【作者】 刘晓雯；

【导师】 赵跃然；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2012， 博士

【摘要】 系统性红斑狼疮(systemic lupus erythematolsus, SLE)是一种弥漫性,全身性的自身免疫病,好发于青年女性,发病高峰为15-40岁,同龄男女发病比例约为1：7到1：10。SLE发病存在明显的种族地区因素差异性,世界各地报道SLE的发病率约在10-100/10万之间,我国的发病率约为70/10万。SLE患者的主要特征是血清中出现多种针对自身组织成分的自身抗体,通过免疫复合物等途径造成多种器官,多个系统的损害。其临床表现复杂多样,主要累及皮肤黏膜,骨骼肌肉,肾脏及中枢神经系统。自身免疫病的发病受遗传,环境,激素水平等因素的共同影响。既往研究发现,感染是诱发自身免疫病的最重要环境因素之一。自身免疫病的发生发展与感染有密切关系,各种病原体(细菌,病毒,寄生虫和真菌)均可使具有潜在免疫失调的人诱发自身免疫病。同一种病原体可诱导产生多种自身免疫病,但是不同的病原体也可能诱发同一种自身免疫病。而最新的研究发现,部分感染性疾病对自身免疫病还存在“保护”作用。但二者之间相互作用的具体机制尚未明确。乙型肝炎病毒(hepatitis B virus, HBV)属于嗜肝DNA病毒科,是人类乙型肝炎的特异性病原体。据WHO估计,目前感染过HBV的人约有20亿,慢性感染的的超过3.5亿,其中75%发生在亚洲。我国是HBV感染的高发区,乙肝表面抗原(hepatitis B surface antigen, HBsAg)携带率约占总人口的7.18%,由此推算,全国约有9300万人携带乙肝病毒,其中乙肝患者超过3000万。多项研究表明HBV感染与自身免疫病发病关系密切,其机制可能是通过分子模拟,旁观者效应,抗原表位扩增,刺激非免疫细胞表达主要组织相容性复合体(majorhistocompatibility complex, MHC)分子等刺激机体产生自身抗体和激活自身反应性T淋巴细胞,从而导致自身免疫病的发生。因此理论上HBV可能促进SLE疾病的发生与发展。但最近的临床研究发现,SLE患者HBV感染率明显低于正常人,同时,SLE患者HBeAg的阳性率明显低于对照人群,并且慢性乙肝合并SLE患者的肾脏损害程度、IFN-α、IFN-γ、抗ds-DNA抗体滴度和狼疮指数均明显低于非合并慢性乙肝SLE患者。提示HBV感染可能对SLE发生发展具有拮抗(保护)作用,但迄今缺乏HBV对SLE保护作用的实验依据,其机制尚不清楚。本文通过Pristane诱导,建立HBV转基因BALB/c小鼠和同基因型BALB/c小鼠实验性狼疮模型。通过实验性狼疮鼠自身抗体水平、肾脏损伤,探讨HBV对SLE发生的保护作用；用液相悬浮芯片技术,检测狼疮模型形成过程中相关细胞因子表达水平的变化,分析相关细胞因子水平和狼疮病理改变的相关性,探讨HBV抑制SLE病理进程的机制。通过上述研究探讨HBV对SLE的保护作用及其机制,为该疾病的临床治疗及药物靶点的选择提供理论依据及指导。第一部分HBV对实验性狼疮的保护作用目的利用BALB/c小鼠与HBV转基因鼠建立狼疮模型,并比较不同组间小鼠SLE的发病率及肾脏损伤程度。方法21只雌性BALB/c-HBV转基因小鼠及雌性同基因型BALB/c小鼠随机分为SLE造模组和对照组。造模组一次性腹腔注射降植烷0.5mL,对照组注射生理盐水0.5mL。四组小鼠分别为：一组,BALB/c-HBVTg-saline组；二组,BALB/c-HBVTg-Pristane;三组,BALB/c-saline;四组,BALB/c-Pristane组。注射前及注射后每月一次剪尾取血50μL,分离血清置于-80℃待用。腹腔注射6个月后,采用摘眼球法处死小鼠,同时收集血液至Eppendorf管(EP管)中,分离血清-80℃保存待用。间接免疫荧光法检测造模前小鼠尾静脉血清和造模后6个月血清中ANA及抗ds-DNA抗体。小鼠处死后,取肾脏组织中性甲醛固定1周后,脱水石蜡包埋后制备组织切片。应用HE染色及PAS染色后,显微镜下观察比较不同组间肾脏损伤程度差别,同时计算肾小球细胞外基质(ECM)堆积指数。各组间ANA滴度的比较采用非参数检验。各组间抗ds-DNA抗体阳性率的比较采用x2检验fisher确切概率法。肾小球ECM堆积指数比较使用采用完全随机设计的单因素方差分析方法(ANOVA),组间两两比较采用LSD检验,应用SS18.0分析软件,检验水准α=0.05。结果(1)模型小鼠存活情况：BALB/c-HBVTg-saline组,存活9只,死亡2只；BALB/c-HBVTg-Pristane组,存活8只,死亡2只；BALB/c-saline组,存活11只,死亡0只；BALB/c-Pristane组,存活10只,死亡0只。(2)HBV转基因狼疮模型鼠的ANA水平明显低于同基因型BALB/c狼疮模型鼠：BALB/c-Pristane组造模前后ANA阳性率的比较有显著性差异,p<0.001,说明造模成功。而BALB/c-HBVTg-Pristane组,及两组对照在造模前后阳性率变化均无统计学差异。造模后第6月时,BALB/c-HBVTg-Pristane组ANA滴度显著低于BALB/c-Pristane组,P=0.001。(3)HBV转基因狼疮模型鼠的抗ds-DNA抗体阳性率显著低于同基因型BALB/c狼疮模型鼠：BALB/c-Pristane组造模前后抗ds-DNA抗体阳性率的变化明显,P=0.02。BALB/c-HBVTg-Pristane组造模前后抗ds-DNA抗体阳性率的变化无统计学差异,P=0.444,两组对照组造模前后均无明显变化。造模后第6月时,HBV转基因狼疮模型鼠抗ds-DNA抗体阳性率明显低于同基因型BALB/c狼疮模型鼠,P=0.025。(4)肾小球病变::肾小球HE染色显示,BALB/c及BALB/c-HBV对照组肾小球肾小球大致正常；BALB/c-Pristane组肾小球变小,系膜区增宽,系膜细胞和血管内皮细胞明显增多,炎细胞浸润,肾小球毛细血管袢开放不良,包曼囊壁有增厚；HBV转基因狼疮模型鼠仅有轻度系膜增生,肾小球病变明显较同基因型BALB/c狼疮模型鼠轻。(5)基底膜/系膜糖原沉积：用PAS染色检测基底膜/系膜糖原沉积情况。HBV转基因鼠和同基因型BALA/c鼠对照组均未见明显糖原沉积。BALB/c-pristane(模型)组糖原沉积明显增多,主要分布于肾小球基底膜及增生的系膜；BALB/c-HBVTg-pritane (HBV转基因鼠模型组)组糖原沉积轻度增多；主要分布于肾小球基底膜。