Effect Of Rats Bone Marrow Mesenchymal Stem Cells On Hepatic Stellate Cell Proliferation

Objective: We observed the effect that bone marrow mesenchymal stem cells(MSC) on the hepatic stellate cells (HSC) of cell cycle and the expression of regulating protein in order to explore the mechanism that bone marrow mesenchymal stem cells regulat the proliferation of hepatic stellate cells.Methods: Bone marrow was sterilely collected from the bilateral thighbone of rat. MSC were isolated, purified and amplified by adherence method. We used the MSC at the four passage. Rat hepatic stellate cells (HSC-T6) and fibroblast cell lines were subcultured after freezing and thawing used. MSC and HSC-T6 were cultured in plastic plates(6 holes).That the MSC were cultured in the transwell insert (2×105cells/ well) and the HSC-T6 were cultured in the plastic plate(2×105cells/ well) establish the upper and lower double-cell co-culture system. Three groups were divided randomly:①experimental group: The MSC were cultured in the transwell insert and the HSC-T6 were cultured in the plastic plate establish the upper and lower double-cell co-culture system;②control group: The fibroblast cell replaced MSC;③blank group: HSC-T6 were cultured alone. The above system were cultured and observed for 72 h. Living cells were dynamic observed the cell morphology with an optical inverted microscope. WST-8 detected in hepatic stellate cell growth inhibition rate. Cell cycle was analysised by flow cytometry. The mRNA expressions of CyclinD1 and P27 in HSC were detected by RT- PCR . The protein expressions of CyclinD1 and P27 in HSC were evaluated by Western blot. Each expression parameters of the above-mentioned were calculated according to correlation analysis.Results: In the experimental group,the inhibitory rate of HSC proliferation with MSC coculture were significant higher than the control group at different pointed time for 24 h,48 h,72 h (P < 0.01 ). The G0/G1 phase cells increased significantly (P < 0.01 )and S phase cells significantly reduced (P < 0.01 )at the co-cultured for 72 h . The MSC can block HSC from G0/G1 phase to S phase conversion. Co-cultured for 24 h, the mRNA and protein expression of CyclinD1 began to come down in experimental group and was significantly lower than the control group and blank group at 72 h (P < 0.01 ). The mRNA expression of P27 in each group of co-culture was no significant difference ( P > 0.05 ). Co-cultured for 24 h, the protein expression of P27 in the experimental group were significantly raised than the control group and blank control group (P < 0.01 ). The results confirmed that the MSC can inhibit the proliferation of HSC.Conclusions: (1)The MSC can inhibit the proliferation of HSC in vitro. (2)The mechanism may be through inhibiting CyclinD1 and increasing the expression of P27 protein to cause cell cycle arresting in G0 / G1 phase.As a result ,the proliferation of the rat hepatic stellate cell has been inhibited.