| The study was supported by Guangdong Nature Science Foundation (No:990386) , by Guangdong Province Medical Research Foundation (No:A1999326) and by the Foundation of Wuhan General Hospital of Guangzhou Command(2005).Immune privilege concept were proposed for more than 100 years.In vivo,brain,eyes, cronea, ovaries and placenta were the immune privilege organs,whiche were not interfered and rejected by the body. The research of immune privilege could make transplanted cells, tissure and organs break the immune barry of homogeneity and heteroplasty,which has wide perspective in clinical application and large economic benefit.Enhancing the research of immune privilege will make organ transplantation not need immunodepressant,but patients will get long-term surviving.In our country or abroad the research about immune privilege of sertoli cell started in recent years.The chief research was concentrated on the immune privilege of sertoli cells.Sertoli cells cound produe two kinds of cytokine ,FasL and TGF-β1,which was the chief reason of which sertoli cells had the function of immune privilege.So we carried the research on the assessment of sertoli cells immune privilege mechanisms and the effect to cardiac allograft of rats.Objective: 1.To investigate the purification method of sertoli cells and detect the expressing rate of FasL and TGF-β1 in sertoli cells.2.To obersve the toxic function of sertoli cells to T lymphocyte,when they were cocultured.3.To explore the apoptosis effect of sertoli cells to T lymphocyte.4.To make cardiac allograft animal models of myocardium transplanted with or without sertoli cells.5.To contrast the acute rejection,myocardium apoptosis,the expressing of Fas mRNA,FasL mRNA and TGF-β1 mRNA in the animal models' transplanted hearts of myocardium with or without sertoli cells transplanted.To explore whether sertoli cells have immune privilege function to allografted hearts.Experiment 1: Isolation and purification of sertoli cells and identification immune privilege mechanisms of sertoli cells1. Objective1) To investigate isolation and the purification method of sertoli cells and detect the expressing rate of FasL and TGF-β1 in sertoli cells;2) To obersve the toxic function of sertoli cells to allogene T lymphocytes, when they were cocultured.3) To explore the apoptosis effect of sertoli cells to allogene T lymphocytes.2. Methods1) Preparing of Wistar rats' sertoli cells Testes of 2-3 weeks Wistar rats were digested by digestive enzyme and then filtrated by 500um grid.The digested cells were incubated in 39℃for 48 hours, which temperature could remove the spermatogenic cells and remove the injured sertoli cells.2) Preparing of SD rats' T lymphocytes To grind the SD rats' spleen on 200 mesh wire gauze and collect the cells suspension.To separate high purity T lymphocytes by using lymphocyte isolation medium and nylon fiber column.3) Detection of the expression of FasL,TGF-β1 in sertoli cells by SABC method.4) Toxic function of different quantitive Wistar rats'sertoli cells to SD rats' T lymphocytesAdding target cell 1*105 T cells and 20ul phytohemagglutinin (PHA, 20ug/ml) in each well of 96 wells cultivation plate, and adding effector cell sertoli cells which exposed to 15Gy ray for 1 hour. To separate 6 groups(T0～T5) according to the rate of effector cell target cell, and one by one the rate was 0/1,0.001/1,0.01/1,0.1/1,1/1,10/1 ; T0 group:T cells+Sc 0(100ul)+ DMEM/F-12 100ul; T1 group: T cells+Sc 1*103/ml (100ul)+ DMEM/F-12 100ul; T2 group: T cells+Sc 1*104/ml (100ul)+ DMEM/F-12 100ul; T3 group: T cells+Sc 1*105/ml (100ul)+ DMEM/F-12 100ul; T4 group: T cells+Sc 1*106/ml (100ul)+ DMEM/F-12 100ul; T5 group: T cells+Sc 1*107/ml (100ul)+ DMEM/F-12 100ul;TO group was blank group,and every group had 6 duplicated wells.To culture for 72 hours and detecte the toxic effect of sertoli cells to T lymphocytes by MTT method.5) Toxic function of Wistar rats'sertoli cells to SD rats' T lymphocytes for different cultured timeAdding target cell 1*105 T cells and 20ul phytohemagglutinin (PHA, 20ug/ml)in each well of 96-wells cultivation plate and adding effector cell 1*105 sertoli cells (100ul) exposed by 15Gy ray.The rate of effector cell and target cell was 1/1. To group according to the culture time : T0d group:T cells+Sc 0(100ul)+ DMEM/F-12 100ul; T1d group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul; T2ds group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul; T3ds