The Differentiation Of Rat Mesenchymal Stem Cells Into Low Immunogenic Hepatocyte-like Cells And Research On Other Characteristics

Objective:1. To approach the feasibility and methods of bone marrow mesenchymal stem cells (BM MSCs) differentiate into hepatocyte-like cells (HLCs) in vitro.2. Concanavalin A (ConA)-induced splenic mix lymphocytes were co-cultured with BM MSCs or HLCs to investigate the lymphocyte proliferation and expression of transforming growth factor (31(TGF-β1) and interleukin-6(IL-6).3. To establish the model of80%hepatectomy in rats, then investigate the effects of BM MSCs injected via portal vein on regeneration and repairment of remnant liver after80%hepatectomy.Methods:1. BM MSCs were isolated, purified and identified by density gradient centrifugation combined with adherent method, then induced differentiation to HLCs in modified hepatic differentiation culture system in vitro. After4weeks of induction, change of cell morphology was observed, albumin (ALB) and urea in the HLCs supernatant were measured by EILSA, and expression of marker genes mRNA in liver cells including alpha-fetal protein, ALB and cytokeratin18(CK18) was measured by RT-PCR.2. Splenic mixed lymphocytes were extracted by density gradient centrifugation in Lewis rats. ConA-induced splenic mix lymphocytes were co-cultured with the3generation of purified BM MSCs and HLCs, respectively for36hours and72hours. Then the supernatant was collected to investigate the lymphocyte proliferation by MTT, measure the expression of TGF-β1and IL-6by ELISA, and detect the percentage of lymphocytes in S phase by flow cytometry.3. To establish the model of80%hepatectomy in rats:6-8weeks old with body weight200-220g Wistar rats were divided into experimental group (BM MSC injected via portal vein after hepatectomy immediately in16rats) and control group (same volume of saline injected in20rats). Liver function was tested by biochemical methods, cell cycle was detected by flow cytometry, and proliferation cell nuclear antigens (PCNA) was detected by immunohistochemistry.Result:1. BM MSCs were isolated, cultured and proliferated successfully in vitro.2. Cultured in the HLCs-induced differentiation culture system, BM MSCs in spindle-shaped gradually transformed to polygonal or quasi-circular, expressed AFP, CK-18and ALB mRNA, with albumin and urea secretion detected in the supernatant of HLCs-induced group. Compared with the group of lymphocytes+ConA, expression of TGF-β1was increased and IL-6was decreased in the group of HLCs+lymphocytes+ConA, both the difference was statistically significant (P<0.01). OD value measured by MTT in the group of HLCs+lymphocytes+ConA was lower than that in the group of lymphocytes+ConA, and the difference was statistically significant (P<0.01). As for the group of HLCs+lymphocytes+ConA and BM MSCs+lymphocytes+ConA, there was no significant difference in the TGF-β1and IL-6expression and OD value between two groups. Percentage of lymphocytes in S phase measured by flow cytometry was31.20%±2.36%in the group of lymphocytes+ConA,3.45%±1.87%in the group of BM MSCs+lymphocytes+ConA, and6.27%±0.62%in the group of HLCs+lymphocytes+ConA, the latter was lower than that in the group of lymphocytes+ConA, and the difference was statistically significant (P<0.01).3. Effects of BM MSCs on regeneration and repairment of remnant liver after80%hepatectomy:survival rate was93.7%in the experimental group and75%in the control group. HE stain showed that hepatocyte necrosis, inflammatory cells proliferation, cellular edema and ballooning degeneration was markedly reduced in the experimental group compared with that in the control group. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) decreased significantly in the experimental group than those in the control group on the1st,3rd and7th day after hepatectomy (P<0.05). Percentage of lymphocytes in S phase in the experimental group was higher than that in the control group (P<0.05). Immunohistochemistry of the remnant liver showed that PCNA expression was higher in the experimental group than that in the control group (P<0.05).Conclusion:1. BM MSCs had the potential to differentiate into hepatocytes.2. Both HLCs and BM MSCs could promote the secretion of TGF-β1, inhibit the secretion of IL-6, and inhibit the proliferation of ConA-induced splenic mix lymphocytes, which confirmed that BM MSCs and differentiated HLCs had low immunogenicity.3. BM MSCs injected via portal vein could promote regeneration and repairment of the remnant liver after80%hepatectomy and improve survival rate of the rats, which showed that BM MSCs did promote proliferation and repairment of liver cells.