siRNA沉默Rel-A基因表达诱导大鼠肝移植免疫耐受的实验研究
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摘要
背景肝移植是终末期肝功能衰竭患者的最佳治疗方案,移植外科技术和现代免疫抑制药物治疗都取得了重大发展,急性排斥反应的发生而造成器官丢失的情况已明显减少,但对于慢性排斥反应的控制还是显得无能为力。术后,器官移植会逐渐因慢性排斥反应性血管病变,以及免疫抑制剂长期使用引起的毒性反应和心血管疾病等原因而进入终末期状态,宿主将面临再次移植甚至死亡。因此,人们致力于研究肝脏移植的免疫耐受机制,希望寻找出新的诱导免疫耐受的方法,使移植患者能够脱离长期服用免疫抑制剂所带来的机会感染和肿瘤的危险。
免疫耐受在许多动物试验和一些临床研究中取得了可喜的结果,也有一些器官移植后的患者,停用免疫抑制剂后长年无排斥反应的报道。目前面临的问题是如何诱导稳定而持久的免疫耐受,以及如何确定已形成的免疫耐受。近年来,树突状细胞在肝移植免疫耐受中的关键作用逐渐受到重视。已知树突状细胞具有诱导机体免疫耐受的形成和延长移植物存活时间的能力。研究表明:刺激DC成熟几乎都是通过激活核转录因子κB(NF-κB)途径实现的。其中,p50/Rel-A(p65)组成的异源二聚体是其发挥生物学功能的主要形式。最近发展起来的RNAi技术为阻断NF-κB发挥生物学功能提供了新的手段,有效的基因治疗方案需要将目的基因稳定、高效地转入移植物的细胞。腺病毒载体因具有嗜肝性、能够转染处于非增殖期的实质细胞而在肝脏疾病研究方面具有优势。
第一部分大鼠髓系来源的树突状细胞的体外诱导及功能鉴定
目的探讨建立合适的大鼠骨髓源性未成熟树突状细胞的培养方法,并对其生物学特性进行评价。
方法分别以不同剂量的粒-巨噬细胞集落刺激因子和IL-4诱导分化获得大鼠骨髓源性DC,比较收获率及OX62阳性的DC数量,获得最佳细胞因子浓度。并用流式细胞仪分析DC表型,混合淋巴细胞反应检测其刺激同种T细胞增殖能力,ELISA测定细胞培养上清IL-12、MLR上清IL-12、IFN-γ水平。
结果GM-CSF 5ng/ml可诱导出更高的收获率及OX62的阳性率。GM-CSF 5ng/ml培养7d的DC,表达中等水平的MHC II和低水平的CD86;分泌少量IL-12;刺激同种T细胞增殖能力极低。LPS刺激后MHC II、CD86表达明显增加;分泌IL-12和IFN-γ增加;刺激同种T细胞增殖能力增强。
结论GM-CSF 5ng/ml为诱导大鼠骨髓源性未成熟DC的最佳剂量,培养7-9d可以获得足够数量和纯度的未成熟DC。
第二部分针对Rel-A的siRNA重组腺病毒载体的构建及表达验证
目的筛选出高效抑制Rel-A表达的siRNA序列,构建针对Rel-A的siRNA重组腺病毒。
方法(1)设计制备4对针对大鼠Rel-A的siRNA,结合对照共分为7个实验组,采用体外转染培养的BRL细胞,逆转录PCR和Western blotting检测siRNA的基因沉默效果。
(2)将Rel-A shRNA表达框插到穿梭质粒pShuttle H1的启动子下游,鉴定包含目的基因的正确克隆,将其线性化后与腺病毒骨架质粒pAdEasy-1体外同源重组,共转化感受态细菌,筛选重组子并扩增。重组子线性化后,转染293A细胞并进行病毒的包装,同时大量扩增病毒,氯化铯密度梯度离心纯化。重组腺病毒转染BRL细胞检测Rel-A基因沉默效率。
结果(1)设计合成的第二对siRNA mRNA和蛋白水平的抑制率分别为90%和80%。(2)穿梭载体插入序列及重组腺病毒质粒的鉴定均完全正确。病毒扩增纯化后滴度为3.0×1010 pfu/ml,并能在体外抑制Rel-A蛋白表达。结论(1)第二对siRNA抑制效率最高。(2)成功构建了针对Rel-A的siRNA重组腺病毒。
第三部分Ad-shRNA-Rel-A对大鼠树突状细胞生物学行为的影响
目的探讨腺病毒介导Rel-A基因体外转染大鼠树突状细胞对其生物学行为的影响。
方法将表达shRNA-Rel-A腺病毒载体转染大鼠骨髓源性DC,采用Westernblotting检测转染基因表达,获得高表达Rel-A的DC。并用流式细胞仪分析DC表型,混合淋巴细胞反应检测其刺激同种T细胞增殖能力,ELISA测定MLR上清IL-10、IFN-γ水平。
结果Western blotting检测结果显示转染Ad-shRNA-Rel-A病毒质粒的DC稳定高表达Rel-A基因,和转染前相比,CD86的表达水平无明显改变(P>0.05)。和负载BN大鼠抗原的未转染DC相比,转染Rel-A并负载抗原的DC刺激同种T细胞增殖能力低下(P<0.01),MLR上清中IL-10水平升高(P<0.01),而IFN-γ水平降低(P<0.01)。
