Bone Marrow Mesenchymal Stem Cells Modulate Cell Proliferation And Apoptosis And TGFβ1Expression In HSCs

Objective:Liver transplantation is the only effective way of treating end-stage liver disease.We need to find new treatments because of a variety of reasons such as the shortage of donor, transplant rejection.If not, we can not be able to improve the treatment of end-stage liver disease.A number of studies have shown that liver stem cells autologous transplantation can be achieved relieving symptoms and improving the effect of the disease. Bone marrow mesenchymal stem cells as one ideal source of liver stem cells because of its convenient drawn. In this study, non-contact cell co-culture system was employed to explore the treatment mechanism of the bone marrow mesenchymal stem cells (BMSCs) in liver cirrhosis in vitro.Methods:human bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs), human hepatic stellate cells (LX2) were passaged after recovery. Reveal1-9days OD value using Tetramethyl azo wow blue (MTT) assay and draw the growth curve of two types cells.We established vertical double cell co-culture system with6holes plastic cell culture box, and put BMSCs(1×105cells/mL)2mL in the semipermeable membrane (transwell insert) upper vaccination and HSCs(1×105cells/mL)2mL in the lower vaccination.Then conventional cultured. Experimental groups:(1)control group:HSCs cultured alone;②experimental group:BMSCs co-cultured with HSCs. The above culture systems were observed at24h and48h.We observed HSCs cell morphology through inverted phase contrast microscope; used MTT assay to determine HSCs proliferation capacity at co-culture system Oh,24h,48h,72h; used flow cytometry Annexin V-FITC/PI double staining to determine HSCs apoptosis at co-culture system24h and48h; observed HSCs apoptotic bodies by Hoechst staining; then used the ELISA to detect HSCs secretion of TGFβ1and RT-PCR to detect TGFβ1and Smad7mRNA expression at co-culture system48h and Western Blot to detect TGFβ1and Smad7protein expression at co-culture system24h,48h.Results:BMSCs at24h did not appear to inhibit HSCs proliferation, but inhibited the HSCs proliferation at48h,72h significantly and rendered time-dependent manner. BMSCs at24h did not appear to promote HSCs apoptosis but at48h.We detected TGFβ1concentration in the supernatant at24h and48h time period. The result was that TGFβ1concentration of the experimental group was significantly lower than HSCs cultured alone group(P＜0.05). The experimental group were co-cultured for48h.We fond out TGFβ1mRNA expression levels lower in the control group but Smad7mRNA expression level higher in the control group.The data comparison resulted a significant difference(P＜0.05). Also we fond out TGFβ1expression in protein levels lower in the control group but Smad7expression levels higher in the control group.The data comparison resulted a significant difference(P＜0.05).Conclusion:BMSCs can inhibit the proliferation of HSCs, induce apoptosis, the mechanism may be that BMSCs suppressed TGFβ/Smad pathway in HSCs through paracrine, up smad7and down TGFβ1, thereby inhibiting hepatic fibrosis.