【作者】 蒋廷旺；

【导师】 仲人前； 邓安梅； 孔宪涛； 韩焕兴；

【作者基本信息】 第二军医大学 ， 临床检验诊断学， 2009， 博士

【摘要】 原发性胆汁性肝硬化(primary biliary cirrhosis, PBC)是一种好发于成年女性的自身免疫性肝病,病因不明且发病率逐年上升。PBC患者的免疫系统对部分线粒体及细胞核抗原的免疫耐受遭到破坏,致使大部分患者的血清中出现抗线粒体抗体和抗核抗体,其中95%的患者血清中存在抗线粒体内膜PDC-E2的自身抗体。此外,多数患者外周血存在PDC-E2自身反应性T细胞,而正常人外周血无此类细胞。然而,什么原因导致PBC的免疫耐受平衡破坏及T细胞的免疫耐受调节机制发生了怎样的改变目前尚不清楚。被自身抗原活化的T细胞通常经由凋亡途径被机体清除。在众多的凋亡机制中,活化诱导细胞凋亡(Activation induced cell death, AICD)是机体维持外周免疫耐受的重要机制,外周免疫系统通过诱导自身反应性T细胞活化后凋亡可有效的控制其对人体的器官、组织等造成免疫损伤,阻止自身免疫病的发生。器官特异性自身免疫病是由逃逸于外周耐受清除的自身反应性T细胞引起的组织损伤和功能异常。PBC的病变特征即是肝内汇管区大量的淋巴细胞浸润,自身反应性T细胞不断破坏小胆管上皮细胞,导致胆管逐渐减少,胆汁淤积并最终引起进行性的肝脏损伤。研究表明在PBC汇管区浸润的T细胞中以CD4+T细胞居多,大约是CD8+T细胞的2.5倍,由此推断CD4+T细胞可能在免疫炎症中发挥重要作用。原位杂交分析发现PBC患者汇管区表达IFN-γmRNA的细胞明显增加,说明Th1细胞介导的免疫炎症反应在致PBC肝脏病变过程中具有重要作用。聚肌苷酸胞苷酸(polyinosinic polycytidylic acid, polyI:C)是Toll样受体3(TLR3)的配体,可以诱导I型干扰素(IFN-α)的分泌。研究表明polyI:C与自身免疫病的发生和进展具有相关性。而IFN-α能够刺激体内的细胞产生多种细胞因子、趋化因子等,同时还可以刺激机体分泌抗核抗体、抗甲状腺抗体、抗胰岛细胞等多种自身抗体。1995年D’Amico等报道一例慢性丙肝(chronic hepatitis C, CHC)患者在接受IFN-α治疗的过程中出现PBC样病变,血清碱性磷酸酶水平达到正常人的3倍以上,且出现抗线粒体抗体,同时汇管区出现单个核细胞浸润。另外,Takii等通过免疫组化研究发现PBC患者汇管区单个核细胞中IFN-α表达较自身免疫性肝炎(autoimmune hepatitis, AIH)和CHC患者显著升高。在临床治疗方面主要依靠药物治疗缓解病人的瘙痒、骨质疏松、门脉高压等并发症,但无法延长患者的生存年限,更不可能从根本上治愈该病。因此有必要从免疫耐受的角度出发,寻找更为有效的治疗手段,从而在根本上治疗PBC。基于以上研究现状,我们拟通过注射M2蛋白及polyI:C诱导建立PBC小鼠模型,并在此基础上对其外周血、脾脏、肝组织的CD4+T细胞活化诱导细胞凋亡情况进行分析,了解其外周免疫耐受机制,最后将PBC自身抗原M2与交联剂ECDI及脾脏淋巴细胞共同作用,经尾静脉注射PBC小鼠诱导重建外周免疫耐受,观察PBC小鼠的疾病进展情况。旨在为进一步研究PBC的免疫耐受机制及其免疫治疗的探索提供思路。第一部分M2三联体抗原的基因工程生产PBC的主要自身抗原是位于线粒体内膜的丙酮酸脱氢酶复合体(PDC)、侧链二氧酸脱氢酶复合体(BCOADC)、2-氧戊二酸脱氢酶复合体(OGDC)的E2亚基。本科室将以上三种主要自身抗原免疫优势表位基因重组后经克隆表达出三联体抗原。为了建立PBC小鼠模型并进行免疫耐受研究,我们通过大量发酵生产获得该抗原。首先以IPTG在试管及烧瓶中进行小量诱导表达,条件成熟后进行发酵罐大量发酵,最后以安玛西亚蛋白纯化仪纯化获得的蛋白。结果表明,小量诱导时IPTG 1mg/ml诱导4小时蛋白表达量明显升高。之后以3.5L发酵罐大量发酵,2.5小时后溶氧量下降,此时开始添加补料。我们分别以葡萄糖和甘油作为补料提供碳源,结果发现以甘油作为碳源时蛋白的表达量显著高于葡萄糖碳源。发酵罐IPTG诱导6小时菌体密度最高,此后不再有明显变化,溶氧控制在30%以上,最大转速达到500rpm/min,pH为6.8-7.4,最终菌体密度OD600达到12.7,湿菌重约30g。经蛋白纯化仪纯化后得到的蛋白浓度为1.5mg/ml。第二部分原发性胆汁性肝硬化动物模型的建立PBC是一种慢性迁延性疾病,病程很长,且患者往往伴有病毒感染等其他病变,更为重要的是病变组织局限于肝脏,很难获得足够的组织用于研究。因此,短期内建立一个有效的动物模型能够从根本上解决以上问题。我们分别以M2抗原50μg和polyI:C 5mg/kg、10mg/kg剂量免疫C57BL/6雌性小鼠,在不同时间段处死分离血清、肝、肾、脑、小肠、胃等组织进行抗线粒体抗体、抗核抗体、碱性磷酸酶(AKP)、病理分析,观察小鼠病变情况。同时对小鼠外周血细胞进行流式分析,了解T细胞亚群的变化。结果发现,蛋白免疫组ANA阳性率为30%,AMA阳性率则高达80%,且滴度均>1:100,对照组未见阳性结果;PolyI:C免疫后,阴性对照组、5mg/kg、10mg/kg组ANA分别在第12、8、4周时阳性率达到100%;对照组及5mg/kg组小鼠从注射第8周开始出现滴度>1:100的ANA,10mg/kg组小鼠则从第4周开始出现>1:100的ANA,而且polyI:C组小鼠均从注射第8周开始出现滴度为1:10000的高滴度ANA;血清AMA检测发现,对照组AMA均为阴性,5mg/kg组随着注射时间延长阳性率逐渐增加,16周达到80%并保持不变,10mg/kg组小鼠在注射后的第4周AMA阳性率即达到80%,但是随着注射时间延长AMA阳性率却出现下降,第12周降至20%并保持不变。AMA抗体滴度:5mg/kg组高滴度抗体的小鼠数量随药物注射逐渐增加,而10mg/kg组则呈现下降趋势。肝组织H.E染色并对浸润细胞数大于50的汇管区进行统计分析,结果以百分率表示,发现5mg/kg组小鼠浸润的汇管区数量逐渐增加至第16周,16周与20周比较差异无统计学意义,而10mg/kg组第12周即达到稳定水平,与16周和20周比较均无统计学意义,阴性对照组小鼠肝脏汇管区均未发现明显的炎症细胞浸润。对肾脏、胃、大脑、小肠和心肌组织进行H.E染色,显微镜观察均未发现炎症反应;polyI:C组小鼠的血清ALP水平随着药物注射逐渐增加,5mg/kg组和10mg/kg组ALP水平均>60U/L,而阴性对照组则在60U/L以下,且随着时间的变化无明显改变,5mg/kg组和10mg/kg组两组间比较差异无统计学意义(P>0.05)。M2免疫组小鼠未出现汇管区炎症改变及血清AKP变化。各组小鼠的外周血CD4+、CD8+ T细胞分布比例各组之间没有出现显著差异(P>0.05),同时各组内也没有随着时间发生变化。polyI:C 5mg/kg剂量注射得到的模型符合PBC实验室诊断指标,具有良好的代表性,可用于后续研究。第三部分PBC模型中细胞增殖及活化诱导凋亡研究体内T细胞活化诱导凋亡(activation induced cell death, AICD)是清除自身反应性T细胞维持外周免疫耐受的重要机制,效应性T细胞AICD缺陷可能会导致自身免疫性疾病的发生。研究表明在PBC患者的汇管区存在着大量的Th细胞,尤以Th1细胞为主。