结论转染Ad-shRNA-Rel-A病毒质粒的DC能够稳定高表达Rel-A基因,并使其维持于未成熟状态。负载BN大鼠抗原后可以抑制同种T细胞增殖反应,为诱导体内免疫耐受提供了实验依据。
第四部分大鼠原位肝脏移植模型的建立
目的用改良二袖套法建立稳定的大鼠原位肝脏移植模型。
方法在Kamada“二袖套法”的基础上,在取肝、肝上下腔静脉的吻合、围手术期处理等方面进行改良,共施行大鼠原位肝移植动物手术120例,观察术后并发症及存活率。
结果建立了稳定的大鼠原位肝脏移植模型。在正式实验阶段,受体无肝期平均12.3分钟,手术成功率94.3%,两周存活率90%。
结论改良的二袖套法具有无肝期短、手术成功率高、大鼠术后存活时间长的优点,是大鼠原位肝移植的理想术式。娴熟细致的外科操作、受体无肝期的长短是决定动物存活的关键。
第五部分Ad-shRNA-Rel-A转染树突状细胞诱导大鼠肝移植免疫耐受
目的探讨Rel-A基因转染并负载供体抗原的受体未成熟DC诱导肝移植免疫耐受的作用及机制。寻求一种应用受体DC诱导肝移植免疫耐受更切实可行的方法。方法以BN大鼠为肝移植供体,Lewis或Wistar大鼠为受体;Lewis大鼠骨髓源性DC转染Rel-A后负载BN大鼠脾脏细胞抗原。将上述DC于肝移植术前7d经阴茎背静脉注入受体作为治疗组,并设立对照组(急性排斥组);DC组;空载体组(转染Adv-null并负载BN抗原);第三方供体组(转染Rel-A并负载BN抗原,但供体采用Wistar大鼠)。术后观察各组大鼠生存时间,检测肝功能,ELISA法检测血清IL-2以及IFN-γ水平,术后第5d单向MLR检测供体脾细胞刺激受体脾脏细胞的增殖反应,移植肝的病理学检查,判断排斥反应程度。
结果与对照组以及其它各组相比,治疗组移植肝生存时间显著延长(29.9±10.76d,P<0.01);术后第5d,治疗组大鼠的TB、ALT均低于其它各组(30.12±9.8,210.6±48.3,P<0.01);其受体脾脏细胞对供体抗原刺激的反应明显低于其它各组(0.055±0.007,P<0.01);血清IL-2、IFN-γ水平降低(73.1±12.3,95.8±34.5,P<0.01);排斥反应轻。
结论成功建立稳定的大鼠同种异体肝移植的急性排斥反应动物模型;Rel-A基因转染并负载供体抗原的受体未成熟DC可以诱导针对供体的特异性免疫耐受,其机制可能与抑制T细胞增殖能力有关。应用受体DC诱导肝移植免疫耐受为临床尸肝移植提供了一种切实可行的方法。
Background liver transplantation is the best therapeutic regimen for Liver function failure in terminal stage. Both surgical technic and contemporary immunosuppressive drug treatment for transplanting have been developed greatly. The occurrence of organ loss by acute rejection has been decreased. But it is helpless for the chronic rejection. Organ transplantation will be state of final stage after operation, which was induce by vasculopathy of chronic rejection ,long-term using immunosuppressant leeding to toxic reaction and cardiovascular disease,then the parasitifer will be faced with retransplantation,even death. Therefore,the mechanism of immunotolerance has been studied for searching new approach of inducing immunotolerance,then the patients with transplantion could depart from risk of opportunistic infection and tumor occurred by long-term using immunosuppressant.
