Effects Of Combined HGF Gene Transfection With Hypoxia Culture On The Expression Of Apoptosis Related Proteins In BMSCs

Objective: To investigate the optimal time of Ad-HGF BMSCs for transplantation,BMSCs Ad-HGF was cultured under hypoxic condition and the expression of apoptosis related protein was detected under different time of hypoxia.Methods:(1)Ad-HGF and Ad-Lac Z adenovirus were amplified by 239 A cells in vitro, and the virus titer was detected by plaque assay.(2)Isolation, culture, virus infection, identification of BMSCs: BMSCs were isolated by preplating method, cultured under normal oxygen condition and hypoxia condition(5%CO2 + 95%N2). X-gal staining was used to detect the infection efficiency. Cell phenotype was identified by flow cytometry.(3)ELISA was used to detect HGF expression in Ad-HGF BMSCs.(4)RT-PCR and Western blot were used to detect Bax, Bcl-2 and Caspase-3 expression in Ad-HGF BMSCs for cultured different time(0, 3, 6, 9, 12h) under hypoxia.Results:(1)The titers of Ad-Lac Z, Ad-HGF was 2.5×109pfu/ml, 1.2×109 pfu/ml respectively.(2)When MOI was 150, the infection efficiency reached a peak was 95.6%.(3)A maximum production of HGF was observed at 7 day after infection(P<0.05).(4)Ad-HGF BMSCs minimum expressed Bax / Bcl-2 ratio and Caspase-3 protein when cultured 6h under hypoxia(P <0.05).Conclusions: Ad-HGF BMSCs minimum expressed Bax/Bcl-2 ratio and Caspase-3 protein when cultured 6h under hypoxia. This time point may be the optimal time point of Ad-HGF BMSCs transplantation for the treatment.