(6)HBV转基因狼疮模型鼠的ECM堆积指数显著低于同基因型BALB/c狼疮模型鼠：狼疮模型鼠的ECM堆积指数均明显高于其对照组；而HBV转基因狼疮模型组显著低于同基因型BALB/c鼠模型组,P<0.001,即HBV转基因小鼠模型组肾脏损伤较同基因型的BALB/c小鼠模型组轻。结论1.建立了pristane诱导狼疮鼠模型。2.HBV转基因狼疮模型鼠的自身抗体(ANA和抗ds-DNA抗体)水平明显低于同基因型的BALB/c狼疮模型鼠,前者的狼疮肾炎样的病理改变也明显轻于后者。提示HBV对实验性狼疮具有保护作用。第二部分HBV对实验性狼疮保护作用的机制目的研究SLE相关细胞因子变化,探讨HBV与SLE相互作用的具体机制。方法小鼠多通道细胞因子芯片检测造模后第1个月和造模后第6个月时的小鼠血清中IL-2、IL-4、IL-6、IL-12p70、IL-17、TNF-a六种细胞因子。ELISA法检测造模后第2个月和造模后第6个月时的小鼠血清BAFF水平。各组之间细胞因子表达水平的比较使用单因素方差分析(ANOVA),组间两两比较采用LSD检验。分析软件为SPSS18.0,双侧P<0.05认为具有统计学差异。结果(1)HBV转基因狼疮模型鼠的IL-17水平低于同基因型BALB/c狼疮模型鼠：BALB/c-Pristane组的IL-17水平在造模后第6个月时较造模后第1个月显著升高,P<0.001。其他各组的IL-17水平在造模后第6个月与造模后第1个月相比,差别无统计学差异。造模后第6个月,BALB/c-Pristane组与BALB/c-HBVTg-Pristane组IL-17水平均比其相应对照组显著提高,(P<0.001,P=0.014)。但HBV转基因鼠造模后IL-17水平明显低于普通BALB/c小鼠造模后水平(P=0.006)。(2)HBV转基因狼疮模型鼠的TNF-a水平显著低于同基因型BALB/c狼疮模型鼠：造模后第1个月,BALB/c-HBVTg-Pristane组TNF-a水平显著高于BALB/c-HBVTg-saline组(P=0.045)。BALB/c-Pristane组在造模后第6个月时血清TNF-a水平显著高于造模后第1个月,P<0.001。其他各组造模后第6个月与造模后第1个月相比,差别无统计学意义。HBV转基因鼠造模后TNF-a水平虽有显著升高,但仍明显低于普通BALB/c小鼠造模后水平(P<0.001)。(3)HBV转基因狼疮模型鼠的BAFF水平显著低于同基因型BALB/c狼疮模型鼠：造模后第1个月,造模组BAFF水平均较对照组明显升高。BALB/c-Pristane组BAFF水平较BALB/c-HBV+SLE显著升高,P=0.009。造模后第6个月BALB/c-Pristane组BAFF水平较BALB/c-HBV+SLE显著升高,P=0.042,但升高程度不如造模后第2个月时显著。(4)IL-6在狼疮模型组中显著升高,但BALB/c-HBVTg-saline组升高更为显著；HBV转基因鼠的IL-2、IL-12p70水平较普通BALB/c鼠高；IL-4水平在各组中差别不明显。(5)IL-17与TNF-α(r=0.647,p<0.001)、IL-6(r=0.529,P<0.001)、IL-4(r=0.296,P=0.009)和BAFF(r=0.360,P=0.001)有明显的相关性,而与IL-2、IL-12无明显相关性。(6)IL-6与TNF-a(r=0.685,P<0.001)、IL-17(r=0.529,P<0.001)、IL-12p70(r=0.515,P<0.001)、IL-4(r=0.473,P<0.001)有明显的相关性。结论IL-17,BAFF, TNF-α三种炎性因子在狼疮模型组中均显著升高,但其在HBV转基因狼疮模型鼠中的水平显著低于同基因型BALB/c狼疮模型鼠。相关性分析显示IL-17、IL-6与其他细胞因子之间存在密切联系。提示HBV对实验性狼疮的保护作用中,细胞因子网络发挥重要作用。更多还原

【Abstract】 Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease, which occurring predominantly in women aged15-40years. And the incidence rate in women is6-to10-fold higher than man. The prevalence of SLE is variable, from10cases per100,000populations to100cases per100,000populations. In China, the prevalence of SLE is about70cases per100,000populations. SLE is characterized by the presence of autoantibodies, formation of immune complexes, and immune dysregulation, which affects several organs, for example, neurological, dermal, haematological, musculoskeletal, and renal.Though the initiation and progression of SLE is still unclear, Genetic factors, environmental factors and hormones may be involved in the disorders. A growing number of evidence supported the vital role of infections in the induction or progression of autoimmune diseases. Sometimes the same kind of pathogens can induce a variety of autoimmune diseases, on the other hand, different pathogens may also induce the same kind of autoimmune disease. Recent researches showes that infections may sometimes protect individuals from specific autoimmune diseases. But till now, the relationship of the2diseases is still unclear.Hepatitis B virus (HBV)is a hepatotropic