杆状病毒介导肝生长因子在兔骨髓间充质干细胞中的调控表达

Controllable HGF expression in bone marrow mesenchymal stem cells through baculovirus system

摘要:

目的：建立转染高效可调控的携带肝生长因子（hepatocyte growth factor，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（bone marrow mesenchymal stem cells，BM-MSCs）后的最佳诱导浓度。方法：用转基因的方法构建重组杆状病毒vAcrtTA2s-Ptight-HGF，随后转染BM-MSCs，用ELISA和Western blot检测不同浓度强力霉素（doxycycline，DOX）诱导体外培养兔BM-MSCs分泌的HGF浓度。结果：构建了高效可调控重组杆状病毒vAcrtTA2s-Ptight-HGF，并且成功转染兔BM-MSCs，用系列浓度DOX诱导时ELISA和Western blot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 d持续表达，且实验组的HGF的表达量明显高于对照组（P<0.001）。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，并实现对HGF表达的调控，当DOX诱导浓度为1 μg/ml时为其体外最佳诱导浓度。

Abstract:

Objective：To construct an efficient and controllable recombinant baoulovirus vAcrtTA2s-Ptight-HGF and to investigate the optimum dose of deoxycycline（DOX） for the induction of hepatocyte growth factor（HGF） expression in bone marrow mesenchymal stem cells（BM-MSCs） infected with vAcrtTA2s-Ptight-HGF. Methods：vAcrtTA2s-Ptight-HGF was built by gene recombination. BM-MSCs of New Zealand white rabbits were isolated and cultured by adhe－sion method and then were infected with vAcrtTA2s-Ptight-HGF. Concentration of HGF secreted by infected rabbit BM-MSCs was verified by ELISA and Western blot with different induction doses of DOX. Results：An efficient and controllable recombinant baoulovirus vAcrtTA2s-Ptight-HGF was successfully constructed by gene recombination and rabbit BM-MSCs was successfully infected. Different levels of HGF expression under different doses of DOX were quantitatively verified by ELISA and Western blot. Expression of HGF was in dose-dependent relationship with DOX doses and was consistently lasted for 7 d. Expression of HGF was higher in experiment group than in control group（P<0.001）. Conclusions：BM-MSCs/vAcrtTA2s-Ptight-HGF demonstrates higher HGF expression and the optimum dose of DOX to induce HGF expression in infected rabbit BM-MSCs was 1 μg/ml.