It has been greatly developed in immunotolerance for many animal experiment and some clinical research., no rejection has been reported in some patients,which stopped using immunosuppressant all the year round. At present,the question to be faced with is how to induce stabilizing, lasting immunotolerance and determine formative immunotolerance. Recently, dendritic cell’s critical role in liver transplantation immunotolerance has been thinked highly of gradually. Dendritic cells have capability of inducing form of immune tolerance and prolonging survival time of transplant. Research has indicateded that stimulating maturity of dendritic cell has been implemented by means of activation of nuclear factor kappa B, Among heterodimer, p50/Rel-A(p65)was major form to educe biological function. Developed technique of RNAi recently has provided original means to educe biological function, Effective gene therapy requires a reliable gene transfer tools to efficiently insert target genes into the cells of the grafts. Adenovirus vectors have been favored because of their propensity to infect hepatic cells and their ability to infect proliferation and nonproliferation cells.
Part I: Generation of Rat Bone Marrow-derived DendriticCells in Vitro and Evaluation of Their Biological Characterization
Objective To establish a suitable method for the generation of Lewis rat immature dendritic cells and evaluate their biological characterization.
Methods Bone marrow cells were developed under the concentration of granulocytemacrophage colonystimulating factor , and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization were analyzed with flowcytometry,the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction (MLR),and the levels of IL-12 secreted by DC and interferon-γ(IFN-γ) and IL-12 levels in 5-day mixed leukocyte culture supernatants was detected by ELISA.
Results With 5% GM-CSF concentrations resulted in a higher number of cell yield and higher percentage of OX62 at day 7. Under the condition of GM-CSF(5ng/ml), DC at day 9 expressed an intermediate levels of MHCII and low levels of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHCII and CD86 and a stronger IL-2 and IFN-γproduction and higher stimulatory capacity of allogeneic T cell.
Conclusions GM-CSF at a concentration of 5ng/ml is the best method to generate large number of lewis rat immature dendritic cells, and this make it possible to induce immunological tolerance by DC.
Part II: Construction of Adenoviral Vector Encoding Rel-A Gene and Identification of Their Expression
Objective To choose the Rel-A siRNA with the highest gene silence efficacy from the 4 pairs of siRNA and construct a recombinant Ad containing Rel-A specific siRNA expression cassette.
Methods (1)4 pairs of siRNA targeting rat Rel-A mRNA were designed. Combined with control group there were 7 experiment groups altogether. Then those siRNA were transfected into the cultivated rat BRL cells use the way we conformed in part 1. The Rel-A gene expression effectiveness was tested by reverse transcription-PCR and Western blotting. (2) The Rel-A specific siRNA expression cassette was cloned behind the sequence of H1-RNA promoter, and the positive clones containing target gene were selected and appraised. A recombinant adenovirus was produced by a double-recombination event between cotransformed adenoviral backbone plasmid pAdEasy-1 and a linearized shuttle vector. Positive clones were selected and confirmed. Then they were linearized and transferred into the 293A cell to amplification, and then were purified by density gradient ultracentrifuge and titrated. The desired Ad vectors were transfected into the BRL cells to detect the gene silence efficiency.
Results (1)The second siRNA was the most efficient one and the Rel-A mRNA expression was decreased 90%, the protein expression was decreased 80% compared with the blank group. (2) Both the plasmid pShuttle-H1-p65 and the recombinant Ad were confirmed correct. The recombinant Ad were generated in a high titer which reached 3.0×1010 pfu/ml, and could efficiently knock down the Rel-A expression in vitro.
