Hamsc_s To Liver Cell Differentiation And With Tiopronin Transplantation In The Treatment Of Acute Liver Injury In Mice

Objective To establish the co-cultured without contact system of human amniotic mesenchymal stem cells (hAMSCs) differentiated into liver-like cells. Compared with the levels of gene and protein in different induce culture systems and hAMSCs.Methods HAMSCs were isolated from by enzyme digestion and mechanical separation. Cultured cells were identified by flow cytometry and immunocytochemistry. hAMSCs were differentiated into liver-like cells by co-culture without contact and cytokine induction. The morphology change was observed, periodic acid-Schiff reaction, oil red staining and immunocytochemistry were used to identify liver-like cells, the levels of gene and protein were detected by gene chips and ELISA method.Results The flow cytometry examination showed the expressions of CD34, CD45, in the P3, P4generation cells were negative, but CD44, CD73, CD166and CD49d were positive, especially vimentin. The P4generation hAMSCs and normal human liver cell HL-7702were co-cultured without contact, HGF and bFGF-2induce hAMSCs in vitro culture. After21d, the cells were polygonal regular nest morphology. Glycogen staining were posttive. Oil Red staining showed a lot of lipid droplets exist in cells. Compared with hAMSCs untreament, the expressions of HNF-3β were obviously higher in liver-like cells which were induced, but CK-18were no changed. The levels of alpha-fetoprotein were increased in liver-like cells of co-cultured group. The expressions of albumin were posttive in both hAMSCs and induced liver-like cells, but undetected in cell culture fluid. Gene expression profiles analysis revealed that13genes expressions of88genes were difference between untreated hAMSCs and induced liver-like cells. But between liver-like cells in co-cultured and cytokines induced groups,12genes expressions were difference. None of them expressed WNT3A、NOTCH1、TERT、ANPEP、NGFR and CCNA2genes.Conclusions It has been found that co-culture without contact of hAMSCs and normal human liver cell HL-7702can induce hAMSCs differentiation into liver-like cells. There were difference between hAMSCs untreament, hepatocyte-like cells in co-cultured group and cytokine induced liver-like cells. None of them expressed WNT3A. NOTCH1、TERT、 ANPEP、NGFR and CCNA2genes Objective To observe the therapeutic effect of hepatoprotective tiopronin combine with hAMSCs transplantation on acute hepatic injury and tiopronin on the field planting and distribution of hAMSCs.Methods The acute liver injury model was established in mice by intraperitoneal injection carbon tetrachloride (CCL4). P3-4generation hAMSCs were injected by tail vein. Then the general condition of mice were observed, hepatic function were detected by automatic biochemical analyzer. The ratio of hepatic function recovery was calculated. Liver pathological sections were observed by HE staining under light microscope. After transplantation, the liver tissues were obtained and frozen-fixed. The field planting and distribution of DAP I labeled hAMSCs was observed using fluorescence microscope.Results The labeling rate of DAPI labeled P4generation hAMSCs was reach up to90%. After acute liver injury48h, hepatic function recovery in all group were not obvious. Compared with normal control group, the levels of ALT, AST and TBil were significantly high in model group, hAMSCs transplantation group and tiopronin combine with hAMSCs transplantation group. The levels of AST and ALB protein were obviously lower than model group, but the levels of TBil were much higher than model group, especially in hAMSCs transplantation group. After hAMSCs transplantation3d, the levels of ALT. AST and TBil were strikingly low in model group and hAMSCs transplantation group, however, still higher than normal group, the ratios of hepatic function recovery of hAMSCs transplantation group and model group were57.14%and33.33%, respectively. But in tiopronin combine with hAMSCs transplantation group, the ratio of hepatic function recovery was100%. At transplantation7th day, the hepatic function recovery of both in hAMSCs transplantation group and model group nearly reached the normal levels, especially in hAMSCs transplantation group. Histopathology results showed that both the area of necrosis of liver tissues and Ballooning change in the other three groups were lower compared with model group after hAMSCs transplantation Id. There were a large area necrosis of liver tissues and Ballooning change, congestion and cholestasis in model group. After transplantation3d, the extent of liver tissue damage in all groups were alleviated obviously, especially in tiopronin combine with hAMSCs transplantation group. After transplantation7d, the recovery of liver tissue damage both in hAMSCs transplantation group and model group nearly reached the normal levels. Under fluorescence microscope, after transplantation Id. DAPI labeled hAMSCs mainly field planting around necrosis tissues of portal area. After transplantation3d-7d, hAMSCs well-distributed diffuse to surrounding.Conclusion Early stage in acute liver injury, both hAMSCs transplantation and tiopronin combine with hAMSCs transplantation can alleviate liver tissue damage at different levels. Tiopronin combine with hAMSCs transplantation could obviously shorten the recovery time of acute liver injury induced by CCL4. The field planting of hAMSCs may have been involved in the processes of alleviating liver tissue damage and promoting liver tissue repair.