| Objective: To observe the regulatory effects of bone marrow mesenchymal stem cells(BMSCs) stimulated by TNF-αon apoptosis of hepatic stellate cells (HSCs) in co-culture system and to explore the possible mechanisms involved.Methods: Bone marrow was isolated from rat femur and grown, purified and amplified by adherence method, passage 3-6 were used. HSCs and fibroblast cell lines were subcultured after freezing and thawing. HSCs were cultured in the transwell insert and BMSCs were cultured in the plastic plate establish the transwell co-culture system. Cells were divided into the following groups:①HSCs blank group: HSCs cultured alone;②HSCs control group: HSCs co-cultured with fibroblast cells ;③BMSCs blank group: BMSCs cultured alone;④BMSCs stimulated culture group: BMSCs + TNF-α;⑤Normal co-culture group: BMSCs + HSCs;⑥Stimulated co-culture group: BMSCs +TNF-α+HSCs. The cultured period was maintained and cells were performed at 24h and 48h. At the different pointed time, dynamic HSCs morphous was observed under the inverted phase contrast microscope; a-SMA expression of HSCs was immunohistochemically evaluated, HGF inhibiting the proliferation of HSCs was tested by MTT assay, and the apoptosis rate of HSCs were detected by Annexin-V-FITC/PI, ; The expression of RhoA mRNA /HGF mRNA and protein in HSCs/BMSCs was determined by reverse transcription-poly- merase chain reaction (RT-PCR) and Western blot respectively. Cell supernatants were harvested to determine the concentration of hepatocyte growth factor (HGF) by enzyme-linked immunosorbent assay (ELISA).Results: 1. No effect on HSCs proliferation after stimulated by HGF alone was observed as compared to the HSCs blank croup. 2. Apoptosis rate of HSCs in Stimulated co-culture group(24,48h: 6.583±0.091%,29.960±0.223%) was significa ntly higher than that of Normal co-culture group(24,48 h: 4.700±0.168%,23.140±0.115%) (P<0.01) with time-dependent manner. 3. The expressions of RhoA protein and mRNA in HSCs were shown time-dependent and significantly lower in the co-cultured group than those in other groups (P< 0.01), and those of co-culture group stimulated by TNF-αwas significantly higher than normal co-culture group(P<0.01). The expressions of HGF protein and mRNA in BMSCs were shown time-dependent and significantly higher in the co-cultured group than those in BMSCs blank group(P<0.01), and those of co-culture group stimulated by TNF-αwas significantly higher than normal co-culture group(P<0.05). The expressions of BMSCs HGF protein and mRNA were shown time-dependent and significantly higher in the BMSCs stimulated cultured group than the BMSCs blank group(P<0.01). 4. Supernatants of the HSCs were determined by enzyme-linked immunosorbent assay (ELISA): HGF level secreated by HSCs at 24 hours(34.573±0.071 ng/ml) was higher than that at 48 hours(21.733±0.133 ng/ml); HGF level screated by BMSCs at 48 hours was higher than that at 24 hours, furthermore, the HGF level in cultured group stimulated by TNF-αwas significantly higher than that in BMSCs blank group (P<0.01); whereas the HGF level in Stimulated co-culture group[24,48 h:(18.791±0.080),(25.123±0.074) ng/ml) ] was significantly higher than that in normal co-culture group[24,48 h:( 11.897±0.045),( 11.297±0.031) ng/ml] (P<0.01) at the corresponding time point.Conclusions: The expression and secretion of HGF is upregulated in co-culture system, associate with the decrease of RhoA and apoptosis of HSCs, The effect is enhanced in BMSCs stimulated by TNF-α.; through synergistic action of HGF and others'factors paracrine by BMSCs may be the underlying mechanism which BMSCs promote the apoptosis and reduce RhoA expression in rat activated HSCs .
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