【作者】 侯炳旭；

【导师】 冯丽英；

【作者基本信息】 河北医科大学 ， 内科学， 2011， 硕士

【摘要】 乙型肝炎病毒(hepatitis B virus,HBV)易致慢性化感染,但其慢性化机制尚不完全清楚。许多研究已经证实慢性乙型肝炎(chronic viral hepatitis B,CHB)的发病机制与机体的细胞免疫功能低下密切相关,但CHB患者细胞免疫功能低下的机制尚未完全阐明,近年来有研究提出与淋巴细胞的激活诱导细胞死亡(activation induced cell death,AICD)有关。AICD是指已活化的成熟淋巴细胞(T或B)再次受到激活信号激活后(特别是TCR/CD3复合体)所发生的细胞凋亡,这是维持机体免疫稳定的一种生理机制,也可能是形成外周免疫耐受的重要原因。外周血单个核细胞(peripheral blood mononuclear cells ,PBMC)主要由淋巴细胞组成,还包括单核巨噬细胞、自然杀伤细胞等免疫活性细胞,在机体的免疫反应特别是细胞免疫反应中起重要的作用,检测PBMC凋亡状况可大体反映外周血淋巴细胞凋亡状况。有研究发现CHB患者外周血淋巴细胞和PBMC发生AICD的比率比正常对照高,提示淋巴细胞可能在HBV特异性抗原刺激下被激活,再发生自身凋亡(即AICD),使淋巴细胞的数量减少或活性下降,从而导致CHB患者细胞免疫功能低下,这可能是导致HBV持续慢性感染的一个重要因素,也是形成HBV慢性感染免疫耐受的一个重要机制。因此如何通过调节AICD来减少淋巴细胞凋亡,改善CHB患者的细胞免疫功能,将是打破免疫耐受状态、清除慢性乙型肝炎患者体内HBV的可能途径。但是关于CHB患者PBMC发生AICD的机制尚未完全阐明,目前关于其凋亡途径的研究主要集中于由Fas/FasL等介导的死亡受体凋亡途径和Bcl-2家族调控的线粒体凋亡途径,认为死亡受体途径和线粒体途径可能在CHB患者PBMC的AICD过程中起着重要的作用。c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)家族是丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)超家族成员之一,也被称为应激活化蛋白激酶(stress-activated MAP kinase,SAPK),其上游双特异性激酶MEK4/7通过双磷酸化JNK的Thr183和Tyr185位点而激活JNK,磷酸化JNK(p-JNK)是其活性形式。JNK能介导多种胞外刺激(如应激、Fas、TNFα等)诱导的细胞凋亡,参与了许多细胞凋亡的发生,在多种疾病的发生发展中起重要作用。目前认为JNK介导凋亡的机制主要是能通过转录依赖或非转录依赖的方式调控下游凋亡相关靶基因的表达或靶蛋白的活性而介导死亡受体途径及线粒体途径的细胞凋亡。c-Jun和Bim都是JNK介导的细胞凋亡信号通路中的重要下游分子。c-Jun是核内转录因子AP-1蛋白的一员,是第一个被发现的JNK核内底物,磷酸化c-Jun(p-c-Jun)是其活性形式。活化的JNK(p-JNK)转位到细胞核后能够使c-Jun第63、73位丝氨酸双磷酸化而激活,从而提高其转录活性,促进下游凋亡相关靶基因的转录和凋亡蛋白的表达(如FasL、Bim、c-myc等),分别启动死亡受体途径及线粒体途径的细胞凋亡。Bim是Bcl-2家族中BH3-only亚家族的成员,是一种重要的凋亡调节蛋白。JNK被活化后可以从胞质转位入核通过激活转录因子c-Jun而上调Bim的表达,也可以在胞质内直接使Bim蛋白磷酸化而诱发线粒体途径的细胞凋亡。近年来研究证明JNK信号通路参与介导了多种疾病淋巴细胞的AICD过程。Mehrotras等发现应用JNK特异性抑制剂SP600125可以对抗流感表位特异性CTL发生的AICD,说明JNK参与介导了流感表位特异性CTL的AICD过程。有研究发现JNK参与介导了小鼠脾脏T细胞的AICD过程。但是关于JNK信号通路是否也参与介导了CHB患者PBMC的AICD过程还未见报道。为此我们以CHB患者PBMC为研究对象,体外模拟AICD过程,观察JNK通路的p-JNK、总JNK及其下游转录因子p-c-jun和下游蛋白Bim的表达情况和PBMC的凋亡率,以及在JNK特异性抑制剂SP600125抑制JNK后,p-JNK、总JNK及其下游p-c-jun、Bim蛋白的表达和PBMC凋亡率的变化,来探讨JNK信号通路在CHB患者外周血单个核细胞AICD过程中的作用。目的:研究JNK信号通路在慢性乙型肝炎患者外周血单个核细胞AICD过程中的作用,进一步探讨AICD发生的机制,为慢性乙型肝炎的治疗提供理论依据。方法:1入选标准和排除标准选取慢性乙型肝炎患者共40例,其中男性22例,女性18例,年龄在18岁-60岁,平均年龄35岁。随机分为两组,慢性乙肝组20例,慢性乙肝+抑制剂组20例。所有病例诊断标准均符合2005年中华医学会制定的“慢性乙型肝炎防治指南”, HBVDNA大于1×103copies/ml小于1×107copies/ml,ALT两倍以上升高。排除应用过抗病毒药物的患者,排除HAV、HCV、HDV、HEV和HIV感染,排除近3个月应用糖皮质激素、干扰素等影响免疫功能药物的患者,除外有近期急性感染患者。健康对照组20例,男12例,女8例,年龄在18岁-45岁,平均年龄30岁,取自健康献血员。2 PBMC的提取和培养无菌条件下抽取肝素抗凝血4mL,应用密度梯度离心法分离PBMC。分离成功的PBMC加入植物血凝素(Phytohemagglutinin,PHA)100μg/ml刺激培养16-18小时,弃去PHA,加入外源性IL-2 1000U/L进行培养至少7天后,用抗CD3抗体500ug/ml重新刺激48小时。慢性乙肝+抑制剂组在加抗CD3抗体前用JNK特异性抑制剂SP600125 20μmol/l预处理2小时。3细胞凋亡的检测培养的细胞用70%乙醇固定,4℃离心去掉固定液,并用冷PBS洗2次,加入含有RaseA的PI染液约1000μL,室温静置30分钟后上流式细胞仪检测凋亡率。4 p-JNK、总JNK、p-c-jun、Bim蛋白的检测:剩余细胞,提取蛋白后,-80℃保存,待收集够标本后集中行Western blot检测p-JNK、总JNK、p-c-jun、Bim蛋白的表达情况。结果:1经抗CD3抗体刺激48h后,慢性乙肝组PBMC凋亡率(28.27%±4.61%)高于健康对照组(19.36%±5.90%),差异有统计学意义(P<0.05),也高于慢性乙肝+抑制剂组(23.37%±5.52%),差异有统计学意义(P<0.05)。慢性乙肝+抑制剂组PBMC凋亡率(23.37%±5.52%)高于健康对照组(19.36%±5.90%),差异有统计学意义(P<0.05)。2慢性乙肝组PBMC中p-JNK蛋白的表达量高于健康对照组(1.09±0.16 vs 0.83±0.12,t=5.885,P<0.01),也高于慢性乙肝+抑制剂组(1.09±0.16 vs 0.53±0.15,t=11.502,P<0.01),差异有统计学意义。总JNK蛋白在三个组的表达差异无统计学意义。慢性乙肝组PBMC中p-c-jun蛋白的表达量高于健康对照组(1.21±0.15 vs 0.96±0.13,t=5.477,P<0.01),也高于慢性乙肝+抑制剂组(1.21±0.15 vs 0.62±0.14,t=12.899,P<0.01),差异有统计学意义。慢性乙肝组PBMC中Bim蛋白的表达量高于健康对照组(0.96±0.14 vs 0.58±0.12,t=9.133,P<0.01),也高于慢性乙肝+抑制剂组(0.96±0.14 vs 0.52±0.13,t=10.086,P<0.01),差异有统计学意义。3慢性乙肝组PBMC凋亡率与PBMC中p-JNK蛋白的表达成正相关(r=0.823,P<0.01)。结论:1慢性乙型肝炎患者PBMC存在AICD现象,且凋亡率较正常人高。2慢性乙型肝炎患者PBMC中存在JNK信号通路的过度激活。3慢性乙型肝炎患者PBMC凋亡率与PBMC中p-JNK蛋白的表达呈正相关,JNK特异性抑制剂SP600125可明显降低p-JNK、p-c-jun、Bim蛋白的表达,下调PBMC凋亡率。提示JNK信号通路参与介导了慢性乙型肝炎患者PBMC的AICD过程,是其凋亡增多的机制之一。更多还原

