| End-stage liver diseases caused by virus, toxic injury, inherited metabolic liver diseases are threatening human health seriously. Epidemiological investigation indicated that there were 12 million Hepatitis B virus carriers in China, and more than 110 thousand patients are died of end-stage liver diseases each year. Today, liver transplantation is the only established treatment strategy for these diseases. More than 50,000 patients have benefited from liver transplantation worldwide with an actuarial one-year, 3-year, 5-year survival of 90%, 80%, and 70% respectively, and now, more and more patients have chose this therapic method to save their lives. The rapid development of liver transplantation brings hopes to patients with these diseases.Immunorejection has been one of the main barriers for organ transplantation. Spontaneous tolerance ofen happened after liver transplantation, and the rejection of liver is not so seriously that the major histocompatibility complex (MHC) matching is not always considered in clinical liver transplantation. However, the immunoprivileges of liver are not absolutely, especially in human, there are ofen serious rejection taking place after liver transplantation, and even deadly GVHD.In recent years, although the use of immunosuppressive agents resolved the problem of rejection and promotes the development of liver transplantation significantly, long-life outcomes of liver transplantation are still limited by serious side effects of these drugs. Infection and recurrence of HCC and HCV post-transplantation, which is the main reasons of transplant death, is also considered to be associated with life-term immunosuppression administered。In addition, the complications, such as diabetes, hypercholesterolemia, hypertension and osteoporosis, may be associated with immunosuppression, which reduce the quality of life of patients.It would be a significant advancement for liver transplantation to seek new tolerogenic strategies, which can prolong the allograft survival and improve the patients'life quality in the absence of continuing immunosuppressive therapy. Athough rapid progresses were made in this field, such as mixed lymphohematopoietic chimerism induced by donor stem cells infusion, T cell costimulatory pathways blockade and dendritic cells therapy, which, to a certain extent, can induce immunosuppression or even tolerance in animal models, the results were not stable and lasting. Now, to overcome the weaknesses of one therapic way, it is a trend to choose several methods to obtain a Permanent tolerance.Bone-marrow-derived cells are presently considered to be the most promising cell source of the cell therapy of various human disorders including solid organ transplants. In these cells, one ideal potential candidate for treating allograft rejection after liver transplantation is mesenchymal stem cells (MSCs), which, different from hematopoietic stem cells (HSCs), are rare residents of the bone marrow, and can be isolated by their characteristic adherence to plastic and expanded rapidly in vitro without loss of their multipotential to differentiate into various cell types. In recent years, the immunomodulatory properties of MSCs have gained great interest. MSCs are of inherently low immunogenicity and have an immunoregulatory activity. MSCs express low levels of major histocompatibility complex (MHC) class I and negligible levels of MHC-class II; they express adhesion molecules involved in T-cell interaction, such as VCAM-1, ICAM-1, and LFA-3, and are absent of costimulatory molecules including B7-1, B7-2, CD40, or CD40L. In addition, MSCs display immunosuppressive properties on T-lymphocyte proliferation in vitro and exert a suppressive effect on (NK) cell activity, dendritic cell (DC) differentiation and maturation and decrease the production of inflammatory cytokines by various immune cell populations. Intravenous administration of MSCs also leads to in a modest but significant prologation of skin graft survival in baboons or results in decreased bone marrow graft rejection in mice.With the development of transgene techniques, gene therapy has become an important means to study induction of transplant tolerance. Blocking the CD28/B7 costimulatory pathway by transgene techniques has become a routine way. CTLA4, only expresses on activated T cells, has a significantly higher binding efficiency for the B7 molecules than that of CD28, so that it can block the CD28/B7 pathway to suppress full T cell activition. It is taking advantage of this property of CTLA4 that the recombinant fusion protein CTLA4Ig acts as a competitive antagonist of CD28 to block the CD28/B7 pathway.Our study was based on immunosuppressive properties of MSCs and combined with the CD28/B7 pathway blockade by adenovial-mediated CTLA4Ig gene transfer to MSCs, and determined whether these CTLA4Ig modified cells can induce tolerance after liver transplantation in rats, which aimes to provide a cell therapy to induce immune tolerance and avoid using immunosuppressive agents.Firstly, extracellular domain gene of rat CTLA4 and a gene fragment of the Fc portion of rat IgG were cloned and then this two genes were used to construct the recombinant fusion gene CTLA4Ig. Because the signal peptide of CTLA4 has been not studied clearly, we cited the Oncostatin M peptide gene into the 5'site of CTLA4 gene which ruled out the signal peptide sequence. Recombinant adenovirus vector harboring CTLA4Ig was constructed by homobgous recombination in E. coli BJ5183, and then packaged and propagated in 293 cells and its titer was 6.1x107PFU/ml determined by TCID50 method. Bone marrow MSCs were isolated and expanded in