转化生长因子β_3基因转染兔骨髓间充质干细胞的实验研究

EXPERIMENTAL STUDY ON TRANSFORMING GROWTH FACTOR β_3 GENE TRANSFECTING INTO MARROW MESENCHYMAL STEM CELLS IN RABBITS

目的构建转化生长因子β3（transforming growth factor β3，TGF-β3）的腺病毒载体，并通过腺病毒载体将TGF-β3基因转染骨髓间充质干细胞（marrow mesenchymal stem cells，MSCs），使其在细胞内得到表达。方法将人TGF-β3质粒定向克隆进入穿梭质粒pAdTrack—CMV中，与pAdEasy-1共同转入细菌并重组，获得TGF-β3重组腺病毒质粒，用脂质体法导入包装细胞HEK293中，48～96h后荧光显微镜下观察绿色荧光蛋白的表达。取1月龄新西兰大白兔5只，取其胫骨骨髓。采用密度梯度离心法分离纯化MSCs，并进行传代。取传至第3代细胞采用TGF-β3腺病毒载体转染，10h后荧光显微镜观察绿色荧光表达，转染TGF-β3重组腺病毒4d后的MSCs用免疫细胞化学方法观察TGF-β3在细胞内的表达。结果pAdEasy—TGF-β3转染HEK293细胞48～96h后，荧光显微镜可见明显的绿色荧光表达。兔骨髓分离培养10d后，培养MSCs融合达70％～80％、贴壁紧密，细胞形态均一，似成纤维细胞；14d完成原代细胞培养。TGF-β3重组腺病毒转染MSCs17h后，荧光显微镜下出现少量绿色荧光，48～72h荧光加强，荧光与MSCs轮廓一致。免疫细胞化学观察，转染TGF-β3重组腺病毒MSCs胞浆内有棕黄色阳性颗粒表达。结论TGF-β3基因重组腺病毒载体的成功构建并在MSCs内的表达，为创伤愈合的基因治疗提供了研究基础。

Objective To construct recombinant adenovirus vector containing human transforming growth factor beta 3 （TGF-β3）, which was transfected into marrow mesenchymal stem cells （MSCs） and to observe its expression. Methods The cDNA TGF-β3 was intergraded into the shuttle vector of pAdTrack-CMV and recombinated with adenovirus skeleton vector pAdEasy-1 by homologous recombination. Then the product was transfected into package cell HEK293 by lipofedtamine and the recombinant adenovirus expressing the TGF-β3 gene was generated. The rabbit＇s MSCs were isolated, cultivated, purified, and then transfected with recombinant adenovirus containing the TGF-β3 gene. The green fluorescence protein expression was observed after 10 days, and the TGF-β3 expression was observed in MSCs transfected by recombinated adenovirus with TGF-β3 gene after 4 days. Results PCR showed that TGF-β3 cDNA was inserted into the recombinant adenoviral plasmid. The recombinant virus vectors with TGF-β3 gene were collected by the packaging HEK293 cells. The fusion rate of MSCs was 70%-80% with an intensive adhesion and uninform shape after the cultured 10th day. Fluorescent microscopy and immunocytochemistry demonstrated that TGF-β3 was expressed in MSCs. Conclusion Successful construction of human TGF-β3 recombinant adenovirus and its expression in MSCs provide a basis of research for the gene therapy of wound healing.