The Mechanism Of Bone Marrow Mesenchymal Stem Cells Modulate The Proliferation, The Apoptosis And The Expression Of RhoA In Rat Hepatic Stellate Cells

Objective To observe the bone marrow mesenchymal stem cells(BMSCs) regulate the proliferation and apoptosis and the expression of RohA in rat hepatic stellate cells(HSCs).The research explore the underlying mechanism of rat BMSCs paracrine HGF to modulate HSCs.Methods BMSCs were isolated from bone marrow in SD rats and cultured and purified in vitro. HSCs and fiberoblast cells were recoveried and activated morphologically, a-SMA expression in HSCs was evaluated with immuno-histochemically. An indirect co-culture system was established using a Transwell membrane system (diameter:24 mm; pore size:0.4μm). The HSCs were seeded in the lower chamber with MSCs or fiberoblast cells (1×105) cells /ml were seeded onto the Transwell membrane of the inner chamber with the ratio of 1:1.Cultures were maintained in HSCs in medium for 24 hour and 48 hour. Rat normal fibroblast cell lines and HSCs (HSC-T6) were respectively cultured as a control group. HSCs were randomly divided into four groups: blank control group (HSCs alone), negative control group (HSCs co-cultrue with fibroblasts), and experimental group (HSCs co-culture with BMSCs). At some conditions, rabbit polyclonal antibody of C-met was pre-administrated 6h before co-cultrue as pre-treatment group according to experiment need. Cell proliferation was determined by MTT and cell apoptosis was determined by flow cytometry. The expression of RohA mRNA in HSCs was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of RohA protein expression by Western blotting respectively. Supernatants were harvested and stored at-70℃until they were analyzed. The HGF concentration in the supernatants of culture was determined by ELISA method.Results 1. BMSCs inhibited the HSCs proliferation. After 24hour,48hour co-clutrue, the inhibition rates of HSCs were (12.21±2.55)%, (35.43±6.17) % respectively. The inhibited rate of HSCs in the experimental group was significantly higher than those in the other three groups (P< 0.01).2.The apoptosis of HSCs was detected by Annexin-V-FITC/PI, the rate of apoptosis of the BMSCs with HSCs group were significantly increased at time of 24 hour and 48 hour,which were significantly higher than those in the other three groups (P<0.01). Flow cytometry found that compared with the other three groups, BMSCs could promote the apoptosis of HSCs.3. The expression of HSCs RohA mRNA in each group after co-cultured was determined by RT-PCR. At time of 48 hour, the expression of RohA mRNA in HSCs was significantly lower on the MSCs Co-cultured with HSCs group than those in the other three groups (P<0.01).BMSCs inhibited the expression of HSCs mRNA.4. By Western blotting detection system and digital image analysis software, we found that BMSCs inhibited the expression of HSCs RohA protein after BMSCs co-cultrued with HSCs at time of 48 hour.Compared with the other three groups,the difference had statistical significance (P<0.01).5. The HGF concentration in the supernatants of BMSCs co-culture with HSCs was higher than MSCs culture and HSCs culture, the difference had statistical significance(P<0.01).Conclusions The rat bone marrow mesenchymal stem cells inhibit the proliferation and promote the apoptosis and decrease the expression of RohA in rat activated hepatic stellate cells in vitro. The underlying mechanism of rat bone marrow mesenchymal stme cells may be by way of paracrine HGF pathway.