Conclusions (1)The second pair of siRNA was the most efficient function of gene silence.(2) The recombinant Ad which containing Rel-A specific siRNA expression cassette was successfully constructed.
Part III: Effect of Biological Characteristics of Rat Bone Marrow-derived Dendritic Cells Transfected by Ad-shRNA-Rel-A in Vitro
Objective To study the effect of biological characteristics of rat bone marrow-derived dendritic cells transfected by Ad-shRNA-Rel-A gene in vitro.
Methods Recombinant adenovirus expression plasmid Ad-shRNA-Rel-A was transfected into rat bone marrow-derived dendritic cells to arrest their maturation, construct tolerenence DC. The expression of transfected gene was detected by western blotting analysis, surface molecules of Ad-shRNA-Rel-A-DC were detected by FCM. Autologous T cell proliferation stimulated by Ad-shRNA-Rel-A -DC was detected by MTT assay, and the level of IFN-γand IL-10 secreted by DC was analyzed by ELISA.
Results Western blotting analysis detected high and stable expression of Ad-shRNA-Rel-A gene in Ad-shRNA-Rel-A transfected DC, and compared with the untransfected DC, DC transfected by recombinant adenovirus vector encoding Rel-A didn’t up-regulate the expression of CD86(P>0.05). In contrast to untransfected DC, allogeneic T cells proliferation induced by Ad-shRNA-Rel-A-DC was obviously lower(P<0.01), and a stronger IL-10 secretion level(P<0.01) and a weaker IFN-γsecretion level(P<0.01) was detected in the Rel-A transfected DC.
Conclusions Rel-A gene can be highly and stably expressed in Ad-shRNA-Rel-A transfected DC, and it can restrain their maturation. DC transfected by Ad-shRNA-Rel-A and loaded with BN antigen can lower the allogeneic MLR, which provided an experimental base for immunological tolerance induction by DC.
PART IV: The establishment of a model of rat orthotopic liver transplantation
Objective To establish a reliable animal model of rat orthotopic liver transplantation with modified cuff technique.
Methods 120 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, the anastomoses of suprahepatic vena cava and the perioperative treatment.
Results Of the last 30 cases, the mean time of anhepatic period was 12.3min, The 24 hours and 2 week survival rates were 94.3% and 90% respectively.
Conclusions The modified two-cuffed technique is convenient and easy to repeat. It can enhance the stability and survival rate of rat orthotopic liver transplantation models. Skilled surgical techniques and shortening the anhepatic phase of recipient as much as possible are the keys to animal survival.
PART V: Tolerance in Rat Allograft liver transplantation Induced by Dendritic Cells Transfected by Rel-A Gene
Objective To establish rat allograft liver transplantation model of acute rejective reaction and investigate the effect and mechanisms of recipient-derived imDC transfected by Rel-A and loaded with donor antigen to induce immune tolerance. To develop a more applicable approach that uses recipient-derived dendritic cells to induce tolerance for clinical liver transplantation.
Methods Liver transplantations were performed from BN or Wistar donors to Lewis recipients. DC were cultured from recipient rats (Lewis) bone marrow with low dose GM-CSF and IL-4. At day 7, they were transfected by Rel-A to arrest maturation, and at day 9, they were plused with BN splenocyte lysate for another 2 days. Then, this modified recipient bone marrow derived DC were injected into recipient rats 7 days before transplantation. Null treatment, DC-treatment, Adv-0 transfected and plused BN spleen cell lysate, and a third party donor (Wistar) were served as controls. The survival of liver allografts were observed, cytokine levels were analyzed by ELISA. Their allostimulatory activity was assessed in vitro by one-way MLR. Pathological examination was performed to identified the grade of rejection.
Results Compared with Null treatment, DC- treatment, and the third party donor(Wistar) controls, Rel-A transfected DC plused with BN splenocyte lysate markedly prolonged the survival of liver allografts in an antigen-specific manner(26.8±1.76d, P<0.01). Rel-A-DC loaded with BN antigen elicited markedly lower proliferative responses and reduced IL-2 and IFN-γproduction. In contrast to other groups, Rel-A-DC loaded with BN antigen show a lower grade as estimated by pathological examination.