本部分我们以polyI:C 5mg/kg剂量注射C57小鼠重新建立PBC小鼠模型,16周后分离淋巴细胞并以磁珠纯化获得肝脏、脾脏CD4+T细胞,以M2蛋白及ConA结合anti-CD3刺激细胞增殖及活化后凋亡实验,并对凋亡相关基因及信号蛋白FLIP、caspase-8进行检测。此外,为了了解M2蛋白是否对T细胞功能产生影响,我们按照第二部分的方法同时注射M2抗原。结果发现:1)M2蛋白刺激后,空白对照组、PBS组、M2蛋白组之间的细胞增殖能力差异也无统计学意义(p>0.05),但是PBC组小鼠细胞增殖能力与以上三组相比显著增强,且PBC小鼠肝内淋巴细胞增殖能力强于脾淋巴细胞,差异有统计学意义(p<0.001);2)空白组、PBS组、M2蛋白组AICD之间差异无统计学意义(p>0.05),但是这三组细胞的凋亡率均显著高于PBC组的脾脏和肝脏CD4+T细胞(p<0.001)。比较PBC组小鼠的肝脏和脾脏CD4+ T细胞凋亡结果表明,肝脏CD4+ T细胞的凋亡率显著低于脾脏细胞,差异有统计学意义(p<0.001);3)模型组小鼠肝脾淋巴细胞FasL基因的表达较对照组均明显降低(p<0.01),而两组小鼠淋巴细胞Fas表达水平无明显改变。TRAIL在模型组小鼠肝脾淋巴细胞中的表达也显著低于对照组小鼠(P<0.01);4)模型组caspase-8表达较对照组无明显变化,而FLIPL表达则明显升高,大部分在升高5倍以上,且肝脏中CD4+ T淋巴细胞FLIPL表达高于脾脏,差异有统计学意义(P<0.001)。以上结果说明,PBC组小鼠体内存在大量自身反应性淋巴细胞,且肝内居多;CD4+T淋巴细胞的抗凋亡能力增强使得PBC小鼠模型淋巴细胞AICD明显降低,最终导致外周免疫耐受平衡被破坏;FLIPL的表达增高可能是AICD受到抑制的重要原因并参与汇管区炎症反应,同时FasL和TRAIL mRNA表达降低可能也起到一定的作用。第四部分重建外周免疫耐受抑制原发性胆汁性肝硬化原发性胆汁性肝硬化目前尚无十分有效的根治手段,只能针对患者出现瘙痒、骨质疏松、高脂血症、门脉高压等并发症进行对症治疗,而对于晚期患者肝脏移植则是最佳治疗措施。因此,从免疫学角度出发寻找更为有效的根治手段已经迫在眉睫。我们将M2蛋白与新生小鼠的脾淋巴细胞及交联剂ECDI共培养,形成抗原负载的淋巴细胞,随后通过尾静脉注入小鼠体内诱导免疫系统对PBC自身抗原的免疫耐受,同时注射polyI:C诱导PBC病变,对照组注射与牛血清白蛋白(BSA)共培养的脾细胞,16周后观察小鼠的各项实验室指标,分析重建PBC小鼠外周免疫耐受是否能够有效的阻止病变产生或缓解病程。结果发现:1)AMA:免疫耐受组与BSA对照组、模型对照组相比阳性率显著降低,差异有统计学意义(P=0.007, P=0.003);BSA对照组和模型对照组间无明显差异(P=0.74);2)免疫耐受组、BSA对照组、模型对照组的AKP水平分别为80.5±9.8U/L、93.8±15.7U/L、92.5±17.7U/L;其中BSA组和模型组差异无统计学意义(P=0.83),而免疫耐受组低于BSA组(P=0.0095)和模型对照组(P=0.029);3)免疫耐受组、BSA对照组、模型对照组的胆管阳性浸润率分别为:42.67±12.3%、57.07±11.35%、51.53±9.96%;其中免疫耐受组阳性浸润率低于模型对照组,差异有统计学意义(P=0.039),同时显著低于BSA对照组(P=0.0024),而且BSA对照组和模型对照组比较差异无统计学意义(P=0.167)。综上,本研究以M2抗原在体外与初始状淋巴细胞相互作用后,经尾静脉注射小鼠诱导外周免疫耐受,在一定程度上抑制了PBC小鼠的病变程度,为今后进一步研究PBC的免疫治疗提供思路。更多还原

【Abstract】 Primary biliary cirrhosis is a slowly progressive autoimmune disease of the liver that primarily affects women. The pathogenesis of PBC is unknown and the incidence is growing with the rapid development of diagnostic technologies. PBC is characterised immunologically by the breakdown of immune self-tolerance to highly conserved mitochondrial and nuclear antigens. In over 95% of patients, anti-mitochondrial antibodies are directed at the members of the 2-oxoacid dehydroganese complex family of multi-enzyme complexes. Besides, PDC-E2 specific autoreactive T cells can be detected in most patients with PBC but not in normal ones. However, the reason for the breakdown of immune tolerance and the regulatory mechanisms are still unclear.After T cells are activated by responding to antigenic stimuli, they are generally killed by apoptotic mechanisms. Among the apoptotic mechanisms, activation-induced cell death (AICD) plays a central role, especially in killing autoreactive T cells and in preventing autoimmune responses. AICD in T cells in vivo has been proposed to limit the expansion of an immune response by eliminating effector cells that are no longer needed. Organ-specific autoimmune diseases are characterized by tissue destruction and functional decline due to autoreactive T cells that escape self-tolerance. Primary biliary cirrhosis is characterized immunologically by lymphocytes infiltration in portal areas, destruction of biliary epithelial cells and cholestasis, which lead to progressive liver damage.The portal cellular infiltrate in PBC contained a preponderance of CD4 cells in