The Investigation Of Rat Bone Marrow Mesenchymal Stem Cells On Apoptosis Of Hepatic Stellate Cell Line In Vitro

Objective To investigate the effect of rat bone marrow mesenchymal stem cells (MSCs) on apoptosis of hepatic stellate cells (HSCs) in vitro, and explore the underlying mechanism of MSCs on HSCs.Methods MSCs were isolated from the femur bone marrow in SD male rats, cultured and passaged to 4 in culture flask. Rat hepatic stellate cells line (HSC-T6) and fiberoblast cell line were recoveried and activated morphologically. The co-culture of MSCs with HSCs was performed by using a 6-well transwell membranes (24mm diameter,0.4μm pore size). The HSCs (2×105cells/ml) were seeded in the lower chamber, meanwhile MSCs and fiberoblast cells (2×105cells/ml) were seeded onto the inner chamber of the transwell membrane. Cultures were maintained in HSCs medium for 24h,48h and 72h. They were divided into three groups randomly:①HSCs control group: HSCs cultured alone;②Fiberoblast control group:fibroblasts and HSCs were cultured;③MSCs group:MSCs were cultured with HSCs. At the different pointed time, dynamic HSCs morphous was observed in the inverted phase contrast microscope, a-SMA expression of HSCs was evaluated by immuno-histochemical method, the inhibitory rate of HSCs proliferation was tested by the method of WST-8, and the apoptosis rate of HSCs were detected by Annexin-V-FITC/PI and DNA Ladder, the mRNA expressions of Caspase-3, Bax in HSCs were detected by RT-PCR, and the protein expressions of Caspase-3, Bax in HSCs were evaluated by Western blot.Results (1) MSCs and HSCs were cultured 24h,48h and 72h, HSCs show significant growth inhibition and presented time-dependent. The growth inhibition of MSCs group was significantly higher than HSCs control group and Fiberoblast control group (P<0.01). (2) The apoptosis rate of MSCs group by Annexin-V-FITC/PI was increased significantly after 24h and showed time-dependent, which were significantly higher than control groups at different pointed time (P< 0.01). (3) The apoptosis rate of MSCs group by DNA Ladder showed the strip of DNA Ladder, but the other groups did not show DNA Ladder. (4) Compared with the control groups, the mRNA expression of Caspase-3 and Bax by RT-PCR in MSCs group was increased significantly at time 24h (P< 0.01), and the same effection was taken place at the following times. (5) Compared with the control groups, the protein expression of Caspase-3 and Bax by Western blot in MSCs group was increased significantly at time 24h (P< 0.01), and the same effection was taken place at the following times.Conclusions (1) MSCs could inhibit the proliferation of HSCs and induce apoptosis in vitro. (2) MSCs may induce apoptosis of HSCs through the paracrine pathways. (3) MSCs induce apoptosis of HSCs by up-regulating the expression levels of Caspase-3 and Bax.