，用系列浓度DOX诱导时ELISA和Westernblot均检测到BM-MSCs中HGF的表达有DOX诱导剂量依赖关系并且连续7天持续表达，，且实验组的HGF的表达量明显高于对照组（P0.001），转染后的BM-MSCs仍然呈涡旋形生长，诱导产生序列浓度的HGF的BM-MSCs的增殖速度明显高于空白对照组（P0.001）。结论：成功构建一次性转染、高效可调控的重组杆状病毒vAcrtTA2s-Ptight-HGF,并且实现对HGF表达的调控,当强力霉素诱导浓度为1ug/ml时为其体外最佳诱导浓度，并证实HGF可促进BM-MSCs的增殖,转染后BM-MSCs的细胞形态没有明显改变。
[Abstract]:Objective: to establish an efficient and controllable hepatocyte growth factor carrying hepatocyte growth factor. The recombinant baculovirus vAcrtTA2s-Ptight-HGF with HGF as the target gene. To explore the DOX induction concentration after transfection of bone marrow mesenchymal stem cells (BM-MSCs) into bone marrow mesenchymal stem cells (BM-MSCs). Changes of cell morphology and proliferation rate after transfection. Methods: BM-MSCswere cultured by whole bone marrow culture. Recombinant baculovirus vAcrtTA2s-Ptight-HGFwas constructed by genetic engineering method and then transfected into BM-MSCs. ElISA and WesternBlot were used to detect the concentration of HGF secreted by rabbit BM-MSCs in vitro induced by doxycycline doxycycline doxycycline (doxycycline doxycycline dox). MTT method was used to detect the proliferation of BM-MSCs after transfection. Results: the fifth passage of rabbit BM-MSCs cells were successfully obtained and the recombinant baculovirus vector vAcrtTA2s-Ptight-HGF was successfully constructed. Rabbit BM-MSCs was transfected successfully. The expression of HGF in BM-MSCs was found to be in a dose-dependent manner by DOX induction in both ELISA and Westernblot induced by serial concentrations of DOX and continued for 7 days. The expression of HGF in the experimental group was significantly higher than that in the control group (P 0.001), and the BM-MSCs still grew in a vortex shape after transfection. The proliferation rate of BM-MSCs induced by sequence concentration of HGF was significantly higher than that of control group (P 0.001). Conclusion: the recombinant baculovirus vAcrtTA2s-Ptight-HGFwas successfully constructed, and the expression of HGF was regulated by the recombinant baculovirus vAcrtTA2s-Ptight-HGF. The optimal concentration of doxycycline was 1ugrml in vitro, and HGF could promote the proliferation of BM-MSCs, but the cell morphology of BM-MSCs was not changed after transfection.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2

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