【Abstract】 Hepatitis B virus( HBV) often cause chronic infection,but the mechanism has not been completely clear.Many studies have already confirmed that the pathogenesis of chronic viral hepatitis B(CHB) is closely related with the lower cellular immunity function of the patients,but the mechanism of the lower cellular immunity function of the CHB patients has not been thoroughly expounded.In recent years,Studies have proposed that it was related with the activation-induced cell death(AICD).The apoptosis of activated mature lymphocytes(T or B)after activated again by stimulation(especially the TCR/ CD3 complex)is called AICD,Which is a physiological mechanisms of maintaining the stability of immunity and may be an important reason of peripheral immune tolerance.Peripheral blood mononuclear cells(PBMCs)are composed mainly by lymphocytes,and also include monocyte-macrophage and natural killer cells and other immune cells. PBMCs play an important role in body’s immune response especially the cellular immune responses,and detection of apoptosis of PBMC can roughly reflect the apoptosis of peripheral blood lymphocytes.Studies have found that the ratio of AICD of peripheral blood lymphocyte and PBMC in chronic hepatitis B patients was higher than normal control group.This phenomenon indicate that lymphocytes may be activated by HBV-specific antigen,and then apoptosis(AICD)happen.AICD may cause the quantity and the activity of lymphocytes to reduce,so that lead to cellular immunity response of CHB patients decrease,which may be an important factor of persistent infection of HBV,and an important mechanism of immune tolerance.So how to adjust AICD to reduce the apoptosis of lymphocyte and improve cellular immunity function of the CHB patients will be a possible way to break the state of immune tolerance and eliminates HBV in CHB patients.But the mechanism of AICD of PBMC in CHB patients has not been completely clear.The studies about the apoptosis pathway of AICD of PBMC in CHB patients mainly focused on the death receptor pathway which was mainly mediated by Fas/ FasL and the mitochondria pathway which was mainly regulated by Bcl-2 family,and proved that this two pathway might play an important role in the AICD of PBMC in CHB patients.c-Jun N-terminal kinases(JNKs)family,also referred to as stress-activated kinases(SAPKs), is a subfamily of the mitogen-activated protein kinases (MAPKs) superfamily.Its upstream dual specificity kinase MEK4/ 7 can phosphorylate the Thr183 and Tyr185 sites to activate JNK,and phosphorylation JNK(p-JNK) is its active form.A variety of apoptosis induced by extracellular stimuli (such as stress, Fas, TNFα) were mediated by JNK,and JNK signal pathway was involved in many apoptosis,and palyed an important role in the development of many diseases.The mechanism was that through the transcription-dependent or transcription-independent pathway, JNK could regulate the expression of downstream apoptosis-related target gene or the activity of target proteins,and mediated the apoptosis of