Conclusions Successfully and surely established rat allograft liver transplantation model of acute rejective reaction. Recipient-derived imDC transfected by Rel-A and loaded with donor splenocyte lysate could induce immune tolerance in a donor-specific manner, it might be associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Our successful induction of tolerance by DC in a recipient manner provides a more feasible strategy for deceased-donor liver transplantation.
免疫耐受在许多动物试验和一些临床研究中取得了可喜的结果,也有一些器官移植后的患者,停用免疫抑制剂后长年无排斥反应的报道。目前面临的问题是如何诱导稳定而持久的免疫耐受,以及如何确定已形成的免疫耐受。近年来,树突状细胞在肝移植免疫耐受中的关键作用逐渐受到重视。已知树突状细胞具有诱导机体免疫耐受的形成和延长移植物存活时间的能力。研究表明:刺激DC成熟几乎都是通过激活核转录因子κB(NF-κB)途径实现的。其中,p50/Rel-A(p65)组成的异源二聚体是其发挥生物学功能的主要形式。最近发展起来的RNAi技术为阻断NF-κB发挥生物学功能提供了新的手段,有效的基因治疗方案需要将目的基因稳定、高效地转入移植物的细胞。腺病毒载体因具有嗜肝性、能够转染处于非增殖期的实质细胞而在肝脏疾病研究方面具有优势。
第一部分大鼠髓系来源的树突状细胞的体外诱导及功能鉴定
目的探讨建立合适的大鼠骨髓源性未成熟树突状细胞的培养方法,并对其生物学特性进行评价。
方法分别以不同剂量的粒-巨噬细胞集落刺激因子和IL-4诱导分化获得大鼠骨髓源性DC,比较收获率及OX62阳性的DC数量,获得最佳细胞因子浓度。并用流式细胞仪分析DC表型,混合淋巴细胞反应检测其刺激同种T细胞增殖能力,ELISA测定细胞培养上清IL-12、MLR上清IL-12、IFN-γ水平。
结果GM-CSF 5ng/ml可诱导出更高的收获率及OX62的阳性率。GM-CSF 5ng/ml培养7d的DC,表达中等水平的MHC II和低水平的CD86;分泌少量IL-12;刺激同种T细胞增殖能力极低。LPS刺激后MHC II、CD86表达明显增加;分泌IL-12和IFN-γ增加;刺激同种T细胞增殖能力增强。
结论GM-CSF 5ng/ml为诱导大鼠骨髓源性未成熟DC的最佳剂量,培养7-9d可以获得足够数量和纯度的未成熟DC。
第二部分针对Rel-A的siRNA重组腺病毒载体的构建及表达验证
目的筛选出高效抑制Rel-A表达的siRNA序列,构建针对Rel-A的siRNA重组腺病毒。
方法(1)设计制备4对针对大鼠Rel-A的siRNA,结合对照共分为7个实验组,采用体外转染培养的BRL细胞,逆转录PCR和Western blotting检测siRNA的基因沉默效果。
(2)将Rel-A shRNA表达框插到穿梭质粒pShuttle H1的启动子下游,鉴定包含目的基因的正确克隆,将其线性化后与腺病毒骨架质粒pAdEasy-1体外同源重组,共转化感受态细菌,筛选重组子并扩增。重组子线性化后,转染293A细胞并进行病毒的包装,同时大量扩增病毒,氯化铯密度梯度离心纯化。重组腺病毒转染BRL细胞检测Rel-A基因沉默效率。
结果(1)设计合成的第二对siRNA mRNA和蛋白水平的抑制率分别为90%和80%。(2)穿梭载体插入序列及重组腺病毒质粒的鉴定均完全正确。病毒扩增纯化后滴度为3.0×1010 pfu/ml,并能在体外抑制Rel-A蛋白表达。结论(1)第二对siRNA抑制效率最高。(2)成功构建了针对Rel-A的siRNA重组腺病毒。
第三部分Ad-shRNA-Rel-A对大鼠树突状细胞生物学行为的影响
目的探讨腺病毒介导Rel-A基因体外转染大鼠树突状细胞对其生物学行为的影响。
方法将表达shRNA-Rel-A腺病毒载体转染大鼠骨髓源性DC,采用Westernblotting检测转染基因表达,获得高表达Rel-A的DC。并用流式细胞仪分析DC表型,混合淋巴细胞反应检测其刺激同种T细胞增殖能力,ELISA测定MLR上清IL-10、IFN-γ水平。
