| PartⅠThe expression of HLA-G5 on umbilical cord mesenchymal stem cells and negative immune-regulation on T cell in vitroBackground Mesenchymal stem cells(MSCs) has great ability of self-renewal and multilineage differentiation potential, in addition, MSCs inhibit the allogeneic immune response and reduce graft versus host disease (GVHD), so MSCs were described as an ideal seed cells for tissue engineering. But the exact mechanism has not been completely clarified. Some report showed MSCs express human leukocyte antigen (HLA) class I molecules at low levels but do not express either HLA class II antigens or CD80, CD86, CD40, or CD40L costimulatory molecules . Therefore, MSCs are not able to trigger T-cell activation.HLA-G5, the nonclassic HLA class I molecule, alters various immune cell functions, such as NK celland cytotoxic T lymphocytes-mediated cytolysis, allogeneicT-cell proliferation, and dendritic cells maturation. In view of HLA- G5 and MSCs have immunomodulatory properties, in our research we aimed to isolate and culture the MSCs from umbilical cord, observed whether HLA-G5 secreted by UC-MSCs and is critical to the suppressive functions of MSCs.Objective Isolating and culturing mesenchymal stem cells from umbilical cord,and identifying them.To study expression of HLA-G5 in UC-MSCs and the negative co-stimulation on T-cells in vitro.Methods UC-MSCs were isolated from human umbilical cord by trypsin-digesting. After 3 passages, UC-MSCs were observed under inverted phase contrast microscope, the flowcytometer assay were used to detect the surface markers on MSCs. UC-MSCs were cultured under different induced condition to show their multiable differentiation potential respectively. Western blot analyses were performed on UC-MSCs lysates using a specific anti-HLA-G5 antibody,HLA-G5 detected by enzyme-linked immunosorbent assay(ELISA) using the supernatants of UC-MSCs stimulated with rhIL-10.3H-TDR insert was used to detect the effect of UC-MSCs on proliferation of PHA-stimulated T cells, HLA-G5mAb block were used to analysis the role of HLA-G5 in the immune -regulation of UC-MSCs on T cell proliferation. The expression level of cell cytokine with or without HLA-G5mAb blocking were tested by ELISA.Result (1) UC-MSCs showed plastic adherence and typical fibroblastic morphology by light microscopy,the surface maker expressions as CD73, CD90, CD105were positive, while CD34,CD45,HLA-DR were negative. The differentiation potential of UC-MSCs could be induced into adipocytes and osteocyte in vitro. (2) Western blot analyses were performed on cell lysates using a specific anti-HLA-G5 antibody recognizing the intron-4 retaining sequence. A37-kDa protein corresponding to the HLA-G5 isoform was detected in MSCs extracts, decrease of intracellular HLA-G5 in MSCs according to passages in culture. (3) Interleukin-10 has previously been shown to upregulate the expression of HLA-G5. (4)UC-MSCs could inhibit T cell proliferation in vitro,and regulated the secretion of IL-4, IFN-γ, blocking HLA-G5 by specific Abs was able to reverse the MSC-mediated inhibition of allogeneic T-cell proliferation.Conclusion MSCs could be successfully isolated and cultured by trypsin-digesting. UC-MSCs could inhibit T cell activation and proliferation, HLA-G5 secreted by MSCs is critical to the suppressive functions. PartⅡTransplantation of umbilical cord mesenchymal stem cell to treat rat acute liver failureBackground Acute liver failure has become the hotspot and difficulty of the liver disease research, because of its fast expanding, lots of complications, difficult treatment high mortality,bad prognosis. Its pathogenesis is complex,we considered that the immune response is strong,short-term T cell response rapid destruction of live cells necrosis.Liver transplantation is an effective therapeutic method.However,liver transplantation is limited by a lot of problems, such a insufficient liver donor, severe surgical injury, immunity rejection, high cost and so on. So it is urgent to explore a new effective feasible treatment for acute 1iver failure. Recent study found that umbilical cord mesenehymal stem cells(UC-MSCs) can be induced and differentiated into hepatocytes in vivo and vitro. MSCs transplantation could provide liver function support and stimulate the regeneration of hepatocytes by proliferation and differentiation. The study on the mechanism of MSCs transplantation for acute liver failure focused on the potential differentiation ability of stem cell. The differentiation of MSCs into hepatocytes needs 6~12 days in vivo and4~5 days in vitro. However, MSCs transplantation showed obvious effects during the early stage when MSCs were far from differentiation into hepatocytes or hepatoeyte-like cells. What were the effects of UC-MSCs due to acute liver failure and its mechanism? These were the focus of the part.Objective To evaluate the effect of UC-MSCs transplation on liver function, survival and cytokines of acute liver failure rats, the importance of immune regulation.Methods Establish the rat model of acute liver failure induced by D-gal and LPS. MSCs cultivated in vitro as the first part and were labeled by CFSE. 48 rats with liver failure induced by D-gal and LPS were divided into 3 groups. Group A(UC-MSCs with HLA-G5 high expressed transplantation group); Group B(UC-MSCs transplantation group): labeled UC-MSCs were suspended in 1.5ml saline at a concentration of 1×106 cells and transplanted by tail vein inection. Group C (control group): 1.5 ml saline were injected by tail vein (n=16). Blood samples were taken from the rats on 1d, 3d ,5d and 7d after transplantation, survival rats were executed and the liver tissues were sectioned into slices, used Fluorescence microscope to detect the UC-MSCs in the liver parenchyma. The contents of IL-4 and IFN-γin plasma were measured by ELISA.Results About 99% of the cells were labeled by CFSE, Fluorescence microscope showed the cytoplasm of cell is green.Liver failure Liver failure was observed in the rats induced by D-gal and LPS.The liver failure was characterized with decreaed activity, downcast, hypnody exanimation, low response to stimulus and urinary incontinence,liver function was severely damaged, and the histological examination shows hepatocyte necrosis along with the inflammatory cell infiltrate. All the above have aproved that the establishment of the rat model of acute liver failure induced by D-gal and LPS is successful. There were significantly decrease in the ALT, AST, T-BIL levels on 3d and 7d after transplantation in group A and B when compared to group C. Group A has a better improvement of liver function. Compare with the normal rat, the levels of IFN-γwas significant increased in C group.After transplantation of UC-MSCs the levels of IL-4 was significant increased (P<0.05), the level of IFN-γwas significantly lower than C group (P<0.05), especially in group A.Conculsion Transplantation of UC-MSCs through the tail vein which can hasten the recovery of liver function and relieve the pathological changes of hepatic tissue, UC-MSCs transplantation can protect the injured liver in the early stage of transplantation and adjust anti-inflammatory and pro-inflammatory cytokines to reach a new equilibrium.It must be one of the mechanism of UC-MSCs transplantation on the treatment of acute liver failure. |
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