comparison with CD8 cells, with a CD4/CD8 ratio of 2.45:1, which indicate that CD4+T cells play important roles in the immune inflammation process. In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis showed that IFN-γmRNA-positive cells were detected primary around damaged bile ducts. The data indicate that Th1 cells are the more prominent T cell subset in the lymphoid infiltrates in PBC.Polyinosinic polycytidylic acid (polyI:C) is ligand for toll like receptor 3, which can induce the secretion of IFN-α. Prolonged courses of IFN-a have been associated with the onset of autoimmune events. These ranged from the appearance of autoantibodies such as antinuclear, anti-thyroid, and anti-islet-cell antibodies in 80% of patients treated to the exacerbation, or, in some cases, to the development of a variety of autoimmune diseases, including hyper- or hypothyroidism, type I diabetes, psoriasis, and autoimmune hepatitis. In 1995, D’Amico reported for the first time on a case of primary biliary cirrhosis (PBC) induced by IFN-a in a patient with chronic hepatitis C. Alkaline phosphatase levels reached about three times. Further evaluation revealed the constant presence of high titers of serum anti-mitochondrial antibody.The patient underwent a second liver biopsy that showed infiltration of the portal areas with mononuclear cells. Besides, Takii found that the expression levels of type I IFN-αwas significantly higher in portal tract and liver parenchyma as compared to AIH and CHC.In clinical treatment for PBC, some medicines can be used to relieve pruritus, osteoporosis, hyperlipidemia and portal hypertension. However, survival is not adversely affected by the treatment and can not cure PBC fundamentally. According to the reasons underlined above, it is of great importance to investigate an effective method for PBC treatment from immunology aspect.Based on the facts showed above, we plan to develop a kind of PBC animal model by M2 and polyI:C injection and study activation induced cell death of CD4+T cells from spleens and livers to understand peripheral immune tolerance mechanism in PBC model. In order to prevent PBC, we injected spleen cells which were incubated with M2 antigen at the presence of ECDI though caudal vein to reconstruct immune tolerance. All the studies underlined above aim to provide clues for advanced investigation of PBC pathogenesis and clinical treatment.Part I. Fermentation and purification for M2 autoantigen E2 subunit of PDC, OGDC and BCOADC are the predominant self-antigen for patients with PBC. Immunodominant epitopes of PDC, OGDC and BCOADC have been cloned together and expressed in our laboratory. In order to develop PBC animal model and to investigate its immune tolerance, we prepared M2 protein through fermentation. At first, the protein was induced by IPTG to express in tubes and flasks. Second, a lot of M2 was obtained through 3.5L fermentor. At last, the protein was purified by Amersham protein purification system.Results showed that high expression of protein was identified 4h after induction by 1mg/ml IPTG. In the 3.5L fermentor, dissolved oxygen decreased 2.5h later and then glycerol was used as carbon source. We