the death receptor pathway and the mitochondrial pathway.C-Jun and Bim are important downstream molecules of the JNK-mediated apoptosis signal pathway.C- Jun is one of the nuclear transcription factor AP-1 proteins and the first discovered nuclear substrates of JNK, and phosphorylation c-Jun(p-c-jun) is its active form.Activated JNK(p-JNK) can phosphorylate the Ser63 and Ser73 sites to activate c-jun after translocation into the nucleus,thereby enhancing its transcription activity,and promote the transcription of downstream apoptosis-related target gene and the expression of apoptosis proteins(FasL,Bim,c-myc,et al),and mediate apoptosis of the death receptor pathway and the mitochondrial pathway.Bim is a member of BH3- only subfamily in Bcl-2 family and an important apoptosis regulatory protein. Activated JNK can transfer from the cytoplasm into the nucleus to increase the expression of Bim through activating the transcription factor c-jun;and can also directly phosphorylate Bim in the cytoplasm and mediate the apoptosis of mitochondrial pathway. In recent years, studies have shown that JNK mediated AICD of lymphocytes in a variety of diseases.A study found that application of JNK inhibitor SP600125 could fight the AICD of influenza epitope-specific human cytolytic T lymphocytes(CTL),and proved that JNK was involved in mediating AICD of the influenza epitope-specific human CTL.Another study found that JNK was involved in mediating AICD of the mouse spleen T cells.But whether the JNK signal pathway is also involved in mediating AICD of PBMC in CHB patients have not been reported.So we took the PBMC of CHB patients as the research object and simulated the process of AICD in vitro.We observed the expression of p-JNK,total-JNK and its downstream transcription factor p-c-jun and downstream protein Bim and the apoptosis rate of PBMC,and the change of the expression of this proteins and the apoptosis rate of PBMC after the application of JNK inhibitor SP600125,to research the role of JNK signal pathway in the process of AICD of PBMC in CHB patients.Objective:To study the role of JNK signal pathway in the process of AICD of PBMC in CHB patients,and further explore the mechanism of AICD to provide a theoretical basis for the treatment of CHB.Methods:1 Inclusion criteria and exclusion criteriaWe selected a total of forty CHB patients, including twenty-two males and eighteen females,aged from eighteen to sixty years, mean age thirty-five years,and they were randomly divided into two groups:twenty patients in CHB group and twenty patients in CHB+inhibitor group.All subjects were diagnosed according to the guide of prevention and treatment of chronic hepatitis B which was established in 2005 by Chinese Medical Association. HBVDNA greater than 1×103copies/ml and less than 1×107copies/ml,and ALT more than twice higher.All patients had not used antiviral drugs.We excluded HAV,HCV,HDV,HEV,HIV and acute infections.The patients who had used the drugs that can affect the immune function such as glucocorticoid and interferon nearly 3 months were ruled out.Twenty individuals obtained from healthy blood donors were included in the healthy control group, including twelve males and eight females,aged from eighteen to forty-five years,mean age thirty years.2 PBMCs extraction