Genetically Modified Bone Marrow Mesenehymal Stem Cells Can Inhibit The Coagulation In The Xenotransplantation

BackgroudLiver transplantation therapy is one of the most effictive treatment for hepatic disease in terminal stages. With the widely use of immunosuppressant, the problem of grafts rejection following allogeneic liver transplantation has been to some extent solved. Consequently, owing to the use of immunosuppressant, the survival time of patient has been greatly extended. Nonetheless, the most urgent barrier of liver transplantation therapy is the shortage of donor. According to previous research, it is about a hundred million HBV carriers all over China, and three hundred thousand dead because of terminal-stage hepatic disease every year, most of them could live longer if receive liver transplantation therapy, however, less than four thousand are preformed as a result of insufficient liver donors. Recently, lots have been done by Chinese government to promote organ donations, yet after acts on banning liver donors from executed prisoners taking effect, the problem of liver donor shortage is urgent to be solved. Consequently, it is a possible way to perform liver xenotransplantation therapy using organs from animals instead of human donors. Nowadays,xenotransplantation therapy still has some barriers to be overcome, such as immunological rejection and coagulation disorders. Fortunately, the hyperacute rejection(HAR) has been some extently solved because the α-1, 3-galactosyl transferase knock-out pig(GTKO pig). However, the acute humoral xenograft rejection(AHXR) after operation some days to some weeks still remains as the main factor of inactivation of xenograft and patient death. AHXR is a immune reaction process having multifactor and multipath, and its main pathological characteristics is thrombotic microangiopathy or disseminated intravascular coagulation(DIC). The previous research showed that the incompatibilites between the porcine and primate blood coagulation play a important role in coagulation disorders. Antithrombin- 3(AT- III) has species barrier between porcine and primate, therefore, intravascular thrombus arosed in transplanted organ, resulting from pig AT-III can’t anticoagulate effectively in human body, which leads to transplant failure. Producing by liver and vascular endothelial cells, AT- III is one of the most important anticoagulant in plasma, and plays anticoagulation role when combined with FXa and blood coagulation enzymes under the synergy of heparin. This present study aimed to identify the anticoagulation effect of genetically modified bone marrow mesenchymal stem cells(MSCs) with human AT-III in a blood coagulation model of xenotransplantation in vitro.ObjectiveThe purpose of the study was to test whether coagulopathy could be inhibited in liver hetero-xenotransplantation model. using lentivirus vector transform human antithrombin III(AT- III) gene into GTKO pig bone marrow mesenchymal stem cells(BMMSCs), we want to verify that the AT- III transfected BMMSC could secret human AT – III protein and have a good function in a in vitro model.Methods1.The primary cell isolation and analysis: The iliac bone marrow of GTKO pig were harvested aseptically. Then we tested the BMMSCs surface makers using flow cytometry and the capacity of differentiation into adipogenic, osteogenic and chondrogenic using oil Red O, Alizarin Red and Gomori staining.2. The BMMC immortalization: Transfect GTKO pig BMMCs with SV40 T lentivirus vector, then test SV40 T expression of the transfected cells by rt-PCR.3. Introduce the human AT-III gene into GTKO BMMCs: Introduce the human AT-III gene into GTKO BMMC with h AT-III lentivirus vector, then confirm h AT-III expression of the transfected cells by immunofluorescence, Western Blot, and rt-PCR.4. The co-culture system: Culture BMSCs and human serum and porcine endothelial cell to create the xenotransplantation coagulopathy model in vitro.5. Grouping: GTKO BMMCs,GTKO/h AT-III BMSCs, recombination AT-III with GTKO BMMCs, and anti-h AT-III GTKO/h AT-III BMSCs. All of the groups were cultured with human serum and porcine endothelial cell.6. Recalcification test and ELISA: detect the clotting time of each group through recalcification test, and the thrombin-antithrombin complex expression of each group by ELISA.Results:1.The cells of Passage 3 from GTKO pig bone marrow were used in the following experiments. Flow cytometry showed that the expression of CD34 and CD45 was low, and the expression of CD29, CD90, CD105 was high. Meanwhile, the cells has the capacity of differentiation into adipogenic cells, osteogenic cells and chondrogenic cells.2. The resulsts of rt-PCR showed that the m RNA expression of SV40 T is high in Transfected the GTKO BMMCs with SV40 T lentivirus vector.3.Results of Immunofluorescence, Western Blot, and rt-PCR displayed that the GTKO/h AT-III BMMCs expressed h AT-III.4. Recalcification test showed clotting time of the GTKO/h AT-III BMSCs group and the recombination AT-III group were longer than the GTKO BMMCs group. ELISA indicated that higher TAT concentration were observed in the transfection group than other control groups.Conclusions:The GTKO pig BMMCs transfected with human AT-III could express human AT-III protein,and clotting time and TAT concentration suggests gene modified GTKO pig BMMCs could inhibite coagulopathy in xenotransplantation coagulopathy model in vitro.