vitro, and then were transfected by Ad- CTLA4Ig. Expression of CTLA4Ig was detected at mRNA level, protain level and cell level by RT-PCR,Western Blot and fluorescent immunocytochemistry respectively, and CTLA4Ig was also detected in the culture supernatant from a 7-day-culture system of CTLA4Ig/MSCs, which indicated that CTLA4Ig was secreted out of cells by MSCs. Co-cultured with splenocytes, CTLA4Ig/MSCs did not elicit allogeneic lymphocyte proliferation, which can prove that CTLA4Ig/MSCs have the same low immunogenicity as MSCs. Co-culture of CTLA4Ig/MSCs with ongoing MLR demonstrated that CTLA4Ig/MSCs had stronger immunosuppressive capacities on MLR than those of MSCs in vitro, and there was a dose-effect relationship between this immunosuppression and the number of CTLA4Ig/MSCs in the culture system. In addition, the IL-2 levels detected by ELISA could be descreased by adding culture supernatant of 7-day-culture CTLA4Ig/MSCs in a dose-increasing style. This result demonstrated that CTLA4Ig secreted into the supernatant took part in the immunoregulation of CTLA4Ig/MSCs by blocking the CD28/B7 pathway, through which the immunosuppressive properties of MSCs were enhanced.Next, CTLA4Ig/MSCs were cultured in hepatic differentiation medium containing HGF for 14 days. Theses cells could express hepatocyte-specific markers including AFP, Alb and CK18 verified by RT-PCR and fluorescent immunocytochemistry. Moreover, these differentiated cells from CTLA4Ig/MSCs also had the capabilities of glycogen deposition and ICG uptake and excretion, which are mature hepatocyte-specific functions. Expression of CTLA4Ig in the transfected MSCs cultured in the differentiation medium was confirmed by RT-PCR, Western Blot and fluorescent immunocytochemistry, and the expression at 14d was weaker than that at 7d. In the model of MLR, the transfected MSCs could suppress immune response, and the suppression was statistically stronger than that of MSCs at the same number. Even if cultured in hepatic differentiation medium, the transfected MSCs also had the same immunosuppression function. These results could demonstrated that adenovirus-mediated CTLA4Ig gene transfer into MSCs can not change the potential of MSCs to differentiate into hepatocytes, and on the other hand, this gene transfer can improve the immunosuppressive function of MSCs, which can broaden the applying space of MSCs in the clinical therapy for liver diseases.At last, we establish the orthotopic liver transplantation models in inbred rats in which female DA and LEW rats served as liver donors and recipients, respectively. In this model, the CTLA4Ig-gene modified MSCs from male LEW rats infused into liver graft soon after operation. The observation found that the recipients could survive for a long time (>120days) in the absence of immunosuppressive therapy. The post-transplant evolution of the liver function of recipients (alanine aminotransferase, ALT; aspartate aminotransferase, AST; total bilirubin, Tbil and albumin, Alb) was normal, and histopathologic examination of the liver grafts found that the rejection was disappeared on 30d post- operation and the regeneration of hepatocytes was actively. These founds suggested that the tolerance of recipient to the donor liver was induced without use of Immunosuppressants.Long-term survivors in CTLA4Ig/MSCs group were challenged with full-thickness skin grafts from donor DA rat and the third-party SD rat respectively on day 60 after transplantation. We found that the donor skin graft was accepted, and the black fur of DA rat could be seen on day 10 post operation. On the other hand, the skin graft from the third-part, SD rat, was rejected about one week after placement and then necrosis of the skin was seen so that the graft was lost at last. Specificity of the induced tolerance was determined by MLR assay on day 120 after liver transplantation. Splenocytes obtained from recipients of CTLA4Ig/MSCs group served as responders and the splenocytes isolated from DA or SD rats were used as stimulators. The respondor cells had a significantly lower reactivity to splenocytes from DA rats (p<0.01) and had a normal reactivity to SD rats'splenocytes (p>0.05) compared with the control. The tolerace could be proved to be a donor-specific tolerace by skin grafting and MLR, which induced by immunosuppression function of MSCs and CTLA4Ig secreted by transfected MSCs at the same time. In addition, MSCs did not take part in liver regeneration in this model for there were not seen hepatocytes differentiated from the transplant MSCs.We can make a conclusion that infusion of CTLA4Ig modified syngnic MSCs can induce donor-specific tolerance to liver allografts after liver orthotopic transplantation without using immunosuppresive agents in rats. However, there ia something unsatisfactory. Phenomena of bile duct hyperplasia and liver fibrosis were detected in the liver grafts of 30d and 60d, which might be associated with the transplant MSCs. These phenomena suggested us that we should be more careful in using these cells for organ injury repair although the hepatic differentiation potential of MSCs brings us a new hope, because the side effects of MSCs might act at the same time. In next studies, we can try to pre-treat MSCs with the hepatic differentiation medium before infusion into the liver to avoid the side effects of MSCs and will be able to obtain a satisfactory result. Anyway, our research provided a new cell therapy to induce immune tolerance and avoid using immunosuppressive agents after liver transplantation, which can improve the life quality of the patients. .
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