and cells culturePBMCs were extracted from fresh and heparinized blood by density gradient centrifugation under aseptic condition. PBMCs were stimulated by PHA(100μg/ml) for 16～18h,then removed PHA from nutrient medium and added exogenous IL-2(1000U/L).Cells were cultured at least 7 days and stimulated again by anti-CD3(500ug/ml) antibody for 48 hours.The members of CHB+inhibitor group were pretreated with the JNK specific inhibitor SP600125(20μmol/l) for 2 hours before the use of anti-CD3 antibody.3 Detection of Cell apoptosisCultured cells were fixed with 70% ethanol,and centrifuged at 4℃to remove the fixative.The cells were washed twice with cold PBS(phosphate buffered saline),and then added the PI dye about 1000μl containing RaseA. Placed at room temperature for 30 minutes,and then analyzed the apoptosis rate by flow cytometer.4 Detection of p-JNK,total-JNK,p-c-jun and Bim proteinProteins were extracted from remaining cells and preserved under -80℃.We collected enough samples and detected the expression of p-JNK, total- JNK,p-c-jun and Bim protein by western blot.Result:1 The apoptotic rate of PBMC of CHB group(28.27%±4.61%)was higher than healthy control group(19.36%±5.90%),the discrepancy was statistically significant(P<0.05),and also higher than CHB+inhibitor group(23.37%±5.52%),the discrepancy was statistically significant(P<0.05).The apoptotic rate of PBMC of CHB+inhibitor group(23.37%±5.52%) was higher than healthy control group(19.36%±5.90%),the discrepancy was statistically significant (P<0.05).2 The expression of p-JNK in PBMC of CHB group(1.09±0.16)was higher than healthy control group(0.83±0.12),the discrepancy was statistically significant(t=5.885,P<0.01),and also higher than CHB+inhibitor group(0.53±0.15),the discrepancy was statistically significant(t=11.502,P<0.01).The discrepancy of the expression of total-JNK in the three groups was not statistically significant.The expression of p-c-jun in PBMC of CHB group (1.21±0.15)was higher than healthy control group(0.96±0.13),the discrepancy was statistically significant(t=5.477,P<0.01),and also higher than CHB+ inhibitor group(0.62±0.14),the discrepancy was statistically significant(t= 12.899,P<0.01).The expression of Bim in PBMC of CHB group(0.96±0.14)was higher than healthy control group(0.58±0.12),the discrepancy was statistically significant(t=9.133,P<0.01), and also higher than CHB+inhibitor group(0.52±0.13),the discrepancy was statistically significant(t=10.086,P< 0.01).3 The apoptotic rate of PBMC had positive correlation with the expression of p-JNK in PBMC of CHB patients(r=0.823,P<0.01).Conclusion:1 There is the phenomenon of AICD of PBMC in CHB patients,and the apoptotic rate is higher than healthy persons.2 The JNK signal pathway is excessive activated in PBMC of CHB patients.3 The apoptotic rate of PBMC had positive correlation with the expression of p-JNK in PBMC of CHB patients.The expression of p-JNK and its downstream transcription factor p-c-jun and downstream protein Bim in PBMC of CHB patients and the apoptotic rate of PBMC were decreased after JNK was inhibited with the JNK specific inhibitor SP600125.This shows that JNK signal pathway is involved in mediating AICD of PBMC in CHB patients,and is one of the mechanisms of increased PBMC apoptosis in CHB patients.更多还原