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

Liver transplantation has become the only and the most effective treatment for patients at the end stage of liver disease. However, acute cellular rejection (AR) is still one of the main complications of liver transplantation. Immature dendritic cells (imDC) can induce antigen-specific T cell hyporesponsiveness and prolong the survival time of receptor; however, they may be induced maturity in the complex living microenvironment by receiving mature-signal, which consequently may aggravate transplantation AR. Basic study and research have confirmed that single gene-modified imDC could strengthen immune tolerance induced by imDC, but failed to achieve the desired state of immune tolerance owing to their own limitations. Accordingly,we joint modified imDC with different genes in order to further improve imDC's ability to induce immune tolerance, and access to the long-term survival of recipient ultimately.ObjectiveTo improve imDC's tolerance capability which induces rat allogeneic orthotopic liver transplantation and prolong the survival time of allograft, this research co-modifying imDC with FasL and TGF-β1 gene.Methods1. Established the modified rat model of orthotopic liver transplantation: On the basis of the previous two-cuff-technique, 120 cases of rat orthotopic liver transplantation were performed with fast resecting the donor liver, continuous sutureing suprahepatic inferior vena cavum and stoping bleeding point with hot pin and so on and to observe the survival rate and viability.2. Established the rat liver transplantation model of acute rejection: 60 cases of rat orthotopic liver transplantation were done in the dissimilar strain combination, including SD→SD(control group), SD→Wistar, DA→Lewis, there were 20 cases in each group. Four animals from each group were randomly sacrificed on days 3, 5, 7 and 10 after transplantation. Hepatic function (ALT and TBIL) changes were detected in peripheral blood and the pathological changes of the liver graft were observed. Then the remnant rats were carried out survival analysis, besides rats that were dead for technical factor.3. DC were cultured and derivated in vitro from bone marrow stem cells of DA rats. After their morphology, immunophenotype and function were identified and confirmed, the plasmids of pIRES2-EGFP-hFasL and pIRES2-EGFP-hTGF-β1 were co-transfected into imDC to detect expression of hFasL and hTGF-β1 gene.4. Empty vector-transfected imDC from DA rat were injected to Lewis rats via intravenous injection (IV group, 20) and intraperitoneal injection (IP group, 20). Four Lewis rats from each group were randomly sacrificed on days 1, 2, 3, 5 and 7 after syringing; from which the organs, for instance, thymus, spleen, celiac lymph nodes, liver and kidney were gathered to perform frozen section, and then observed and analyzed through fluorescence microscope.5. One hundred and forty cases of rat allogeneic orthotopic liver transplantation were accomplished in the strain combination of DA to Lewis rats, which were divided equally into 7 groups, including non- injection group(control group), mDC group, imDC group, empty vector group, FasL group, TGF group and cotransfection group (F-T group). Then 2×106 cells were respectively intraperitoneally injected to Lewis rats with distinct cells, such as mDC, imDC, empty vector-transfected imDC, hFasL-modified imDC, hTGF-β1-transformed imDC and co-modified imDC with FasL and TGF-β1 gene. Four Lewis rats from each group were randomly sacrificed on days 3, 7 and10 after transplantation. Hepatic function (ALT and TBIL) and cytokines alteration were evaluated in peripheral blood. The pathological changes of the liver graft were observed. And the DNA fragmentaion characteristic of apoptosis in spleen, celiac lymph nodes and liver were detected by carrying out TUNEL staining. Survival analysis were performed for remnant rats, except rats dead for technical factor.Results1. The median time of major steps in rat orthotopic liver transplantation: Donator operation( 20 min), donator liver trimming( 3 min), suprahepatic inferior vena cava inosculation( 9 min), portal vein recondition( 3 min), anhepatic phase( 15 min), and infrahepatic vena cava reestablishment( 3 min). The operation achievement ratio was 96.7%, and the three-week survival rate was 94.2%.2. There was only a very small number of mononuclear cell infiltration in hepatic graft