Bone Marrow Mesenchymal Stem Cells Modulate The Expression Of RhoA And P27 In Hepatic Stellate Cells

OBJECTIVE To investigate the effects of Bone marrow mesenchymal stem cells (MSCs) on the mRNA and protein expression of RhoA and p27 in hepatic stellate cells (HSCs) by co-cultured, and explore the underlying mechanism of cell cycle regulation of MSCs on the HSCs.METHODS MSCs were isolated from bone marrow in rats and grown, propagated in culture flask. HSCs and fiberblast cells were recoveried and activated morphologically. An indirect coculture system between MSCs/fiberblast cells and HSCs were established using Transwell membrane systems(24mm diameter, 0.4μm pore size). Three groups were divided randomly:①HSCs control group②fiberblast control group③MSCs group. The inhibitory rate of HSCs proliferation with MSCs coculture were tested by the method of WST-8 and cell cycle was determined by flow cytometry, the mRNA and protein expressions of RhoA, p27 in HSCs were determined by reverse transcription-polymerase chain reaction(RT- PCR)and Western blot respectinvely. RESULTS The inhibitory rate of HSCs proliferation with MSCs coculture were significant higher than that of HSCs control group and Fiberoblast control group at times 24h,48h and 72h(P < 0.01). Flow cytometry showed that MSCs blocked the HSCs from G0/G1 period convert to the S phase as compared with the control group, and the percentage of G0/G1 phase cells becomed large and the S phase cells small (P<0.01). Furthermore, the mRNA expression of RhoA in MSCs group were significantly reduced at time 12h(P<0.01)as compared with the controls, and the same effects existed at the following times; but p27 expression in MSCs group was not changed during the whole cocultrue process. Finally, the protein expression of RhoA in MSCs group were significantly reduced at time 12h(P<0.01),and the same effects were taken place at the following times; whereas p27 expression in MSCs group was increased at time 12h(P<0.05), and significantly increased at time 24h and the following times(P<0.01). There were not relationship between the mRNA expression of RhoA and p27(r=0.105);However, there were negative relationship between the protein expression of RhoA and p27(r=-0.943, P<0.01).CONCLUSIONS The mechanism of MSCs inhibit the proliferation of HSCs may modulate the cell cycle process of HSCs through regulating the RhoA-p27 pathway. The underlying reason for the increased expression of p27 protein may be the down-regulated RhoA activity inhibited by MSCs.