virus, which causes acute and chronic liver diseases. In China, chronic infection rate in general population was about10-15%, despite the appliance of hepatitis B vaccination. Several researches reported that the generation of autoantibodies and formation of immune complexes were found in the HBV infectious process through molecular simulation, Bystander Effect, antigenic epitope amplification and major histocompatibility complex (MHC), which may be closely related to the autoimmune diseases. So it is reasonable to suspect a higher frequency of HBV infection in SLE patients. But recent studies show that the HBsAg-positive rate was lower in SLE patients than controls, and the clinical features of HBV infected SLE patients was distinguishable from the SLE patients, especially the renal lesions, serum levels of ANA and anti-dsDNA, IFNα, IFNγ and SLE index. Several researchers suspected that the potential protective role of HBV in SLE patients could be explained by the high levels of IFN, T cells and B cells hyperactivity, and the abnormalities of cytokines. But few research of the molecular association between HBV and SLE has been published.As for investigating the mechanism by which HBV affected the pathogenesis of SLE, we compared the HBV infection mice with and without SLE, from the incidence of SLE in mice models, renal lesions and related cytokines.Part1. The protective role of hepatitis B virus infection from pristane induced lupus miceObjective:To establish the SLE models based on both the BALB/c mice and HBV transgenic BALB/c mice, and compare the prevalence of SLE and the renal changes in the different groups.Methods:21female BALB/c mice and21female homozygous HBV transgenic BALB/c mice (expressing HBV full gene in mice liver) aged two months were randomly divided into4groups:normal control group (11), pristane-induced lupus group (10), HBV transgenic group (11), and pristane treated HBV transgenic group (10). The mice in normal control group and HBV transgenic group were given an intraperitoneal injection of0.5ml normal saline (NS), and the other2groups were given an intraperitoneal injection of0.5ml pristane. Sera were collected from the tail vein before treatment and every month after injection until the mice were killed. ANA and anti-dsDNA levels in serum were detected by indirect immunofluorescence. Twenty-four weeks after pristane administration, all mice were killed. ANA and Anti-dsDNA levels were detected by indirect immunofluorescence. For histopathological examination, kidney were cut and stained with hematoxylin and eosin (H&E) and periodic-acid Schiff (PAS). Glomerulonephritis, renal tubular lesions and interstitial inflammation were observed, and the degree of severity of histological lesions characteristic of lupus nephritis was evaluated as absent (-), mild (+), moderate (++), and severe (+++). The