group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul; T4ds group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul; T5ds group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul; T6ds group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul; T7ds group: T cells+Sc 1*105 (100ul)+ DMEM/F-12 100ul;T0d group was blank group,and there were 8 groups,which every group had 6 duplicated wells.To culture and detecte the toxic effect of sertoli cells to T lymphocytes by MTT method.6) The mechanism of sertoli cells toxic function to T lymphocytes Adding target cell 1*105 T cells and 20ul phytohemagglutinin (PHA, 20ug/ml)in each well of 96-wells cultivation plate and adding effector cell 1*105 sertoli cells (100ul) exposed by 15Gy ray. The rate of effector cell and target cell was 1/1. To group according to the interfering factors: G0 group:T cells+Sc 0(100ul)+DMEM/F-12 100ul; G1 group: T cells+Sc 1*105cultured for 30min within FasL-mAb and TGF-β1 mAb(100ul)+DMEM/F-12 100ul; G2 group: T cells+Sc 1*105 cultured for 30min within FasL-mAb (100ul) +DMEM/F-12 100ul; G3 group: T cells+Sc 1*105 cultured for 30min within TGF-β1 mAb (100ul)+DMEM/F-12 100ul; G4 group: T cells+ supernatant solution of cultured Sc 1*105 for 48hours (100ul)+DMEM/F-12 100ul; G5 group: T cells+Sc 1*105 (100ul)+DMEM/F-12 100ul;G0 group was blank group, and there were 6 groups,which every group had 6 duplicated wells.To culture for 72 hours and detecte the toxic effect of sertoli cells to T lymphocytes by MTT method.7) The mechanism of sertoli cells apoptosis function to T lymphocytes Adding target cell 1*106/ml(0.5ml)T cells and 100ul phytohemaggluti -nin (PHA, 20ug /ml) in each well of 48-wells cultivation plate and adding effector cell 1*106/ml(0.5ml) exposed to 15Gy ray for 1 hour.The rate of effector cell and target cell was 1/1. To group according to the interfering factors: G0 group:T cells+Sc 0(0.5ml)+DMEM/F-12 0.5ml; G1 group: T cells+Sc 1*106/ml(0.5ml )cultured for 30min within FasL-mAb and TGF-β1 -mAb+DMEM/F-12 0.5ml; G2 group: T cells+Sc 1*106/ml(0.5ml )cultured for 30min within FasL-mAb +DMEM/F-12 0.5ml; G3 group: T cells+Scl*106 /ml(0.5ml ) cultured for 30min within TGF-β-mAb+DMEM/F-12 0.5ml; G4 group: T cells+ supernatant solution of cultured Sc 1*106/ml(0.5ml ) for 48hours +DMEM/F-12 0.5ml; G5 group: T cells+Sc 1*106/ml(0.5ml )+DMEM/F-12 0.5ml;GO group was blank group, and there were 6 groups,which every group had 6 duplicated wells.To culture for 72 hours and to detecte the apoptosis effect of sertoli cells to T lymphocytes by TUNEL method.3. Results1) Every testis of Wistar rats can be separated (2.68±0.29)×107 sertoli cells. Survival rate of sertoli cells attained (94.15±2.23)%.The sertoli cells' expressing rate of FasL arrived (91.65±1.63) %. The sertoli cells' expressing rate of TGF-β1 arrived (89.20±2.16) %.2) With increasing of sertoli cells, the toxic function to allogeneic T lymphocytes increased.When the rate of sertoli cells and T cells attained 1/1,the toxic function arrived at the peak effect.When the rate exceeded 1/1,the effect of sertoli cells could not increase,but still in high level.3) With the cocultivation time increasing, the toxic effect of sertoli cells to allogeneic T lymphocytes increased; but when cocultivation time passed 3 days,the toxic effect no longer increasd and still stayed in high level. 4) The toxic effect of sertoli cells to T lymphocytes was blocked by anti-FasL and anti-TGF-β1. The toxic effect could not be blocked by anti-FasL or anti -TGF-β1 respectivly.The culture solution had toxic effect to T lymphocytes, which showed that the culture solution had some cytokine as solubility FasL which had toxic effect.5) The apoptosis effect of sertoli cells to T lymphocytes was blocked by anti-FasL mAb and anti-TGF-β1. The apoptosis effect could not blocked by anti-FasL mAb or anti-TGF-β1 mAb respectivly.The culture solution had apoptosis effect to T lymphocytes,which showed that the culture solution had some cytokine as solubility FasL which had apoptosis effect.The apoptosis and toxic function of sertoli cells to allogeneic T lymphocuye were at the equal place.4. Conclusion1) The testes of 2 weeks rats contained plenty of sertoli cells.2) Sertoli cells expressed FasL and TGF-β1 in high level.3) With increasing of sertoli cells, the toxic function to allogeneic T lympho -cytes increased.When the rate of sertoli cells and T cells attained 1:1,the toxic function