结果Western blotting检测结果显示转染Ad-shRNA-Rel-A病毒质粒的DC稳定高表达Rel-A基因,和转染前相比,CD86的表达水平无明显改变(P>0.05)。和负载BN大鼠抗原的未转染DC相比,转染Rel-A并负载抗原的DC刺激同种T细胞增殖能力低下(P<0.01),MLR上清中IL-10水平升高(P<0.01),而IFN-γ水平降低(P<0.01)。
结论转染Ad-shRNA-Rel-A病毒质粒的DC能够稳定高表达Rel-A基因,并使其维持于未成熟状态。负载BN大鼠抗原后可以抑制同种T细胞增殖反应,为诱导体内免疫耐受提供了实验依据。
第四部分大鼠原位肝脏移植模型的建立
目的用改良二袖套法建立稳定的大鼠原位肝脏移植模型。
方法在Kamada“二袖套法”的基础上,在取肝、肝上下腔静脉的吻合、围手术期处理等方面进行改良,共施行大鼠原位肝移植动物手术120例,观察术后并发症及存活率。
结果建立了稳定的大鼠原位肝脏移植模型。在正式实验阶段,受体无肝期平均12.3分钟,手术成功率94.3%,两周存活率90%。
结论改良的二袖套法具有无肝期短、手术成功率高、大鼠术后存活时间长的优点,是大鼠原位肝移植的理想术式。娴熟细致的外科操作、受体无肝期的长短是决定动物存活的关键。
第五部分Ad-shRNA-Rel-A转染树突状细胞诱导大鼠肝移植免疫耐受
目的探讨Rel-A基因转染并负载供体抗原的受体未成熟DC诱导肝移植免疫耐受的作用及机制。寻求一种应用受体DC诱导肝移植免疫耐受更切实可行的方法。方法以BN大鼠为肝移植供体,Lewis或Wistar大鼠为受体;Lewis大鼠骨髓源性DC转染Rel-A后负载BN大鼠脾脏细胞抗原。将上述DC于肝移植术前7d经阴茎背静脉注入受体作为治疗组,并设立对照组(急性排斥组);DC组;空载体组(转染Adv-null并负载BN抗原);第三方供体组(转染Rel-A并负载BN抗原,但供体采用Wistar大鼠)。术后观察各组大鼠生存时间,检测肝功能,ELISA法检测血清IL-2以及IFN-γ水平,术后第5d单向MLR检测供体脾细胞刺激受体脾脏细胞的增殖反应,移植肝的病理学检查,判断排斥反应程度。
结果与对照组以及其它各组相比,治疗组移植肝生存时间显著延长(29.9±10.76d,P<0.01);术后第5d,治疗组大鼠的TB、ALT均低于其它各组(30.12±9.8,210.6±48.3,P<0.01);其受体脾脏细胞对供体抗原刺激的反应明显低于其它各组(0.055±0.007,P<0.01);血清IL-2、IFN-γ水平降低(73.1±12.3,95.8±34.5,P<0.01);排斥反应轻。
结论成功建立稳定的大鼠同种异体肝移植的急性排斥反应动物模型;Rel-A基因转染并负载供体抗原的受体未成熟DC可以诱导针对供体的特异性免疫耐受,其机制可能与抑制T细胞增殖能力有关。应用受体DC诱导肝移植免疫耐受为临床尸肝移植提供了一种切实可行的方法。
Background liver transplantation is the best therapeutic regimen for Liver function failure in terminal stage. Both surgical technic and contemporary immunosuppressive drug treatment for transplanting have been developed greatly. The occurrence of organ loss by acute rejection has been decreased. But it is helpless for the chronic rejection. Organ transplantation will be state of final stage after operation, which was induce by vasculopathy of chronic rejection ,long-term using immunosuppressant leeding to toxic reaction and cardiovascular disease,then the parasitifer will be faced with retransplantation,even death. Therefore,the mechanism of immunotolerance has been studied for searching new approach of inducing immunotolerance,then the patients with transplantion could depart from risk of opportunistic infection and tumor occurred by long-term using immunosuppressant.
