【Abstract】Objective This study was conducted to build experimental model of orthotopic liver transplantation in rat (ROLT) with the character of acute rejection; and to study the effect of cytotoxic T lymphocyte antigen 4 immunoglobulin G (CTLA4Ig) on prevention of rejection and the induction of immune tolerance of ROLT. Methods Build model of Wistar→SD ROLT(performed by the two cuff method) with character of acute rejection.Recipients were injected with CTLA4Ig 75 μg per ROLT into abdominal cavity after 2 days of operation. Contrast was made with no treatment group, the clinical characters, the liver function, the transplantated liver pathologic character and the concentrations of TNFα in serum were observed and measured on postoperative day 7. In treatment group, all above observation were done on postoperative month 4. Above all, determination of the effect of CTLA4Ig on preventing acute rejection and inducing tolerance in ROLT was observed.Results ①Recipients (no treatment group) died one by one within 6th～14th days; pathologic character of rejection in transplantation liver could be found; ② In treatment group, on postoperative day 7 and month 4, no clinical rejection character and no pathologic character of rejection in transplantation liver were found and serum concentration of cytokins related to TNFα found lower than that of contrast group(P＜0.05), and serum concentration of ALT、AST、TBIL、DBIL found lower too(P＜0.05); But serum concentration of TP and Alb was found higher than that of contrast group(P＜0.05). Conclusion ① CTLA4Ig treatment alone inhibits the rejection responce in ROLT. ② CTLA4Ig treatment can Prevent rejection and induce immune tolerance in ROLT model with characters of acute rejection; the serum concentration of cytokins related to TNFα can probably be used as evaluation standard of rejection in ROLT rejection.
Objective To study the role of chimerism on immune tolerance to cardiac allografts. Methods Male DA rat hearts were transplanted to male Lewis rats using Ono’s model and randomly divided into three groups: normal control group (group Ⅰ), rejection group (group Ⅱ), immune tolerance group (group Ⅲ). Mean survival time (MST), histological changes, mixed lymphocyte reaction (MLR), chimerism of recipients’ spleen and thymus were measured after operation. Results The MST of cardiac allografts in group Ⅲ (85.28±7.48 d) was significantly longer than that in the group Ⅱ (7.33±1.03 d). Only a few inflammatory cells infiltrated in cardiac allografts in group Ⅲ. MLR of group Ⅲ were significantly decreased compared with those of group Ⅰ (Plt;0.01). Conclusion The chimerism of recipient plays an important role on immune tolerance to cardiac allografts.
Objective To introduce the research progress in the immune of composite tissue allotransplantation. Methods The related articles were reviewed to summarize the immune characteristics, experimental developments, and cl inical experiences of composite tissue allotransplantation. Results Composite allogeneic tissue is on the body surface, including the composition of the complex with high antigenicity. There are a lot of differences in the immune responsesbetween composite tissue allotransplantation and organ transplantation, such as immunosuppressant protocol, rejectiondiagnosis, and chronic rejection. Conclusion In the next study, it is urgently needed to learn these experiences and toestabl ish the special standard of composite tissue allotransplantation in induction of immune tolerance, local medication, and rejection diagnosis.
Objective To investigate the expression of transcription factor Foxp3 in the orthotopic liver transplantation by using the inbred rats with spontaneous immune tolerance. Methods The model of orthotopic liver transplantation was established on inbred rats according to double-sleeve technique. The total RNA that was isolated from liver was reversely transcribed into cDNA. The method of real-time fluorescence quantitative PCR (RFQ-PCR) was used to analyze the expression level of Foxp3 mRNA in tolerance group and syngeneic group, respectively. The expression of Scurfin in hepatic tissue was assayed by Western blot and then was analyzed by computer imaging system. Results The expression levels of Foxp3 mRNA and Scurfin in the transplanted liver were significantly lower than those of normal liver within the first week after transplantation. The level of Foxp3 mRNA began to increase on day 7 and reached the peak point on day 14. The expression level of Foxp3 mRNA began to decrease on day 30 but was still higher than the normal value (P＜0.05). The Western blot showed resemble changes on that of Scurfin. Conclusion Transcription factor Foxp3 may play an important role in the spontaneous immune tolerance in the orthotopic liver transplantation of inbred rat.
