ANG-1基因重组腺病毒载体的构建及其转染骨髓问充质干细胞的实验研究

目的构建携带大鼠血管生成素．1（angiopoietin-1，ANG．1）基因的重组腺病毒载体，并检测其转染对大鼠骨髓间充质干细胞（bone marrow mesenchymal stemcells，BMSCs）活力的影响。方法RT-PCR法获取大鼠ANG-1基因并亚克隆至穿梭质粒pAdTrack-CMV，经测序无误后与骨架质粒pAdEasy-1在BJ5183中同源重组。重组质粒pAdEasy-1-ANG-1经鉴定后转染293细胞进行病毒包装扩增。体外转染BMSCs，检测转染后BMSCs中ANG-1的表达。MTT法评估常氧及缺氧环境下ANG-1对BMSCs的影响。结果重组腺病毒载体pAdEasy-1-ANG-1经测序及酶切鉴定正确；BMSCs经转染ANG-1基因后表达ANG-1。MTT法检测提示常氧及缺氧条件下转染前后BMSCs活性的差异均无统计学意义（缺氧组与缺氧下转染组相比，P〉0-05；常氧组与常氧下转染组相比，P〉0-05）。结论成功构建大鼠ANG-1基因重组腺病毒载体，且其转染在体外不影响BMSCs的活性。

Construction of recombinant adenovirus vectors encoding angiopoietin-1 gene and its transfection on bone marrow mesenchymal stem cells

To construct recombinant adenovirus vectors encoding rat angiopoietin-1 （ANG-1） gene and to evaluate the effects of ANG-1 gene transfection on proliferation of bone marrow mesenchymal stem cells （BMSCs） in vitro. Methods The ANG-1 gene sequence was amplified by RTPCR and then subcloned into shuttle plasmid pAdTrack-CMV, and transformed into BJ5183 carrying backbone plasmid pAdEasy-1 to obtain adenovirus plasmid through homologous recombination. The recombinant vector was identified by digestion with Pac I, and was packaged and amplified in 293 cells. The successfully packaged virus were transfected to BMSCs. The expression of ANG-1 protein in BMSCs were monitored by Western Blot assay, and the effects orANG-1 on the proliferation of BMSCs under hypoxia and normoxia conditions in vitro were evaluated by MTT assay. Results The recombinant plasmid pAdEasy-l-ANG-1 was identified to be confirmed by double digestion and DNA sequencing. The expression of ANG-1 protein in BMSCs was detected by Western Blot after the transfection of pAdEasy-l-ANG-1. MTT assay suggested that the expression orANG-1 protein in BMSCs had no significant effect on the proliferation of BMSCs before and after transfection （hypoxia ＋ ANG-1 vs hypoxia： P 〉 0.05; normoxia ＋ ANG-1 vs normoxia： P 〉0.05）. Conclusion The recombinant adenovirus vector named pAdEasy-l-ANG-1 carrying ANG-1 gene have been constructed successfully. The transfection of ANG-1 has no significant effect on the proliferation of BMSCs in vitro.