arrived at the peak effect.When the rate exceeded 1:1,the effect of sertoli cells could not increase,but still in high level.4) With the cocultivation time increasing,the toxic effect of sertoli cells to allogeneic T lymphocytes increased;but when cocultivation time passed 3 days,the toxic effect no longer increasd and still stayed in high level.5) The toxic effect of sertoli cells to T lymphocytes was blocked by anti-FasL and anti-TGF-β1. The toxic effect could not blocked by anti-FasL or anti -TGF-β1 alone.The culture solution had toxic effect to T lymphocytes, which showed that the culture solution had some cytokine as solubility FasL.6) The apoptosis effect of sertoli cells to T lymphocytes was blocked by anti-FasL and anti-TGF-β1. The apoptosis effect could not blocked by anti-FasL or anti -TGF-β1 alone.The culture solution had apoptosis effect to T lymphocytes,which showed that the culture solution had some cytokine as solubility FasL.The apoptosis and toxic function of sertoli cells to allogeneic T lymphocuye were at the epual place. Exeperiment 2.Making cardiac allograft animal models of rats with myocardium transplanted allogeneic sertoli cells1. ObjectiveMaking Wistar to SD rats allograft cardiac animal models with transplanting Wistar rats' sertoli cells into donator's myocardium.Contrast to the group without sertoli cells transplanted into donator myocardium,observing the survival time of transplanted hearts.2. MethodsMaking 40 cases of Wistar rats to SD rats allograft cardiac animal models. 40 cases were divided in two groups.Every group had 20 cases.Contrast group (T1) : When the heart transplantation finished, DMEM/F-12 culture medium(100ul) were injected into donators' myocardium.Experiment group(T2): When the heart transplantation finished, 1*107 Wistar rats' sertoli cells(100ul) were injected into donators' myocardium. If transplanted hearts stoped beating or recipient died,those cases were regarded as unsuccessful cases.The successful cases were sended into the experiment 3.Recorded the time of obtaining donator hearts,perfusing heart,clipping heart,aorta arterial anastomosis,pulmonary arterial anastomosis,cold ischemia,anesthesia and volume of bleeding in operation.3. Results1) Made allografted animal models 40 cases.Survival cases were 34 cases. In T1 group, survival cases were 18, and defeated cases were 2; In T2 group, survival cases were 16, and defeated cases were 4. The achievement ratio were 85.0% in total. In T1 group,one cases was died of anesthesia and one case died of bleeding in arterial anastomosis position. In T2 group, 2 cases was died of anesthesia, one case died of bleeding in arterial anastomosis position,and one case died of diffused peritonitis.All transplanted hearts of 34 cases survived for more than 7days,and went into the experiment 3.2) In T1 and T2 group,with t-test analysis,the time of obtaining donator hearts,perfusing heart,clipping heart,aorta arterial anastomosis,pulmonary arterial anastomosis,cold ischemia,anesthesia and volume of bleeding in operation were not statistically significant(P＞0.05). T1 group and T2 group had no statistically significant in operation wounded and myocardium protection.4. Conclusion1) The microsurgery practice was necessary,which was the basement to make cardiac allograft animal models. Good anesthesia,effective heart perfusion,short ischemic time, gracious surgery skill, good hemostasis andeffective heparinization;which were very importance in making cardiac allograft procedure.2) In T1 and T2 group, the time of obtaining donator hearts,perfusing heart,clipping heart,aorta arterial anastomosis,pulmonary arterial anastomosis,cold ischemia,anesthesia and volume of bleeding in operation were not statistically significant. The operation condition of T1 group and T2 group at was at the equal place in operation wounded and myocardium protection,which eliminated the interfering of human factor.Experiment 3. The effect to acute rejection and apoptosis in cardiac allograft animal models with myocardium being transplanted sertoli cells1. ObjectiveObserved the acute rejection(AR),apoptosis index(AI) and expression of Fas mRNA ,FasL mRNA and TGF-β1 mRNA in the models with or without myocardium transplanted sertoli cells.With correlation analysis, to identify the effect of sertoli cells to transplanted hearts and explored the immune pretection of sertoli cells to cardiac allografts.2. Methods1) Experiment group:The heart