【作者】 沈立萍；

【导师】 毕胜利； 张勇；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2009， 博士

【摘要】 乙型肝炎(以下简称乙肝)是严重威胁人类健康的传染病之一。据不同的感染时机和机体免疫状态,乙肝病毒(Hepatitis B virus,HBV)感染后可发展为恢复、无症状携带、急慢性乙肝、肝硬化和肝癌。我国乙肝表面抗原(Hepatitis B surfaceantigen,HBsAg)阳性者高达9300万,但有关乙肝相关性疾病的患病状况和自然史的研究资料十分欠缺。慢性HBV感染的主要原因是HBV特异性免疫耐受,近期研究发现PD-1/PD-L1信号通路与免疫耐受密切相关,阻断该通路能够刺激效应T细胞增殖、增强细胞毒性T细胞(Cytotoxic T lymphocyte,CTL)效应、促进病原体的清除。因此,为了解我国HBsAg携带者中乙肝相关性疾病的患病状况、HBV感染的发展和转归、探讨PD-1/PD-L1信号通路作为免疫治疗靶位的可能性和方法,本课题通过两个方面开展研究。一是调查慢性乙肝等乙肝相关性疾病的患病状况,二是研制抗-(PD-L1)单克隆抗体、应用于HBV感染小鼠模型,探讨PD-1/PD-L1信号通路作为乙肝免疫调节治疗靶位的途径和效果。一、慢性乙型肝炎患病状况的队列研究通过横断面和回顾性调查的方法在广西隆安县、河北正定县、黑龙江肇东市、青海同德县、河南漯河市收集了2743例HBsAg阳性者;2006年底开展随访调查,调查的内容是:问询填写调查表、采集静脉血标本、临床症状观察和肝部B超检查;血清用固相放射免疫法检测HBV五项感染指标,结果与既往资料进行比较分析。结果为:(1)平均HBsAg年度自然阴转率为10.68%;(2)HBsAg携带者中无症状携带者占91.50%,慢性乙肝占6.98%,肝硬化占1.09%,肝癌占0.43%;(3)平均慢性乙肝患病率为6.98%,30～50岁为高发年龄,20岁以下低年龄组人群的患病高峰消失,男性高于女性;(4)平均肝硬化患病率为10.9‰,远高于普通人群,50岁以上为高发年龄;(5)平均肝癌患病率为4.3‰,远高于普通人群,男性高于女性。调查过程中发现的慢性乙肝患者多数没有明显临床症状和个体不适感,容易被忽略。我们的调查发现HBsAg携带者的乙肝相关性疾病患病现况较为严重,推算我国慢性乙肝现患人数达到650万;定期开展检查、早发现、早治疗是提高患者生活质量和生存几率的重要措施。二、PD-1/PD-L1信号通路作为乙肝免疫治疗靶位的研究第二部分的研究根据实施程序分为四个单元来:首先进行PD-L1蛋白的表达、纯化和生物学活性分析;然后利用杂交瘤技术、以PD-L1重组蛋白为抗原制备单克隆抗体;进一步将纯化的抗-(PD-L1)单抗应用于HBV慢性感染小鼠模型,观察其阻断PD-1/PD-L1信号通路、促进HBV清除的功效。(一)PD-L1重组蛋白的表达纯化根据Gene bank的人源PD-L mRNA基因序列,通过密码子优化、全基因序列合成PD-L1的目的基因片段,成功构建了硫氧还原蛋白-Pet-43b/(PD-L1)重组表达质粒,在BL21(DE3)系统中进行表达,分析表达产物,发现目的蛋白主要以包涵体的形式存在,分子量约为47KD;表达量约占总菌体蛋白的20%;大量表达并用二步法进行纯化后得到纯度为85%、浓度为0.4mg/ml的PD-L1重组蛋白。(二)PD-L1重组蛋白的生物学活性分析根据PD-L1与其受体PD-1能够特异性稳定结合的原理,我们用辣根过氧化物酶(HRP)标记本室前期表达的PD-1蛋白,用PD-L1重组蛋白包被于96孔板,以牛血清白蛋白为对照,进行ELISA方阵滴定法实验,结果证明PD-L1重组蛋白能够与其受体PD-1特异性结合,结合的最佳配比为酶标物稀释度1:400,包被物浓度为4～6μg/ml。进一步利用BIA core分析系统,以PD-1为固定相,PD-L1为流动相分析其特异性结合能力,结果证明PD-L1重组蛋白能与PD-1结合,亲和常数为RU=129,本研究表达的PD-L1重组蛋白有良好的生物学活性。(三)PD-L1单克隆抗体的制备以PD-L1重组蛋白为抗原免疫BALB/c小鼠、利用杂交瘤技术制备单克隆抗体。经ELISA竞争抑制实验和Western-blot印迹实验验证本次制备获得了五株能特异性分泌抗-(PD-L1)抗体的细胞株,编号为5812,5C7,5F12,6A12,6F1;接着将5812,5C7,5F12三株细胞注射到小鼠腹腔进行腹水制备,得到的腹水进行纯化,获得了浓度为1.8mg/ml抗-(PD-L1)纯化抗体。(四)抗-(PD-L1)单克隆抗体应用于HBV感染小鼠模型据水动力注射法的原理,将AAV/HBV DNA 1.2质粒经尾静脉注射到C57BL/6小鼠体内构建HBV慢性感染模型,以抗-(PD-L1)纯化抗体为免疫调节治疗药物经尾静脉、分两次注射到HBV慢性感染模型体内,观察小鼠的HBV感染血清学指标,同时设置对照组。结果显示:(1)第7天对照组的HBV DNA拷贝数不低于1×10~6,模型构建是成功的;(2)给药组的HBV DNA总体显著低于对照组,给药后第7天的HBV DNA拷贝数显著低于对照组,第28天的HBVDNA清除率显著高于对照组;(3)给药组的HBsAg值总体显著低于对照组,第7、14天的HBsAg平均值显著低于对照组;(4)给药组第28天抗-HBs的量低于对照组。本实验结果可以说明抗-(PD-L1)纯化抗体作为免疫调节剂药物应用于HBV感染小鼠模型,能有效促进病毒的清除。总之,从流行病学方面,本研究调查分析了我国慢性肝炎等乙肝相关性疾病的患病现况,为乙肝流行病学防控和临床诊疗提供参考,建立了HBV感染者随访队列,为下一步HBV感染转归和发展研究打下基础;从免疫学方面,本研究成功地通过原核系统表达出PD-L1重组蛋白并研制了单克隆抗体、应用于HBV感染小鼠模型,结果证明抗-(PD-L1)抗体能够阻断PD-1/PD-L1信号通路,促进HBV的清除,说明PD-1/PD-L1信号通路是有效治疗慢性HBV感染的靶位,抗-(PD-L1)作为免疫调节治疗药物有良好的前景。更多还原