It has been greatly developed in immunotolerance for many animal experiment and some clinical research., no rejection has been reported in some patients,which stopped using immunosuppressant all the year round. At present,the question to be faced with is how to induce stabilizing, lasting immunotolerance and determine formative immunotolerance. Recently, dendritic cell’s critical role in liver transplantation immunotolerance has been thinked highly of gradually. Dendritic cells have capability of inducing form of immune tolerance and prolonging survival time of transplant. Research has indicateded that stimulating maturity of dendritic cell has been implemented by means of activation of nuclear factor kappa B, Among heterodimer, p50/Rel-A(p65)was major form to educe biological function. Developed technique of RNAi recently has provided original means to educe biological function, Effective gene therapy requires a reliable gene transfer tools to efficiently insert target genes into the cells of the grafts. Adenovirus vectors have been favored because of their propensity to infect hepatic cells and their ability to infect proliferation and nonproliferation cells.
Part I: Generation of Rat Bone Marrow-derived DendriticCells in Vitro and Evaluation of Their Biological Characterization
Objective To establish a suitable method for the generation of Lewis rat immature dendritic cells and evaluate their biological characterization.
Methods Bone marrow cells were developed under the concentration of granulocytemacrophage colonystimulating factor , and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization were analyzed with flowcytometry,the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction (MLR),and the levels of IL-12 secreted by DC and interferon-γ(IFN-γ) and IL-12 levels in 5-day mixed leukocyte culture supernatants was detected by ELISA.
Results With 5% GM-CSF concentrations resulted in a higher number of cell yield and higher percentage of OX62 at day 7. Under the condition of GM-CSF(5ng/ml), DC at day 9 expressed an intermediate levels of MHCII and low levels of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHCII and CD86 and a stronger IL-2 and IFN-γproduction and higher stimulatory capacity of allogeneic T cell.
Conclusions GM-CSF at a concentration of 5ng/ml is the best method to generate large number of lewis rat immature dendritic cells, and this make it possible to induce immunological tolerance by DC.
Part II: Construction of Adenoviral Vector Encoding Rel-A Gene and Identification of Their Expression
Objective To choose the Rel-A siRNA with the highest gene silence efficacy from the 4 pairs of siRNA and construct a recombinant Ad containing Rel-A specific siRNA expression cassette.
Methods (1)4 pairs of siRNA targeting rat Rel-A mRNA were designed. Combined with control group there were 7 experiment groups altogether. Then those siRNA were transfected into the cultivated rat BRL cells use the way we conformed in part 1. The Rel-A gene expression effectiveness was tested by reverse transcription-PCR and Western blotting. (2) The Rel-A specific siRNA expression cassette was cloned behind the sequence of H1-RNA promoter, and the positive clones containing target gene were selected and appraised. A recombinant adenovirus was produced by a double-recombination event between cotransformed adenoviral backbone plasmid pAdEasy-1 and a linearized shuttle vector. Positive clones were selected and confirmed. Then they were linearized and transferred into the 293A cell to amplification, and then were purified by density gradient ultracentrifuge and titrated. The desired Ad vectors were transfected into the BRL cells to detect the gene silence efficiency.
Results (1)The second siRNA was the most efficient one and the Rel-A mRNA expression was decreased 90%, the protein expression was decreased 80% compared with the blank group. (2) Both the plasmid pShuttle-H1-p65 and the recombinant Ad were confirmed correct. The recombinant Ad were generated in a high titer which reached 3.0×1010 pfu/ml, and could efficiently knock down the Rel-A expression in vitro.
Conclusions (1)The second pair of siRNA was the most efficient function of gene silence.(2) The recombinant Ad which containing Rel-A specific siRNA expression cassette was successfully constructed.
Part III: Effect of Biological Characteristics of Rat Bone Marrow-derived Dendritic Cells Transfected by Ad-shRNA-Rel-A in Vitro
Objective To study the effect of biological characteristics of rat bone marrow-derived dendritic cells transfected by Ad-shRNA-Rel-A gene in vitro.