after orthotopic liver transplantation in the SD→SD group, which belonged to a non-identified type of acute rejection (or no actue that the ALT, TBIL serum concentration of DA→Lewis group at day 7 had reached ( P=0.934, P=0.072 ). Long-term survival of rat receptors were found in SD→SD group without occurring acute rejection in hepatic graft; the median survival time ( MST ) was 8 days in DA→Lewis group, but in SD→Wistar group the MST was 16 days, the survival of rat recipients in DA→Lewis group was significantly shorten compared to that in SD→Wistar group( P=0.000 ).3. The process had been successfully established for culturing, derivating and amplificating DC from bone marrow stem cells of rats. These DC have been validated and identified by the inverted microscope, transmission electron microscope, scanning electron microscope and flow cytometer. The outcome of morphology showed that DC has irregular shape, the unsymmetric nucleus, and slender dendritic processes on cell surface. The result of immunophenotype demonstrated that on the cell surface of mDC and imDC, the expression level of OX62 were both highly; however, the expression level of costimulatory molecules, CD86, on imDC surface was lower than that on mDC surface. The consequence of mixed lymphocyte reaction( MLR ) displayed that the capability of imDC stimulating allogeneic T lymphocytes was significantly weaker than the ability of mDC's( P=0.0005 ). The sequences of hFasL in pIRES2-EGFP-hFasL and hTGF-β1 in pIRES2-EGFP-hTGF-β1 were consistent with those in Pubmed Genebank, which have been confirmed by gene sequencing; and the PCR notified the clear zone in 846bp and 339bp respectively. After transfection, the imDC could emit green fluorescence under fluorescence microscope, the CD86 and CD80 expression on the imDC surface did not be obviously impacted in the result of flow cytometry. The result of MLR demonstrated that the imDC induced lower T-cell proliferatoin in FasL group, TGF group and F-T group than that in imDC group( P=0.006, 0.002 and 0.000 ). The result of ELISA showed that the expression of TGF-β1 in TGF group and F-T group were all markedly higher than that in other groups. Although the differences were statistically significan, the expression differences of FasL between all groups were not statistically significant. And the Westen-Blot had detected the expression of FasL and TGF-β1.4. Result of count in IV Group showed that at day 1, liver contained the largest number of imDC ( 92.2±4.2 ), which followed by the spleen ( 32.6±7.6 ), for abdominal lymph nodes had the least number of imDC ( 3.7±2.0 ). With the extension of time, the numbers of imDC all grew downwards in the liver, spleen and abdominal lymph nodes; on the contrary, the quantity of imDC in thymus grew in number, and reached to peak value( 30.6±14.3 ) at day 5. In IP group, the largest number of imDC ( 40.7±4.0 ) located in abdominal lymph nodes at day 1, and increased at day 2 ( 64.4±17.8 ), afterward gradually declined; the imDC numbers of abdominal lymph nodes in IP group were all more than those in IV group ( P=0.000, 0.000, 0.000 and 0.003 ), respectively at day 1, 2, 3 and 5. In IP Group, the second number of imDC( 24.6±5.0 ) was contained by liver, and gradually increased, reached the peak at day 5( 33.6±15.5 ), which was severally less than that in IV Group ( P=0.000, 0.000, 0.005 ) at day 1, 2, 3. After injection the imDC number in spleen increased progressively in IP group, which was up to the peak ( 35.9±11.3 ) at day 5, was more than that in IV group ( P = 0.000 ). At day 5, the imDC amount of thymus in IP group was less than that in IV group ( P = 0.001 ).5. There was only a slight number of mononuclear cell infiltration in hepatic graft after orthotopic liver transplantation in F-T group and TGF group, which belonged to a"non-identified"or"slight"type of AR with RAI score 2 to 5; and the liver graft showed up regeneration of hepatocyte along with a small number of neutrophile granulocyte infiltration at day 10. In FasL group, histological grading of damage was rated as"slight"or"moderate"type of AR with neutrophile granulocyte infiltration in liver graft at day 7, then there was a great quantity of neutrophile granulocyte infiltration at day 10, which resulted in damage of generous hepatocytes. The outcome of Kruskal-Wallis H tests displayed: At day 3, although the rejection in liver graft of each group was rated as"slight"type