摘要
目的观察携带bFGF基因的重组腺病毒体外转染平滑肌细胞的效率及转染后目的基因的表达。方法应用磷酸钙共沉淀法分别构建携带bFGF基因和大肠杆菌LacZ报道基因的重组腺病毒载体Ad.bFGF和Ad.LacZ,并以Ad.bFGF(组Ⅰ)、Ad.LacZ(组Ⅱ)、PBS(组Ⅲ,对照组)转染体外培养的大鼠平滑肌细胞(SMCs)。X-gal染色检测腺病毒载体的转染效率,噻唑蓝(MTT)检测各组SMCs的增殖情况,酶联免疫吸附(ELISA)检测各组培养液中bFGF蛋白的浓度。结果当MOI值为100时,X-gal核蓝染细胞比率达95%以上;组ⅠSMCs的增殖水平显著高于组Ⅱ和组Ⅲ(P<0.01);ELISA方法检测,在转染后第1天组Ⅰ培养液中即有bFGF蛋白的分泌表达,第3天达峰值后分泌量逐渐下降,第8天仍能检测到少量分泌,而组Ⅱ和组Ⅲ培养液中均未检测到bFGF蛋白。结论成功地将所构建的Ad.bFGF转染体外培养的SMCs,并在培养液中检测到目的基因的蛋白表达。
Objective To investigate the efficiency and the effects of adenovirus-mediated gene transfer of basic fibroblast growth factor （bFGF） to rat＇s smooth muscle cells （SMCs） in vitro. Methods A replication-deficient recombinant adenovirus vector coding for bFGF （Ad. bFGF） was constructed and transferred into SMCs cultured in vitro, which were isolated and acquired from fetal rat＇s stomach. The efficiency of transfection was estimated by X-gal staining. On 1,2,3,5,7d after infection, the effect of Ad. bFGF on SMCs proliferation was evaluated by MTT assay. Also, bFGF protein expression was detected in the cell culture medium by ELISA on 1,2,3,5,8d after transfection. Results The constructed Ad. bFGF was a very efficient vector. When MOI reached 100, the ratio of infected cells arrived at 95% high in vitro. MTT assay revealed a significant increase in cell number of SMCs transfected with Ad. bFGF as compared with the cells fransfected with Ad. LacZ and untransfected （PBS）. After gene transfection, bFGF protein expressed in SMCs and secreted into the cell culture medium, which could be detected by ELISA. Conclusion A high efficient recombinant adenovirus vector coding for bFGF is successfully constructed. After transferred into SMCs in vitro, bFGF could be detected expressing and secreting in culture medium.
出处
《重庆医学》
CAS
CSCD
2006年第22期2053-2054,共2页
Chongqing medicine
