Expressions of apoptosis-related proteins in hepatocyte growth factor-transfected bone marrow mesenchymal stem cells under hypoxia conditions / 中国组织工程研究

ABSTRACT

BACKGROUND:Previous studies have demonstrated that hepatocyte growth factor (HGF) gene transfection can improve the effectiveness of bone marrow mesenchymal stem cel transplantation, but the mechanism is stil unclear. OBJECTIVE:To observe the effects of HGF gene transfection on c-MET, Bax, Bcl-2, Caspase-3 of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions. METHODS:(1) Bone marrow mesenchymal stem cel s were isolated and amplified in vitro by differential adhesion method. The infection efficiency of recombinant adenovirus Ad-HGF in bone marrow mesechymal stem cel s was tested by x-gal staining. (2) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 0, 3, 6, 9, 12 hours. RT-PCR and western blot assays were used to evaluate the expression of Bax, Bcl-2, Caspase-3. (3) Bone marrow mesenchymal stem cel s were cultured under hypoxia and serum-free conditions for 6 hours, and RT-PCR and western blot assays were adopted to detect HGF, c-Met, Bax, Bcl-2 and Caspase-3. (4) Cel scratch test was used to detect the effect of HGF transfection on the migration of bone marrow mesenchymal stem cel s cultured under hypoxia and serum-free conditions for 6 hours. RESULTS AND CONCLUSION:(1) Transfection efficiency of bone marrow mesenchymal stem cel s was increased with multiplicity of infection in a dose-dependent manner. When the multiplicity of infection was 150, the transfection efficiency was 96.4%. (2) Expressions of Bax and Bcl-2 were gradual y increased with hypoxia time (P<0.05). The Bax/Bcl-2 ratio and Caspase-3 expression reach the minimum at 6 hours of hypoxia (P<0.05). (3) Compared with the control and Ad-LacZ groups, the expressions of HGF, c-Met, Bcl-2 increased, and the expressions of Bax and Caspase-3 decreased in the Ad-HGF group after 6 hours of culture under hypoxia and serum-free conditions (P<0.05). There was no significant difference between the control and Ad-LacZ groups. (4) The mobility of bone marrow mesenchymal stem cel s was higher in the Ad-HGF group than the control group and Ad-LacZ groups after 6 hours of culture under hypoxia (P<0.05). These findings indicate that transfection of HGF in bone marrow mesenchymal stem cel s can increase the expression of c-Met, Bcl-2 and decrease the expression of Bax, Caspase-3 under hypoxia and serum-free conditions, which also enhance the mobility of bone marrow mesenchymal stem cel s under hypoxia and serum-free conditions.