had compared glycerol and glucose as carbon source and found that glycerol can promote M2 expression dramatically. Highest cell density (OD600) was determined 6h after IPTG induction. Dissolved oxygen was controlled up to 30%, the maximum rate of rotation was 500rpm/min and pH was adjusted to 6.8-7.4. At last OD600 was 12.7, bacterial weight was 30g and the concentration of M2 we obtained was 1.5mg/ml.Part II. Development of primary biliary cirrhosis animal modelPBC is a kind of chronic persisting liver disease which is usually accompanied by virus infection and some other pathogens. It is so difficult to obtain enough liver tissues from patients for scientific research that a useful animal model is of great importance.M2 and polyI:C were employed to immunize C57BL/6 female mice at dose of 50μg and 5 mg/kg, 10mg/kg. Mice were sacrificed in different times and serum, liver, kidney, brain, intestine and stomach were separated for further determination. Results are as follows: in M2 group, the positivity of ANA and AMA was 30% and 80%, the AMA titers were all more than 1:100. However, both were negative in control group. In polyI:C group, ANA in control, 5mg/kg and 10mg/kg group were up to 100% positive in the 12th, 8th, and 4th week. The titers of ANA in control and 5mg/kg group were more than 1:100 since the 8th week but in 10mg/kg group it was since the 4th week. Besides, ANA titers more than 1:10000 can be found in each polyI:C group since 8th week. AMA in mice of control group were all negative, however, in 5mg/kg group, the positivity increased with time after polyI:C injection and it was up to 80% in 16th week. Differently, in 10mg/kg group, AMA positivity was 80% in the 4th week but decreased to 20% in the 20th week.Liver tissues were collected and stained with H.E and the number of portal areas with more than 100 mononuclear cells was counted as cell infiltrated portal areas. Data showed that the number of infiltrated portal areas increased until 16th week. The difference between 16th and 20th week had no statistic significance. No infiltrated portal areas were found in the other organisms in mice of each group. Serum AKP in both polyI:C groups increased with time and the average levels were all more than 60U/L, however, it was below 60U/L in control group. No infiltration and AKP change was found in M2 group. There was no significant difference for the ratios of CD4+ and CD8+T between each group. In conclusion, the PBC model developed by polyI:C 5mg/kg injection was more typical than the others and can be used for the following research.Part III Proliferation and activation induced cell death of lymphocytes in primary biliary cirrhosis animal model.Activation induced cell death (AICD) of autoreactive T cells in vivo plays a central role in maintaining peripheral immune tolerance. Defective AICD of effective T cells may result in autoimmune diseases. Studies indicated the predominant of Th1 cells around portal tracts.In this part, we develop PBC model again by injecting 5mg/kg polyI:C. Lymphocytes and CD4+ T cells were separated from spleens and livers after 16th week and stimulated by M2, conA and anti-CD3. Cell proliferation was determined by CCK8 assay and AICD was analyzed by flowcytometry (FCM). We also determined the expression of apoptosis related genes (Fas, FasL and TRAIL) and proteins (FLIPL and caspase-8). Results showed that: 1) No significant difference was found between cell proliferation of M2 group and control mice. However, the ability of cell proliferation of lymphocytes in PBC mice was stronger than that in control mice (P<0.001). 