转化生长因子β1基因转染兔颞下颌关节滑膜间充质干细胞及其表达

Construction of the recombined adeno-associated viral vector of TGF-β1 and transfection of rabbit synovial mesenchymal stem cells

目的构建TGF-β1基因的重组腺相关病毒载体并将其转入兔颞下颌关节滑膜间充质干细胞（SMSCs）中，对转染后外源性TGF—β1 mRNA和蛋白的表达进行检测。方法含TGF-β1全长cDNA的质粒pCMV6-XL4和质粒pAAV—MCS用EcoRⅠ＋XbaⅠ进行双酶切后连接，转化大肠杆菌DH5α感受态细胞，获得重组质粒pAAV-MCS—TGF-β1。通过酶切和DNA测序鉴定重组质粒的正确性。采用磷酸钙共沉淀法，以重组质粒pAAV—MCS—TGF-β1和pAAV—RC、pHelper共转染AAV-293细胞，产生具有传染性的病毒颗粒；斑点杂交方法检测重组病毒的滴度，并转染体外培养的SMSCs。以逆转录-聚合酶链反应（RT—PCR）、Western blot分别检测目的基因及蛋白的表达。结果成功地构建TGF-β1基因腺相关病毒重组质粒，病毒滴度约为3．6×10^13vp／ml，感染的SMSCs能稳定、高效地表达外源性目的基因及蛋白。结论重组腺相关病毒载体rAAV-TGF—β1能有效感染SMSCs并表达目的基因。

Objective To construct the recombined adeno-associated viral vector of transforming growth factor-β1 （ TGF-β1 ） and transfect synovial mesenchymal stem cells （ SMSCs ） of rabbit TMJ. Methods The specific TGF-β1 cDNA sequence was cloned into the plasmid of pAAV-MCS of AAV Helper-Free System to construct the expression plasmid pAAV-MCS-TGF-β1. The recombinant plasmids were identified by restriction endonuclease digesting and DNA sequencing. Then the recombinant plasmids were transfected into the AAV-293 cells together with the control plasmids pHelper and pAAV-RC by phosphate-calcium deposit method. Recombinant AAV viral particles were prepared from infected AAV-293 cells and the titer was determined by dot blot hybridization method. After that the viral particles were used to infect the SMSCs in vitro. The expressions of TGF-β1 were detected by RT-PCR and Western blot respectively. Results The recombinant adeno-associated viral vector vectors carrying TGF-β1 gene were constructed successfully. The virus titer was 3.6×10^13 viral particles per mL. The SMSCs transferred by recombinant AAV viral particles expressed abundant exogenous TGF-β1 mRNA and protein. However positive findings were not found in those cells that were not transferred. Conclusion The recombined adenoassociated viral vector of TGF-β1 gene was constructed successfully, which can transfect the SMSCs effectively.