【Abstract】 Hepatitis B virus infection is the serious public health problem.The number of HBsAg carriers is 93 million in China.HBV infection will develop to recovery;asymptomatic HBsAg carrier;chronic hepatitis B（CHB）;acute hepatitis B;cirrhosis and hepatocellular carcinoma（HCC）.However,the information and knowledge about mechanistic of HBV infection and natural history are lacking in China recently.Many studies reported that immune tolerance specialy for HBV is the main course of chronic infection.The PD-1/PD-L1 signal pass way is highly relevant with immune tolerance and blocking this pass way could restore the function of exhausted T cells;improve the ability of viral clearance.Hence,in order to know the prevalence status of HBV relative disease and study the possibility and method about immunity therapy by blocking PD-1/PD-L1 pass way,we do two parts work as follows.1.Cohort study to investigate the prevalence rate status of CHBBy means of cross-section and review investigation,we collected 2743 HBsAg positive cases in Longan-Guangxi;Zhengding-Hebei;Zhaodong-Heilongjiang;Tongde-Qinghai and Luohe-Henan five regions.We carried out the follow survey in 2006 for these cases. The results of survey include:（1） Average HBsAg yearly natural seroconversion rate is 10.68%;（2） In 758 HBsAg carriers,prevalence rate of asymptomatic HBsAg carrier is 91.50%,CHB is 6.98%,cirrhosis is 1.09%,HCC is 4.3‰;（3） Highly age group of CHB prevalence is 30～,49～;male’s is higher than female’s.（4） Prevalence rate of cirrhosis and HCC are higher than normal people.According to those data,we could calculate that the CHB number in China is about 6.5 million.Most of them have been ignoring because of its mild symptoms. 2.The study about PD-1/PD-L1 signal passway to be the immunotherapy target for hepatitis B.（1）Express and purify the PD-L1 recombination proteinAccording to analysis result of the human source PD-L1 mRNA gene sequence,by means of codon optimized and synthesized the whole gene sequence of PD-L1.We constructed the thioredoxin-（PD-L1） recombination expression plasmid successfully and expressed the fusion protein in E.coli.The molecular weight of target protein was 47KD. After purification we obtain the recombination PD-L1 protein with purity is 85%,density is 0.4mg/ml.So we have obtained the PD-L1 recombinant protein.The result proved the basic condition for further study on antibody and mutually action between PD-1/PD-L1 and chronic virus infectious.