重组腺病毒Ad-HGF体外感染成纤维细胞后转染效率及表达的检测

Detection of the transfer efficiency and HGF expression in fibroblast in vitro after infection with recombinant adenovirus carrying human HGF gene

押检测携带人肝细胞生长因子基因的重组腺病毒Ad-HGF在体外对成纤维细胞的感染效率以及感染细胞对目的蛋白的表达。以不同感染复数(m.o.i.)(25,50,100,200)的Ad-GFP感染NIH3T3细胞,48h时用流式细胞仪检测转染效率;以50m.o.i.感染NIH3T3细胞后48h,用ELISA和Western印迹杂交法分别检测感染上清中HGF的表达。分别以50m.o.i.的Ad-GFP和Ad-HGF感染原代培养人瘢痕成纤维细胞,以检测重组腺病毒对原代培养人瘢痕成纤维细胞的转染效率和其对HGF的表达。结果表明,当m.o.i.为50时,重组腺病毒对NIH3T3细胞的转染效率已达95%以上;HGF的表达量可达每2×106细胞249ng;并可检测到HGF蛋白的一特异杂交带。以50m.o.i.的Ad-GFP感染原代培养人瘢痕成纤维细胞,72h时GFP表达达高峰,此时转染效率可高达36.75%。Ad-HGF感染原代培养人瘢痕成纤维细胞后HGF的表达在72h时达高峰,表达量可达每3.3×105细胞66ng。初步认为重组腺病毒可有效地介导HGF基因转染正常或瘢痕成纤维细胞,且感染细胞可有效表达目的蛋白。

To detect the transfer efficiency and expression of aim gene via a recombinant adenovirus carrying human hep-atocyte growth factor(HGF)gene(Ad-HGF)in fibroblast in vitro.NIH3T3cells were infected with Ad-GFP(the recombi-nant adenovirus carrying green fluorescence protein gene)at different m.o.i.(25,50,100,200).At48h after infecting,the transfer efficiency was evaluated by flow cytometry.At the same time point after infecting NIH3T3with Ad-HGF at50m.o.i.,the HGF expression in suspension was detected by ELISA and Western blotting.Primary cultured fibroblasts from hu-man scar were infected with Ad-GFP and Ad-HGF at50m.o.i.,respectively,then the transfer efficiency and HGF ex-pression were detected with above methods.The results demonstrated that the transfer efficiency of the recombinant aden-ovirus for NIH3T3cells was reached to95%when the m.o.i.was50pfu/cell,the level of HGF expression was about249ng/2×10 6 cells.And a specific hybrid band of HGF was observed.On day3after infection,the transfer efficiency of Ad-GFP at50m.o.i.for primary cultured fibroblasts from human scar reached36.75%,and the level of HGF expression was66ng/3.3×10 5 cells.The recombinant adenovirus can infect efficiently fibroblasts from normal or scar tissue and the in-fected cells could express efficiently aim protein.