Methods Recombinant adenovirus expression plasmid Ad-shRNA-Rel-A was transfected into rat bone marrow-derived dendritic cells to arrest their maturation, construct tolerenence DC. The expression of transfected gene was detected by western blotting analysis, surface molecules of Ad-shRNA-Rel-A-DC were detected by FCM. Autologous T cell proliferation stimulated by Ad-shRNA-Rel-A -DC was detected by MTT assay, and the level of IFN-γand IL-10 secreted by DC was analyzed by ELISA.
Results Western blotting analysis detected high and stable expression of Ad-shRNA-Rel-A gene in Ad-shRNA-Rel-A transfected DC, and compared with the untransfected DC, DC transfected by recombinant adenovirus vector encoding Rel-A didn’t up-regulate the expression of CD86(P>0.05). In contrast to untransfected DC, allogeneic T cells proliferation induced by Ad-shRNA-Rel-A-DC was obviously lower(P<0.01), and a stronger IL-10 secretion level(P<0.01) and a weaker IFN-γsecretion level(P<0.01) was detected in the Rel-A transfected DC.
Conclusions Rel-A gene can be highly and stably expressed in Ad-shRNA-Rel-A transfected DC, and it can restrain their maturation. DC transfected by Ad-shRNA-Rel-A and loaded with BN antigen can lower the allogeneic MLR, which provided an experimental base for immunological tolerance induction by DC.
PART IV: The establishment of a model of rat orthotopic liver transplantation
Objective To establish a reliable animal model of rat orthotopic liver transplantation with modified cuff technique.
Methods 120 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, the anastomoses of suprahepatic vena cava and the perioperative treatment.
Results Of the last 30 cases, the mean time of anhepatic period was 12.3min, The 24 hours and 2 week survival rates were 94.3% and 90% respectively.
Conclusions The modified two-cuffed technique is convenient and easy to repeat. It can enhance the stability and survival rate of rat orthotopic liver transplantation models. Skilled surgical techniques and shortening the anhepatic phase of recipient as much as possible are the keys to animal survival.
PART V: Tolerance in Rat Allograft liver transplantation Induced by Dendritic Cells Transfected by Rel-A Gene
Objective To establish rat allograft liver transplantation model of acute rejective reaction and investigate the effect and mechanisms of recipient-derived imDC transfected by Rel-A and loaded with donor antigen to induce immune tolerance. To develop a more applicable approach that uses recipient-derived dendritic cells to induce tolerance for clinical liver transplantation.
Methods Liver transplantations were performed from BN or Wistar donors to Lewis recipients. DC were cultured from recipient rats (Lewis) bone marrow with low dose GM-CSF and IL-4. At day 7, they were transfected by Rel-A to arrest maturation, and at day 9, they were plused with BN splenocyte lysate for another 2 days. Then, this modified recipient bone marrow derived DC were injected into recipient rats 7 days before transplantation. Null treatment, DC-treatment, Adv-0 transfected and plused BN spleen cell lysate, and a third party donor (Wistar) were served as controls. The survival of liver allografts were observed, cytokine levels were analyzed by ELISA. Their allostimulatory activity was assessed in vitro by one-way MLR. Pathological examination was performed to identified the grade of rejection.
Results Compared with Null treatment, DC- treatment, and the third party donor(Wistar) controls, Rel-A transfected DC plused with BN splenocyte lysate markedly prolonged the survival of liver allografts in an antigen-specific manner(26.8±1.76d, P<0.01). Rel-A-DC loaded with BN antigen elicited markedly lower proliferative responses and reduced IL-2 and IFN-γproduction. In contrast to other groups, Rel-A-DC loaded with BN antigen show a lower grade as estimated by pathological examination.
Conclusions Successfully and surely established rat allograft liver transplantation model of acute rejective reaction. Recipient-derived imDC transfected by Rel-A and loaded with donor splenocyte lysate could induce immune tolerance in a donor-specific manner, it might be associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Our successful induction of tolerance by DC in a recipient manner provides a more feasible strategy for deceased-donor liver transplantation.
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