2) Cell apoptosis rates in control groups were lower than that in PBC mice (P<0.001), but no difference was determined in control groups (P>0.05). Furthermore, in PBC mice, the apoptosis rates of T cells from livers were lower than that from spleens significantly (P<0.001). 3) The expression of FasL and TRAIL in spleens and livers of PBC mice was lower than that in controls (P<0.01), but no difference was found with Fas. 4) The expression of caspase-8 in CD4+T cells in PBC mice was not different from that in control ones. However, the expression of FLIPL in PBC mice was 5 times higher than that in controls. Moreover, in PBC mice, the expression of FLIPL in livers was higher than that in spleens (P<0.001).In conclusion, the anti-apoptotic ability of CD4+ T lymphocytes plays an important role in immunological tolerance of PBC mouse model, and the enhancement of the ability was to some degree correlated with the elevated FLIPL expression. Besides, the inhibition of FasL and TRAIL expression may also of help in the enhancement of the anti-apoptotic ability in lymphocytes and in the aggravation of portal area inflammation.Part IV Prevention of primary biliary cirrhosis through immune tolerance reestablishmentThere is no curable method for PBC nowadays and symptomatic treatment methods for pruritus, osteoporosis, hyperlipidemia and portal hypertension are better choices for clinicians. Liver transplantation is the best and only means for patients with PBC in advance stage. Accordingly, it is imminent to find a better way from immunology aspect for PBC treatment. Spleenic cells from na?ve mice were incubated with M2 in the presence of ECDI and the cells were injected into caudal vein of the mice which would be used for development of PBC model. Spleenic cells incubated with BSA were injected as controls. 16 weeks later, AMA, AKP and portal inflammation were assayed for evaluating the prevention effect. Results are as follows: 1) AMA positive rate in tolerance group was lower than that in BSA and PBC groups (P=0.007, P=0.003). The difference between BSA and PBC was not significantly. 2) Serum AKP levels in tolerance, BSA and PBC group were 80.5±9.8U/L, 93.8±15.7U/L and 92.5±17.7U/L, separately. The level in tolerance group was lower than that in BSA and PBC groups (P=0.0095, P=0.029). 3) The rates of portal areas with cell infiltration were 42.67±12.3%, 57.07±11.35% and 51.53±9.96%, separately. The number of infiltrated portal tracts in tolerance group was less than that in PBC group (P=0.039) and BSA group (P=0.0024).In conclusion, primary biliary cirrhosis in mouse model was prevented to some extent by reestablishing immune tolerance to M2 autoantigen. This provides clues for finding a better treatment proposal.更多还原