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目的观察携带肝细胞生长因子(hepatocyte growth factor,HGF)基因的重组腺病毒对BMSCs的转染表达及活性,为研制稳定表达高生物活性细胞因子的创伤修复细胞基因药物奠定基础。方法Percoll分离液体外分离、培养成年Wistar大鼠BMSCs,取第3～5代细胞进行实验。用免疫细胞化学法检测BMSCs中HGF受体c-Met是否表达;以转染强度(multiplicity of infection,MOI)分别为0、25、50、100、200pfu/cell携带绿色荧光蛋白(green?uorescent protein,GFP)基因的重组腺病毒(adenovirus GFP,Ad-GFP)转染BMSCs,流式细胞仪测定转染效率,MTT法检测细胞存活情况,以筛选出最佳MOI;并以最佳MOI将携带HGF基因的重组腺病毒(adenovirus HGF,Ad-HGF)体外转染BMSCs,ELISA法检测转染后HGF表达水平,MTT法检测上清对BMSCs增殖影响。结果BMSCs胞膜表达HGF受体c-Met。MOI为0、25、50、100及200pfu/cell的Ad-GFP转染BMSCs48h后,流式细胞仪测定转染效率分别为0.34%±0.04%、40.72%±0.81%、61.72%±1.04%、85.33%±0.83%及17.91%±0.63%,MOI为100pfu/cell时对BMSCs的转染效率最高(P<0.05);MTT法检测示MOI为200pfu/cell时,BMSCs损伤程度最大。以MOI为100pfu/cell的Ad-HGF转染BMSCs48h后,ELISA法检测示HGF表达量达高峰;MTT法检测显示BMSCs增殖活性随加入上清中HGF含量的增加逐渐增强,加入10%(320pg)时达高峰,之后剂量加大活性不再增加。结论Ad-HGF可有效转染BMSCs并表达HGF蛋白,HGF蛋白可刺激BMSCs增殖,BMSCs可作为一种理想的基因载体细胞用于创伤修复。
Objective To observe the transfection, expression and activity of recombinant adenovirus carrying hepatocyte growth factor (HGF) gene on BMSCs and lay a foundation for the development of gene therapy for wound healing cells stably expressing high bioactive cytokines. Methods Percoll was separated from the liquid and cultured in vitro. BMSCs of adult Wistar rats were cultured, and the cells of passage 3 to passage 5 were cultured. Immunocytochemistry was used to detect the expression of c-Met of HGF receptor in BMSCs. The MOI of 0, 25, 50, 100 and 200 pfu / cell were respectively measured with green fluorescent protein (GFP) gene were transfected into BMSCs. The transfection efficiency was determined by flow cytometry. Cell viability was detected by MTT assay to select the best MOI. At the best MOI, BMSCs were transfected with recombinant adenovirus HGF gene (Ad-HGF) in vitro. The expression of HGF was detected by ELISA. The proliferation of BMSCs was detected by MTT assay. Results BMSCs cell membrane expressed HGF receptor c-Met. After transfection of BMSCs with MOI of 0, 25, 50, 100 and 200pfu / cell for 48h, the transfection efficiencies of FACS were 0.34% ± 0.04%, 40.72% ± 0.81%, 61.72% ± 1.04% 85.33% ± 0.83% and 17.91% ± 0.63%, respectively. The transfection efficiency of BMSCs was the highest at the MOI of 100pfu / cell (P <0.05). The MTT assay showed that the MOI was 200pfu / cell. After transfection of BMSCs with Ad-HGF with an MOI of 100pfu / cell for 48h, the expression of HGF reached a peak by ELISA. The MTT assay showed that the proliferative activity of BMSCs gradually increased with the increase of HGF content in the supernatant. The addition of 10% (320pg) When the peak, after the dose increase activity no longer increase. Conclusion Ad-HGF can effectively transfect BMSCs and express HGF protein. HGF protein can stimulate the proliferation of BMSCs. BMSCs can be used as an ideal gene carrier for wound repair.