Objective To observe the effect of transfer of immature mouse myeloid dendritic cells （DC） generated with low-dose granulocyte-macrophage colony-stimulating factor （GM-CSF） on cardiac allograft survival. Methods Mouse DC were generated with standard doses or low doses GM-CSF from bone marrow cells, the phenotype and functional properties of these DC were compared through fluorescence-activated cell sorting（FACS） analysis and mixed lymphocyte reaction（MLR）, 1. 0 × 106 DC generated with low doses GM-CSF were administered to the recipients 7 days before transplantation, and the cardiac allograft survival were observed. Results In contrast to DC generated with standard doses, DC generated with low doses were phenotypically immature DC （CD11c＋, CD80- , CD86- , MHCⅡlow）, and induced allogeneic T cell unresponsiveness, and administration of these DC to recipients prolonged cardiac allograft survival from 6.3±1.2 days to 14.3±1.9 days. Conclusions DC generated from mouse bone marrow progenitors in low doses of GM-CSF are phenotypically and functionally immature, and prolong cardiac allograft survival when they are administered 7 clays before transplantation.
ObjectiveTo investigate the relationship between peripheral T cell apoptosis and specific immune tolerance induced by T cell vaccination（TCV）. MethodsT cell vaccinations were made from the spleen cells of SD rats, which were induced by ConA and were challenged with the spleen cells of Wistar rats. Normal SD rats were vaccinated intraperitoneally with TCV (experimental group) or RPMI 1640 culture buffer (control group) respectively .Oneway mixed lymphocyte reaction (MLR) were performed,the apoptosis of peripheral T cell were assayed using flow cytometric analysis before and after vaccination.ResultsIn experimental group, the result of MLR showed that the response captivity of SD rat spleen cells were suppressed significantly after vaccination in comparison with prevaccination (Plt;0.01) and the percentage of peripheral T cell apoptosis was increased significantly after vaccination compared with prevaccination (Plt;0.01); In control group, there was no significant difference between prevaccination and postvaccination about MLR and peripheral T cell apoptosis. ConclusionT cell vaccination is capable of inducing Agspecific immune tolerance, the T cell apoptosis of peripheral blood induced by T cell vaccination may result in the depletion of Agspecific reactive T cells, which is vital in inducing specific immune tolerance.
Abstract: Objective To investigate the phonetype and tolerogenic function of semimature dendritic cells (DC) transfected by myeloid differentiation marker 88（MyD88） small interfering ribonucleic acid（siRNA）. Methods Bone marrow of BALB/c mice was inoculated and cultured in vitro，and induced into DC by 10ng/ml recombinated granulocyte macrophage colony stimulating factor (rmGM-CSF) ，then DC was divided into three groups at the 8th day: blank control group: added nothing; lipopolysaccharide （LPS）group: added with 1μg/ml LPS; and experimental group: added with 1μg/ml LPS after transfected by MyD88 siRNA for 4 hours. The phonetype of three groups was analysed by fluorescenceactivated cell sorting (FACS). The concentration of interleukin 10（IL-10）and interleukin 12（IL-12） in culture supernatant was detected by enzyme linked immunosorbent assay（ELISA）.The function of stimulating alloreactive T cell roliferation were evaluated by primary and secondary mixed lymphocyte reaction （MLR）. The cardiac allograft survival time was compared after DC of three groups injected into recipient mice. Results The phonetype of blank control group DC was CD11c＋，CD25-，CD40low，CD80low，CD86low，MHC-Ⅱlow， which could be induced to mature DC by LPS. Experimental group DC was phonetypically semimature DC (CD11c＋，CD25－，CD40mid，CD80low，CD86low，MHC-IIid) and the IL-10/IL-12 ratio of semimature DC increases significantly. yporesponsiveness of alloactive T cells can be induced by experimental group DC and the survive time of heart allograft was prolonged. Conclusion Immature DC could become semimature DC after transfected by MyD88 siRNA and stimulation by LPS. These semimature DC are more tolerogenic than immature DC.