extracellular matrix (ECM) packing index was also calculated. Differences of ANA levels between experimental groups were evaluated by the nonparametric test, while the Anti-dsDNA levels by the x2test. Differences between experimental groups were evaluated by the ANOVA test. All statistical tests were two-sided, with significance defined as P<0.05. Analyses were performed using SPSS version18.0.Results:(1) Survival of mice at6months later:BALB/c-HBVgroup,9survive,; BALB/c-HBV+SLE group,8survive; BALB/c group,11survive; BALB/c+SLE group,10survive.(2) ANA:the positive rates of ANA in pristane-induced lupus group (100%)were much higher than other groups (HBV transgenic group:33.3%; HBV+SLE group:50%; normal control group:9.1%, respectively). The ANA titers in pristane-induced lupus group were significantly higher than normal control group (P <0.001), and HBV+SLE group (P=0.035).(3) The anti-dsDNA:After pristane administration, the anti-dsDNA levels were much higher than before (P=0.02). But in the BALB/c-HBV+SLE group, the changes were not significant after the administration (P=0.444).6months after the administration, the positive rates of anti-dsDNA were the highest (70%) in pristane-induced lupus group, which were significantly higher than in BALB/c-HBV+SLE group (P=0.025).(4) After6months, kidney from normal control group had normal histology. And in the HBV transgenic control group, only slight mesangial proliferation was found. The most severe patterns of renal abnormalities were found in pristane-induced lupus group. Mesangial broadening and cell layers increasing were observed, while some glomeruli showed glomerular atrophy. An infiltration of inflammatory cells was also seen in renal interstitial. Nevertheless, all the changes were less severe in HBV+SLE group than that pristane-induced lupus group. Glomerular atrophy and mesangial broadening were found in a much milder degree. Slight lymphocytes infiltration was also observed.(5) ECM packing index:The index of BALB/c+SLE group and BALB/c-HBV+SLE group were both higher than the control groups. And the index was much lower in the BALB/c-HBV+SLE group than in the BALB/c+SLE group (P <0.001).Conclusions:1.The establishment of pristane-induced lupus models on both the normal mice and HBV transgenic mice now provide a powerful new platform for the study of SLE.2. The result that ANA and anti-dsDNA levels were lower in the HBV+SLE group, promote us to investigate whether HBV infection play a role in the regulation of autoimmunity for SLE.3. HBV mice showed less severe renal changes during SLE progression.4. The basement membrane thickening is not obvious in the BALB/c-HBV+SLE group, comparing to the BALB/c+SLE group.Part2. The mechanism of the protective role of hepatitis B virus infection from pristane induced lupus mice.Objective:To explore the possible roles of several related