联合转染人VEGF_(165)和ANG-1双基因对大鼠骨髓基质干细胞成骨分化的影响

Effect of adenoviruses-mediated human angiopoietin-1 and VEGF_(165) gene over-expression on ex vivo osteogenic potential of rat bone marrow stromal cells

[目的]研究联合转染入血管内皮细胞生长因子165(VEGF165)和血管紧张素-1(ANG-1)双基因对大鼠骨髓基质干细胞(BMSCs)成骨分化潜能的影响。[方法]将本室构建的编码人VEGF165和ANG-1双基因腺病毒质粒pAd-VIA在QBI-293A细胞内进行包装和扩增,获得腺病毒载体pAd-VIA,将其体外转染大鼠BMSCs,应用诱导培养液定向诱导向成骨细胞分化。实验分为4组:A.腺病毒载体pAd-VIA转染BMSCs并诱导组;B.BMSCs诱导组;C.腺病毒载体pAd-VIA转染BMSCs组;D.单纯BMSCs组。通过碱性磷酸酶(ALP)染色和活性的测定、Ⅰ型胶原免疫荧光染色、茜素红染色来评价联合转染双基因对BMSCs成骨分化潜能的影响。[结果]重组腺病毒质粒pAd-VIA在QBI-293A细胞内成功包装和扩增,有大量的绿色荧光蛋白表达,体外转染大鼠BMSCs后,A、B组的ALP染色、Ⅰ型胶原免疫荧光染色、茜素红染色为阳性,而C组和D组为阴性。ALP活性表达具有时间相关性,3 d开始表达,7 d到达高峰,在7、14 d,A、B组与C、D组之间ALP表达均具有统计学差异(P<0.01),A组和B组之间,在各个时间点均无统计学差异(P>0.05)。[结论]腺病毒载体pAd-VIA转染大鼠BMSCs后,未明显影响其体外成骨分化潜能。

[ Objectivel The purpose of this study was to investigate the effect of adenoviruses-mediated human angiopnictin- 1（ ANG-1 ） and VEGFj65 gene nver-expression on ex vivo osteogenic potential of rat bone narrow stromal cells. [ Method] The re-combinant pAd-VIA plasmid constructed by our laboratory was packaged and amplified in QBI-293A cells. The pAd-VIA recombined adenovims was obtained. Rat BMSCs were infected with adenoviruses encoding VEGF165 and ANG-1 at about 80% confluency.the osteogenic potential of transfected and non-transfected cells in osteogenically differentiated or non-diffierentiatcd medium were determined by alkaline phosphate staining,alkaline phosphate activity assay, collagen I immunofluorescence staining and alizarin red staining. [ Result] We packaged and amplified the recombinant pAd-VIA vectors successfully. Highly positive GFP was expressed in the field through fluorescence microscopy after being transfected with recombinant pAd-VIA vectors. Ttle transt^cted and non-trasfected ceils in osteogenieally differentiated medium were positive in alkaline phosphate staining, collagen I innnunofluorescence staining and alizarin red staining. Compared with the cells in non-differentiated medium, ALP activity in transfected or non-trasfected cells in osteogenically differentiated medium was increased significantly（ P 〈 0. 01 ）. After being iuiected with the reeombined adenovirus,there was no significant difference in ALP activity between the cells transfected and non-lranstected in osteogenically diflerentiatcd medium （P 〉0.05）. [ Conclusion] The pAd-VIA recombined adenoviruses were obtained successfully. After transfection,the osteogenic potential of rat bone marrow stromal cells had not been affected.