HLA-G Genetic Modifyingand Magnetic Targeting Of BM-MSCs Induce Immune Tolerance Inkidney Transplantation

BackgroundRenal transplantation is recognized as the best way to treat end-stage renal disease. There are tens of thousands of uremic patients under renal transplant every year.Acute and chronic rejection after renal transplantation as well as a variety of causes of chronic allograft nephropathy clinically restricted the long-term survival of kidney.Current researches suggested that induce immune tolerance for kidney is the ideal way to solve these problems.Bone marrow derived mesenchymal stem cells(BM-MSCs) have unique immune regulation and multi-directional differentiation potential. It is considered to be a deal tool for inducing immune tolerance, and relieving and repairingtransplanted kidney injury.But after applied to animal models, many studies have found that the immunosuppression effectof BM-MSCs is weak in vivo. It can’t significantly reduce the application of immunosuppressant. Due to the lack of targeted mechanism to transplanted kidney, and the retention effect of intravenous pool in lung and liver, only a few can reach transplant kidney area. So BM-MSCs cannot take part in local immune protection and tissue repair. Therefore, there is an urgent need to study how to improve the BM-MSCs’ immunosuppressive effect and increase in the accumulation of the graft local.So we can narrow the gap between BM-MSCs’ basic research and clinical application in renal transplant area.ObjectiveThis studywould transfect HLA-G gene which have specific immunosuppressive effect into BM – MSCs via lentiviral vector. Observe whether the HLA- G molecules can be expressed on the cell surface of BM-MSCs and verify whether the immunosuppressive effect is enhanced. In the aim to make BM-MSCs play their effect in transplanted after infusion into the body, this study intends to make BM-MSCs swallow Super paramagnetic iron nanoparticles(SPIO), and place a local strong magnetic field in the transplanted kidney area, then observe whether the BM-MSCs can be accumulated in kidney and the immunosuppressive effect can be enhanced. This study intends to offer a new thinking on BM-MSCs application in the field of renal transplantation.Method1 、 Puncture rabbit femoral bone to gain bone marrow, using density gradient centrifugation separation of bone marrow mononuclear cells. Application adherent culture method for BM MSCs.Induction of differentiation in the direction of osteogenesis and adipogenesis. Using flow cytometry identify BM-MSCs’ surface moleculars.2 、 Chemical synthesis of HLA- G gene, PCR expand, enzyme digestion and connection with lentiviral vector, Real-time PCR identification and sequencing, construct of HLA-G lentiviral expression plasmid.Collecting supernatant after transfected 293 T cell, then enrichment, Real-time PCR determinate the virus titer. Using the HLA-G lentiviral transfect BM-MSCs, observe green fluorescent expression, obtain the most appropriate MOI values. Determining the cell membrane type HLA- G expression level using WB method.Determiningthe secreted type of HLA- G levelin the supernatant using ELISA method.3、Set up system of mixed lymphocyte culture, verifying the BM-MSCs’ function transfected with HLA-G(later described as HLA-G-BM MSCs) through coculture BM MSCs with lymphocyte culture.Application of immune magnetic beads from mixed lymphocyte culture to isolate CD4 + T cells, testing its apoptosis, and detecting the expression of cell cycle related proteins through the WB method.4、Using SPIO incubating HLA-G-BM MSCs, make itwith magnetism.Establishing the rabbit model of allograft renal transplantation, placing strong magnetic field in transplanted kidney area in vitro, forming the chemotaxis of the magnetic field.Transfusion of the the SPIO labeled BM-MSCs, pathological observation whether BM-MSCs can gathered in transplanted kidney.Observeing BM-MSCs’ immunosuppressive effect comparing with Cs A.Result1、Using density gradient centrifugation and adherent culture method successfully separated rabbit BM MSCs.Cells grow vigorously, can passage continuously 15 times above, and is with a typical spindle, swirling growth. BM-MSCs can be induced to the direction of osteogenesis and adipogenesis.Flow cytometry showed BM-MSCs surface with positive CD44 expression, negative CD14 expression.2、Successful build lentiviral transfection system carried HLA- G gene, the virus titers met 2 x109Tu/ml.lentiviral successfully transfected BM MSCs, and determined the best MOI values was 50.Strong green fluorescence expression after transfection.After above 10 times passage, there is still stable expression of green fluorescent.WB show BM-MSCs abundantly expressed HLA-G.ELISA show BM MSCs does not produce the secretory type HLA- G.3、Successfully establish a rabbit BM MSCs and human PBMC Transwell co-culture system.Results showed that the HLA-G-BM-MSCs could significantly inhibit lymphocyte proliferation response, the inhibition rate of 61.2%.The inhibition effect should in contact with lymphocytes. The lymphocyte proliferation in the upper chamber of Transwell systemwhich did not contact with BM-MSCs is not suppressed.Apoptosis detection display of CD4 + T cells in co-culture system, and no obvious apoptosis is observedand BM-cells were stay in G0 / G1 phase, failed to enter the proliferative stage. WB show that the cell cycle related protein cyclin D2, cyclin D3 and CDK expression decreased significantly.4 、 Successfully labeled HLA-G-BM-MSCs with SPIO, the cell morphology, proliferation, etc are not affected.Electron microscopy shows a large number of iron particles in lysosomes in the cell.Fluorescence microscope shown with green fluorescence of BM-MSCs internal contains a lot of red fluorescence SPIO particles.BM MSCs in vitro can have directional movement in strong magnetic field.After the infusion, application of transplanted kidney local external magnetic field can make more BM-MSCs gathered in kidney.The immunosuppressive effect obvious enhanced, slightly weaker than Cs A’s effect, but much stronger than not applying magnetic targeted groups.Conclusion1、Density gradient centrifugation labeled and direct adherent culture mathod for rabbit BM MSCs.Identified BM-MSCs successfully withinducing differentiation and flow cytometry.2、Application lentiviral vector which carried HLA-G gene to transfect BM-MSCs cells, gene transcription and protein expression is correctand stable.3、In vitro, HLA-G-BM-MSCs can the restrain lymphocyte proliferation through direct contact.The mechanism of inhibition of CD4 + T cell proliferation is not induce its apoptosis, but by inhibiting the expression of cell cycle proteins.4、In vivo, the magnetic targeting strategymade the SPIO labeled HLA-G-BM-MSCs gathering in kidney, and strongly enhance the immunosuppressive effect.