The Study On Inducement Of Rat Liver Transplantation Tolerance By IL-10 And TGF-β1 Gene Modified Dendritic Cells

IntroductionLiver transplantation has evolved into an effective therapeutic option in the management of patients with end-staged liver diseases. Gene transfer is a process of temporary introduction of exogenous functional genes to the patient resulting in transient gene expression of a functional gene, regulation of the tissue respondent, and elimination of the injury to ischemia-reperfusion and rejection.Interleukin 10(IL-10) is a kind of Th2 cytokine. Previous researches have demonstrated that IL-10 can reduce the expression of major histocompatibility complex II (MHCII) molecule on the surface of antigen-presenting cell(APC), decrease the secretion of Thl cytokine, and inhibit the activation of cytotoxic T cells(CTL), macrophages and NK cells. Furthermore, in the organ transplantation field, recent reports have confirmed that the localized immunosuppression in the allograft is parallel to expression level of IL-10. The high expression of IL-10 can act an immunosuppression of allograft rejection.Transforming growth factor-β1 (TGF-β1), a multifunctional cytokine, is one of a group of closely related, multifunctional molecules that play central roles in embryonic development, tumor genesis, wound healing, fibrosis, and immunoregulation. TGF-β1 has been implicated in many biological responses, including cellular growth and differentiation, intercellular adhesion and epithelial mesenchymal transformation of many cell types. It can promote the production and deposition of extra cellular matrix, healing and immunological regulation. The immunomodulator function is expressed by suppressing the proliferation of B and T cells; antagonizing the inflammatory cytokines such as IL-1, tumor necrosic factor-alpha (TNF-a), or interferon-gamma (IFN-γ); and inhibiting natural killer cells. Virus-mediated immunosuppressive cytokine gene therapy prolongs the cardiac allograft survival in rat's heart transplant model, when TGF-β1 expressed much more after recombined virus was injected into cardiac muscle. Most of donor hearts' lives were prolonged. IL-10 and TGF-β1 are important cytokines inducing immune tolerance of dentritic cell (DC), and participating to induce transplantation tolerance. So we construct IL-10 and TGF-β1 recombinant plasmid, transfect immture DC with single or two recombinant plasmid, study their protecting function after liver transplation, and investigate the immune tolerance mechanism on DC transfected with pIRES2-EGFP-hIL-10 and pIRES2- EGFP-hTGFβ1 plasmid.ObjectionTo investigate the effective method of isolate, culture and proliferation in bone marrow-derived dendritic cells in vitro. To investigate the effective method of construction pIRES2-EGFP-hIL-10 and pIRES2- EGFP-hTGFβ1 plasmid and transfection of immature dendritic cells (imDC) with pIRES2-EGFP-hIL-10 and pIRES2- EGFP-hTGFβ1 plasmid respectively or unionly using gene transfer technique. To investigate gene transfer efficiency and expression in allograft in rat liver transplantion model. We also explored whether extrinsic IL-10 and TGFβ1 are able to inhibit the allograft rejection, prolong the survival time, and find their possible mechanism.Method1. Cell culture and identification of the DC: Rat derived immature DC and mature DC was cultured with different cytokines. The phenotype of these DCs was analyzed with morphological assessment structure observing, flow cytometric analysis and immuocytochemistry. The functional properties of these DCs were identified through mixed lymphocyte reaction (MLR) and DC injected into the allogenic Lewis rat.2. To construct pIRES2-EGFP-hIL-10 and pIRES2- EGFP-hTGFβ1 plasmid with gene transfer technique: (1) To prepare human peripheral blood monouclear cells, amplify the IL-10 by RT-PCR, separate and reclaim PCR production with 1% Agarose gel, connect IL-10 with pIRES2-EGFP at the 4oC, then transform DH5a competent cell, extract sequencing. (2) pIRES2- EGFP-hTGFβ1was identified by the restriction enzyme of Bgl II and Hind III, and then sent sequence. (3) To extract plasmid with kit, detect concentration by ultraviolet spectrophotometer. 3. To transfect imDC by IL-10 and TGFβ1gene: Six days cultured ImDCs were were obtained. They were transferred according to Lipofectamine2000 protocal. The infected imDC were divided into four groups: Group I (pIRES2- EGFP), group II (IL-10), group III (TGFβ1), group IV (cotransfection). The expression of EGFP in imDC was detected after 48hr by fluorescent inverted microscope to determin transfection efficency.4.To detect the phenotype and immunological functions of DC after IL-10 and TGF-β1 of gene transfection: (1) To detect IL-10 and TGF-β1 protein in co-culture superants with ELISA kit. (2) To detect IL-10 and TGF-β1 protein in co-culture superants with Western-blot analysis. (3) To detect CD80 and CD 86 surface mocule of DC using FACS analysis. (4) To detect the expression of MHCII with Immunocytochemical stain.