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

Objective: To investigate the effects of IL-10 and FASL,either alone or combination,on immune rejection inhibition in vitro,we established IL-10 and FASL recombinant plasmid and modified dendritic cells by gene engineering.Moreover,we researched the impact of IL-10 and FASL gene modified dendritic cells(DCs),either alone or combination,on allogenic rat liver transplantation rejection and survival time by building the model of rat liver transplantation.We discussed the mechanism of dendritic cells inducing immune tolerance and hoped to find the novel strategy of reducing immune tolerance in rat liver transplantation model.Methods: 1.Cell culture and identification of bone marrow-derived DCs:culturing rat bone marrow-derived hematopoietic progenitor cells by low dose of cytokines(CSF and IL-4),and the DCs of rat was derived into mature dendritic cells(mDCs)and immature dendritic cells(imDCs)based on different cytokines.Inverted microscope was applied to assess these DCs morphological structure and identification,flow cytometric and immunofluorescence technique was used to identify the phenotype of these DCs.The functional properties of these DCs were identified through observing mixed lymphocyte reaction(MLR).2.To construct,screen,identify,amplify and extract the eukaryotic expression plasmid of PCDH-CMV-EGFP-hIL-10 and PCDH-CMV-EGFP-hFasl:(1)amplified the gene of hIL-10 and hFasl by PCR,separated and reclaimed PCR production with 1% Agarose gel,double digested the vector of PCDH-CMV-EGFP and above mentioned production using ECOR1 and BAMH1,connected overnight by T4 DNA ligase,then transformed into DH5 a competent cell.(2)selectedmonoclonal colony PCR,extracted plasmid with kit and validated,then sent sequence.3.To detect the molecule phenotype and immunological functions of DCs after gene transfection of IL-10 and Fasl:(1)ELISA,Western-blot was applied to detect the expression of IL-10 and Fasl after infection.(2)The expression of CD80,CD86 and MHC-II surface molecules of infected imDCs was tested by flow cytometry.(3)The effect of gene modified imDCs on T cell proliferation was tested by MLR.(4)The TGF-?1 and TNF-? in media of mixed lymphocyte were detected by ELISA.4.To construct the model of rat liver transplantation: two-cuff technique was applied.Recipient was BN rat,donor was lewis rat.5.To study the impact of immature DCs infected by PCDH-CMV-EGFP-hIL-10 and PCDH-CMV-EGFP-hFasl IL-10,either alone or combination,on allogenic rat liver transplantation tolerance:(1)transplanted liver was stained by HE and acute liver rejection grades were valued by Baff standard.(2)survival time of different groups was observed.Results: 1.Culture and identification of DCs:(1)morphological characteristic of DCs was observed by inverted microscope that the appearance of DCs was irregular,the nucleus was empty and bright and there was protrusion around.(2)The mDCs expressed higher level of CD80,CD86 and MHC-II than imDCs analyzed by flow cytometry and immunofluorescence.(3)T cell proliferation was more obvious in mDCs than that in imDCs detected by MLR.2.To construct,screen,identify,amplify and extract the eukaryotic expression plasmid of PCDH-CMV-EGFP-hIL-10 and PCDH-CMV-EGFP-hFasl:(1)molecular size of PCR production was consistent with targeted gene which was detected by agarose gel electrophoresis.(2)bacteria colony PCR showed that plasmid was connected to the targeted gene successfully.(3)recombinant plasmid was double digested and appeared two band,the molecular size of which was consistent with that of targeted gene.(4)qualified recombinant plasmid through sequencing anlaysis was equalized to GENBANK.3.To detect the molecule phenotype and immunological functions of DCs after gene transfection of IL-10 and Fasl:(1)the expression of supernatant targeted protein of every infected group was detected by ELISA,which indicated that the sIL-10 expression of group II(IL-10)and group IV(co-transfection)was higher than other groups,while supernatant sFasl expression of group III(Fasl)and group IV(co-transfection)has no statistical significance with other groups.(2)the relevant protein expressed higher in the group of single gene infection and double genes co-infection,the expression of IL-10 was lower and the expression of Fasl was unapparent in Group I(PCDH-CMV-EGFP),which was tested by western-blot.(3)flow cytometry was applied to show that the surface expression of CD80,CD86 and MHC-II of imDCs has no influence by viral infection.(4)lymphopoiesis in the group of single gene infection was weaker than that in imDCs and mDCs group,and lymphopoiesis in the group of double genes co-infection was weaker than that of single gene infection group.(5)infected DCs and lymphocyte mixed,and the media TGF-? was detected by ELISA that TGF-? in group IV(co-transfection)was higher than group II(IL-10)and group III(Fasl),and media TGF-? of group II(IL-10)was higher than group III(Fasl),while media TGF-? of group III(Fasl)was lower than mDC group and it has no statistical significance with imDC group.Similarly,media TNF-? was detected by ELISA that TNF-?in group IV(co-transfection)was lower than group III(Fasl),imDCs group and mDCs group,but has no statistical significance with group II(IL-10).4.To construct the model of rat liver transplantation: two-cuff technique was applied and the model of rat liver transplantation was constructed successfully.5.To study the impact of imDCs infected by PCDH-CMV-EGFP-hIL-10 and PCDH-CMV-EGFP-hFasl IL-10,either alone or combination,on allogenic rat liver transplantation tolerance.The result that group II(IL-10)and group IV(co-transfection)only had a small amount lymphocytes infiltration was demonstrated by pathological examination.That was belonged to uncertainty acute rejection and slight acute rejection with RAI score 2-4.The majority of portal area in group III(Fasl)was expanded and had a lot of lymphocytes infiltration whichwas belonged to mild-moderate acute rejection with RAI score 4-5.Group I(PCDH-CMV-EGFP)and imDCs group had moderate quantity of lymphocytes infiltration and most of the biliary epithelial cells were damaged.It was belonged to mild acute rejection with RAI score 5.The group of mDCs and acute rejective group had a large number of lymphocytes infiltration.Most of the biliary epithelial cells were damaged and degenerated.The inflammation of central veins was serious with surrounding liver cells necrosis,which was belonged to severe acute rejection with RAI score 4-5.Conclusions:1.Enough mDCs and imDCs were induced and cultured successfully by different cytokines,mDCs and imDCs possessed various biological characteristics.2.We constructed the eukaryotic expression plasmid of PCDH-CMV-EGFP-hIL-10 and PCDH-CMV-EGFP-hFasl successfully,and genes could express stably in imDCs.3.hIL-10 and hFasl co-modified imDCs could inhibit allogenic lymphopoiesis obviously,and decrease the secretion of Th1,increased the secretion of Th2 in mixed lymphocyte supernatant and could induce stronger immune tolerance ability than the group of single infection and vector,which manifested that the combination of hIL-10 and hFasl would be a promising therapeutic method for inducing liver transplantation tolerance.4.To construct the model of rat liver transplantation: two-cuff technique was applied and the model of rat liver transplantation was constructed successfully.5.ImDCs modified with single-gene and double-gene could inhibite the acute rejection.But the effect of double-gene was more efficient.