cytokines in the interaction of SLE and HBV.Methods:Interleukin2(IL-2), IL-4, IL-6, IL-10, IL-17and TNF-α were measured by Multiplex immunoanalytic Xmap (LUMINEX) technology, which combines flow cytometry and sandwich immunoassay. The serum was analysis by a custom-built Panel for cytokines. The measurement was performed with the Luminex200instrument and the Luminex200IS software. BAFF levels were detected by the ELISA. The expression levels of cytokines between experimental groups were evaluated by the ANOVA tests. And the correlation analysis was assessed by the spearman Spearman’s rho. All statistical tests were two-sided, with significance defined as P<0.05. Analyses were performed using SPSS version18.0.Results:(1) IL-17:6months after the administration, the IL-17levels were significantly increased in the the BALB/c+SLE group (P<0.001). And the levels The in BALB/c+SLE group and BALB/c-HBV+SLE group were both higher than the control groups. But the increase rate was much lower in the BALB/c-HBV+SLE group than BALB/c+SLE group (P=0.006).(2) TNF-α:The growth trend of TNF-α was similar with IL-17. BALB/c+SLE group and BALB/c-HBV+SLE group both had a significant increase than control, but the increase rate was much lower in the BALB/c-HBV+SLE group than BALB/c+SLE group (P<0.001).(3) BAFF:1month after administration, the level of BAFF in BALB/c+SLE group were much higher that in the BALB/c-HBV+SLE(P=0.009). But after6months, there was no significant differences between the2groups.(4) IL-6levels were significantly increased in the the BALB/c+SLE group, but the increase rate was much higher in the BALB/c-HBV+SLE group than the BALB/c+SLE group. The levels of IL-2and IL-12p70was much higher in the BALB/c-HBV group than the control.(5) IL-17had significant relationship to TNF-α (r=0.647, P<0.001), IL-6(r=0.529, P<0.001), IL-4(r=0.296, P=0.009) and BAFF (r=0.360, P=0.001). While the IL-6also had significant relationship to TNF-α (r=0.685, P<0.001), IL-17(r=0.529, P<0.001),IL-12p70(r=0.515, P<0.001) and IL-4(r=0.473, P<0.001)。Conclusions:The IL-17, TNF-α and BAFF may play an important role in the progression of SLE. And the correlation analysis promoted us that the abnormal cytokine network plays a central role in the pathogenesis and progression of SLE and HBV. However, Further research of the network is still required.In our research, the incidence of SLE was much lower in HBV mice. And the HBV infection help the pristine-induce mice survive from high levels of inflammatory cytokines and severe renal damage. Based on the above research, we finally demonstrated the protective role of HBV in SLE patients via the immunoregulatory networks of the cytokines. At the therapeutic level, analyzing the role of cytokines in the interaction of HBV and SLE may lead to finding new therapeutic approaches for the treatment of both the two diseases.更多还原