transplanted animal models were came from experiment 2.T1 group had survival 18 cases and T2 group had 16 survival cases. A group: To choose 9 cases from T1 group; B group:To choose 9 cases from T2 group; C group: T1 group residued 9 cases; D group:T2 group residued 7 cases;2) Experiment samples gathering and processing of the data. At the 7th day after operation,transplanted hearts were obtained in steriled condition.100mg tissue of every transplanted heart were obtained and preserved in liquid nitrogen for extracting mRNA.Cutting hearts were preserved in 10% neutral formalin away from light.In condition of away from light samples were carried out solidification, embedment and paraffin section.Every paraffin section were incised into 15 slides which were divided into 3 groups .Every group including 5 slides.By turn every group was carried out making pathological section, observing under fluorescence microscope,detecting apoptosis index of transplanted hearts..With QRT-PCR method ,expression of Fas,FasL and TGF-β1 mRNA was detected.Observed the survival time of transplanted hearts in C groups and D group.3. Results1) Observing sertoli cell under fluorescence microscope Sertoli cells were labeled in high quality by CFSE,which labeled rate attained at 100%. Under fluorescence microscope cytolymph and cells' membrane of sertoli cells showed yellow green color.In experimental group,sertoli cells were dispersed from the injecting place to the periphery, which cytolymph and cells' membrane of sertoli cells showed yellow green color.2) Analysis of acute rejection(AR) and apoptosis index(AI) of A group and B groupThe acute rejection of A group and B group displayed statistical significance, P=0.003.The acute rejection of B group was decreased significance than A group.AI of A and B group displayed statistical significance, P=0.003. The apoptosis of B group was decreased signifycance than A group.With Spearman correlation,the relation between the AR and AI displayed positive correlation,which correlation coefficient was 0.881, P＜0.001.3) Expression of cytokine in A group and B groupThe expression of FasL mRNA in A and B group had statistical significance, P=0.022;The expression of FasL mRNA in B group was high than the A group.The expression of Fas mRNA in A group and B group had not statistically significant.The expression of TGF-β1 mRNA in A and B group had statistical signifycance, P= 0.000. The expression of TGF-β1 mRNA in B group was higher than A group.Sertoli cells increased highly the expression of Fas mRNA and TGF-β1 mRNA.4) The corrrelation among acute rejection,apoptosis and cytokine in B group was analyzed with Spearman correlationAR had had inverse correlation with the expression of FasL mRNA, which correlation coefficient was -0.776, P=0.014.AR had inverse correlation with the expression of TGF-β1 mRNA, which correlation coefficient was -0.867, P=0.002.AI had inverse correlation with the expression of FasL mRNA, which correlation coefficient was -0.800, P=0.010. AI had inverse correlation with the expression of TGF-β1mRNA, which correlation coefficient was -0.783, P=0.013.5) To contrast the survival time of the cases of C group and D group In C group transplanted hearts survived 11.00±2.1days. In D group transplanted hearts survived 17.29±1.80 days.With t-test, survival time of C group and D group had statistically significant p＜0.001.The animal models of myocardium with transplanted sertoli cells in D group survived longer than the animal models without sertoli cells in C group.4. Conclusion1) Sertoli cells were labeled in high quality and in high tracing effective by CFSE.2) Transplanted hearts of which sertoli cells were transplanted decreased significance acute rejection and myocardium apoptosis.3) Acute rejection were positive correlation with Apoptosis idex.4) Transplanted hearts of which sertoli cells were transplanted into increased significance the expression of FasLmRNA and TGF-β1 mRNA,but did not increased the expression of Fas mRNA.5) Transplanted hearts of which sertoli cell were transplanted increased significance the expression of FasL mRNA and TGF-β1 mRNA, and the expression of FasL mRNA and TGF-β1 mRNA displayed significance negative correlation with acute rejection and myocardium apoptosis.6) The animal models of myocardium with transplanted sertoli cells survived longer than the animal models without sertoli cells.
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