Objective To investigate the effect of pre-infusion with allogeneic lymphocytes treated by 5-FU on inducting immune tolerance of liver transplantation in rats. Methods Wistar and SD rats were used as liver transplantation donors and recipients, respectively. They were divided into 4 groups as following: control group: liver was transplanted from Wistar to SD rats without any other treatment; lymphocytes group: recipient was pre-infused lymphocytes （5×106 cell/ml, 1 ml） from Wistar rat 7 d and 4 d separately before transplantation; low concentration of 5-FU with lymphocytes group: lymphocytes were treated by 5-FU (7.5 μg) before pre-infusion; high concentration of 5-FU with lymphocytes group: lymphocytes were treated by 5-FU (15 μg) before pre-infusion. Pathological changes were observed on day 7 after liver transplantation. Results Acute slight rejection was observed in low concentration of 5-FU with lymphocytes group: liver cell cords were well-arranged basically, hepatic lobules structures could be observed, a few inflammatory cells infiltrated around central veins, and a few lymphocytes infiltrated around portal area. Acute severe rejection was observed in control group, and acute moderate rejection was observed in high concentration of 5-FU with lymphocytes group and lymphocytes group. Conclusion Pre-infusion of lymphocytes treated with low level 5-FU can induce immune tolerance better in recipients after liver transplantation.
Objective To study the effect of allogeneic canine cord blood mesenchymal stem cells（cbMSC）transplantation on the distribution of CD4+T and CD8+T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells (MSC) were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by βgal expression. CD4+T and CD8+T cells distributed in infarcted area were detected by immunohistochemical staining method. The ImagePlus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4+T in experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4+T/CD8+T ratios were 0.63±0.12, 0.51±0.15 and 0.66±0.08. In control group, the IOD of CD4+T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28.02±2.59, 29.79±6.83, and the CD4+T/CD8+T ratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. The IOD of CD4+T and CD4+T/CD8+T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8+T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51.28±10.01, 20.87±4.50 and 19.91±2.87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4+T cells infiltrated in periphery of infarcted area and maintaining CD4+T/CD8+T ratios at a lower level.
Objective To investigate the effects of nuclear factor kappa B decoy oligodeoxynucleotides ( NF-κB decoy ODN) transfection on biological characteristics of mature dendritic cells ( mDCs) in mice. Methods Immature DCs were harvested from Balb / c mice bone marrow, followed by the incubation with antigen OVA and LPS, and mature DCs were evaluated by the expressions of CD11c and MHC-Ⅱ detected by FACS. Mature DCs were transfected with NF-κB decoy ODN and the changes of NF-κB activity after the transfection were detected by EMSA. The expressions of the costimulatory molecules( CD40,CD80 and CD86) on DCs were detected by FACS and the proliferation of T cells was tested by mixed lymphocyte reaction( MLR) . Results The mature DCs were cultured successfully. The NF-κB activity of NF-κB decoy ODN transfected DCs was decreased significantly( P lt; 0. 05) . There was no difference in the expressions of CD40 and CD80, but the expression of CD86 was decreased significantly in NF-κB decoy ODN transfection group( P lt; 0. 05) . MLR test showed that the proliferation of T lymphocyte cells was inhibited by NF-κB decoy ODN transfected DCs, but was stimulated bly by the DCs of other groups. Conclusions Mature DCs transfected with NF-κB decoy ODN could inhibit the proliferation and activation of antigenspecical T cells, which was probably related to the down-regulation of CD86 on DCs. This modified DCs might be a promising vaccine for the treatment of asthma in the future.