of AR, there was significantly different ( P=0.020 ) between seven groups, and the AR in liver graft in F-T group and TGF group were all lighter than that in FasL group. At day 7, the difference was statistically significant ( P=0.001) between seven groups, and the AR in hepatic graft in F-T group were all lighter than those in TGF group and FasL group. At day 10, there was statistical significance between seven groups ( P = 0.001 ), the AR in hepatic graft in F-T group were all lighter than those in TGF group and FasL group ,further more the AR in hepatic graft in TGF group was lighter than that in FasL group. At day 7, hepatic function of F-T group had reverted to normal and the hepatic function of FasL group took a turn for the better, but got worse at day 10; the concentration of ALT and TBIL of FasL group were obviously higher than those of F-T group at day 10 ( P = 0.000 and P = 0.028 ). At day 10, Liver function of TGF group returned to normal, and ALT and TBIL concentrations of TGF group were significantly lower than those of FasL group ( P = 0.000 and P = 0.025 ). But compared to those of F-T group, the difference was not statistically significant ( P>0.05 ). The apoptosis indexes of liver, spleen and abdominal lymph nodes in FasL group were all significantly more than those in TGF group( P <0.05 ), but compared to those in F-T group , the differences were no statistical significance ( P>0.05 ). The outcome of ELISA notified that at day 7, the serum levels of IL-1, IL-10 and IL-12 in FasL group compared to those in F-T group and TGF group, the differences were all statistically significant( P <0.05 ),on the contraty, the difference was not statistically significant( P>0.05 ) between F-T group ane TGF group. The MST of rat recepients in FasL group was 20 days and compared to that in imDC group ( 23 d ) with no statistically significance of difference ( P = 0.335 ), and the ability of TGF Group was limited in extending the rat receptors'survival time ( 48 d ). There were rat recepients with long-term survival ( > 90d )only in F-T group, in which the survival of rat recepients was significantly prolonged than that in FasL group ( P = 0.000 )and TGF group( P = 0.001 ).Conclusion1. The modified methods shorten the operation time in rat orthotopic liver transplantation model, and enhanced the stability and increased survival rate of rat orthotopic liver transplantation model. It can be applied to foundational studying in the aspect of orthotopic liver transplantation.2. The rat orthotopic liver transplantation model with SD→Wistar rats combination could emerge acute rejection, but the proceeding was relatively slow, and there would be some rats with spontaneous immune tolerance, which were not the ideal liver transplantation animal model in acute rejection. The appearances of liver graft pathology, liver function and survival time in liver transplantation model with DA→Lewis rats combination were consistent with characteristics of acute rejection after liver transplantation in clinical, so liver transplantation model with DA→Lewis rats combination is the ideal animal model to study acute rejection after liver transplantation.3. A large number of DC could be developed, inducted and amplificated in this experiment. After transfection, hFasL or hTGF-β1 gene could be both effective expression, and didn't impact imDC's phenotype and could induce significantly low allogeneic T lymphocyte reactivity of imDC in MLR. In vitro, the ability of imDC with FasL and TGF-β1 gene co-transfection was more powerful than that with FasL or TGF-β1 single-gene transfection in inducing allogeneic immune tolerance.4. After intraperitoneal injection, initiative migration was the main distribution modus of imDC in vivo with a relatively slow speed, which avoided a large number of imDC rapid distribution in the liver via intravenous injection and the imDC lost after receptors'liver resection. This mean is suitable for liver transplantation study on the basis of immunology tolerance.5. ImDC, hFasL or hTGF-β1 single gene-modified imDC all didn't induced long-term allograft survival of receptors in allogeneic rat liver transplantation. The ability of imDC with hFasL and hTGF-β1 co-transfection was significantly enhanced by hFasL and hTGF-β1 in inducing allogeneic rat liver transplantation immunology tolerance, which could significantly extended survival of recipients in allogeneic rat liver transplantation.