（2）Analyze the bioactivityon of PD-L1 recombination proteinAccording the principle that PD-L1 could combine with its receptor PD-1 specially,we conjugated the HRP on the PD-1,coated PD-L1 on 96 wells plate and carried out ELISA direct combine assay.The result proved that PD-L1 can combine with PD-1 specially.The best proportion of PD-L1 is 4～6μg/ml,HRP（PD-1） is 1:400.Biomolecular interaction analysis（BIA） assay system also proved the special combination of PD-1/PD-L1.The affinity is 129 resonance units（RU）.The result shows us that PD-L1 recombination protein has excellent bioactivity.（3） Prepare the monoclonal antibodies against PD-L1 recombination protein.BALB/c mice was immunized with purified PD-L1 protein and its spleen cells were fused with SP 2/0 cell.After identified by ELISA competitive inhibition assay and western-blot,5 strains monoclonal antibody were selected.The serial number of five strains are 5B12,5C7,5F12,6A12,6F1.We took 5B12,5C7,5F12 as samples to prepare ascites.The cells were injected into BALB/c mice’s abdominal cavity.Ascites were collected,identified and purified,we obtain purity anti-PD-L1 antibody with its density is 1.8mg/ml.（4）Block PD-1/PD-L1 pass way by anti-PD-L1 and analyze the effect of HBV clearance.Plasmid named AAV/HBV DNA1.2 was injected into C57BL/C mice by t way of tail vein to construct HBV infection model.Anti-PD-L1 was injected into HBV infection model and observe the HBV DNA copies and HBV infectious markers.The result are（1） Average copies of HBV DNA of control group are not less than 1×10⁶ at 7th days.（2） the whole HBV DNA value of treat group is lower than control group significantly,the 28^th day’s HBV DNA clearance rate of treat group is high than control group significantly;（3） whole value of HBsAg of treat group are less than control group,the 7^th’s,14^th day’s HBsAg of treat group are less than control group;（4） The 28^th day’s the anti-HBs of treat group is lower than control group.We could make a conclusion that anti-（PD-L1） antibody can block the PD-1/PD-Llpass way and enhance the effect of HBV clearance.In short,our study investigated and analyzed the prevalence of HBV relative disease such as CHB,et al;established the HBV infection survey cohort.We also expressed recombination PD-L1 protein and prepared the anti-PD-L1 antibody,Used the antibody as immune-medicine on HBV infective mice model.The results show us that anti-PD-L1 can block the signal passway.PD-1/PD-L1 signal passway is the possible therapy target of HBV chronic infection.Anti-PD-L1 should be prospective as being immune-medicine for HBV infection.更多还原