(5) To detect infected imDC's function by MLR.5.To construct the model of rat liver transplantation. Recipient was Lewis rat, donor was DA rat.6. To study immature DCs modified by IL-10 and TGF induced recipient liver transplantation tolerance. (1) To observe the migration and homing of the transfected DC. (2) Two days cultured DCs (2x106) were injected into the allogenic Lewis rat, which is going to be the recipient of liver transplantation 5 days later. The animals were divided into seven groups: Group control (allogenic transplantation), group imDC, group mDC, group pIRES2-EGFP, group IL-10, group TGFβ1 and group cotransfection. Acute liver rejection grades were valued by Baff standard. We observed several indexes after liver transplantation 3d, 7d and 10d respectively: Liver function, IL-12 level in the blood, Liver pathology by HE staining, apotosis of lymphocyte in recipient spleen, and lymphone node and liver was analyzed by TUNEL staining. (3) To observe the survival time.7. Statistical analysis: All data were expressed as mean±SD. Statistical analysis was performed by SPSS 12.0 software. Kaplan-Meier was used to analyze the survival rate between each group. Mann Whitney U test was used to determine the difference of graft survival time between each group. One-way ANOVA and LSD Test were also used for parametric data analysis between groups, respectively. Probability P values <0.05 were considered different and P values <0.01 05 were considered significantly different. Results1.To culture and identify DC. (1) About 1-2×107 dendritic cells were obtained from bone marrow of one rat after 10 days of culture according to the method established in our lab. The cells possessed morphological characteristic of typical DCs. The appearance of DC was irregular, and presented various length. (2) The DCs of immature group and mature group expressed OX-62 surface molecule mDC showed much higher expression of CD86 than imDC. (2) The DCs of immature group intermediately expressed class II lowly express co-stimulatory molecules, whereas the DCs of mature group reflected highly level of cell surface MHC class II and costimulatory molecules.(3) DCs of mature group induced strong allogeneic T lymphocytes proliferation, whereas DCs of immature group were poor stimulators in MLR (P<0.01) immature group is lower than that in mature group (P<0.01). (4) DC was administered to recipients 5 day before transplantation by intraperitoneal injection, the liver allograft survival time was much longer in the imDC group than in the mDC group and in control group. At the same time, the rat of mDC group showed strongest in the much early time after liver transplantation, but the rat of imDC group showed light acute rejection 7 days after transplantation.2.We contructed pIRES2-EGFP-hIL-10 and pIRES2- EGFP-hTGFβ1 plamid successfully.3. IL-10 and TGFβ1 gene can be transfered successfully to immature DC cells by Lipofectamine 2000.4. To detect the biological functions of immature DC cells modified by IL-10 and TGFβ1 gene. (1) Gene transfection was confirmed by Western blot and ELISA analysis. The results suggested that the IL-10 and TGF-β1 gene had been transduced into the immature BM-DCs successfully and IL-10 and TGFβ1 protein was effectively expressed. (2) By FACS and immunocytochemistry, it was found that the expressions of MHC classⅠ, CD80 and CD86 of IL-10-DC, TGFβ1-DC and cotransfection-DC were significantly down regulated. The capacity of stimulating T lymphocyte proliferation of IL-10-DC, TGFβ1-DC and cotransfection-DC was decreased. 5.We contructed the model of rat liver transplantation successfully.6.We also found that in the group of IL-10 and TGFβ1-DC injected, the number of apoptosis of T lymphocytes were more than that in other control groups. The injected DC was chimerised in the recipient spleen and lymph node 7 days after injection and could induce apoptosis of lymphocytes especially of T cells.7.To study the liver transplantation tolerance induced by imDC modified with IL-10 and TGFβ1.(1) Liver functions were much lighter in the cotransfection group, IL-10 group and TGFβ1 group than in control groups after transplantation of 3 days, 7 days and 10 days. (2) IL-12 level is much higer in control groups than in the cotransfection group, IL-10 group and TGFβ1 group. (3) The liver allograft survival time was much longer in the cotransfection group than in the IL-10 group, TGFβ1 group and in control group. At the same time, the control groups showed strongest rejection in the much early time after liver transplantation, IL-10 and TGFβ1 modified DC group showed light acute rejection 10 days after, and the survival time had significant difference compared with control groups (p<0.01), while the cotransfection group didn't show acute rejection after 10 days transplantation, and survival time surpassed 3 months. (4) Apoptosis of T cells in recipient spleen, lymph node and liver was analyzed by TUNEL staining. We found that apoptosis of T cells is much more in control groups than in gene transfection groupsConclusion1.A method to propagate large number of DC from rat bone marrow in vitro was established. It may be contribute to future study on the role of DCs in the immune tolerance of liver transplantion.2.We contructed pIRES2-EGFP-hIL-10 and pIRES2- EGFP-hTGFβ1 plasmid successfully, and IL-10 and TGFβ1 gene can be transferred successfully to immature DC cells by Lipofectamine 2000. IL-10 and TGF-β1 gene modification obviously inhibited multiple functions and enhanced the tolerogenicity of rat immature DCs.3.In general, this study demonstrated that in vitro, donor liposome-mediated gene cotransfection of IL-10 and TGF-β1 may inhibit the subsequent acute rejection in rat liver allografts, it may be useful in the clinic and be worthwhile to be popularized in the future. The experiments convinced the prospect of therapeutic application of DCs in organ transplantation therapeuties.