【作者】 石晓东；

【导师】 牛俊奇；

【作者基本信息】 吉林大学 ， 内科学， 2011， 博士

【摘要】 乙型和丙型病毒性肝炎是两种呈世界性分布的传染性疾病,是急慢性肝病的主要原因。乙肝感染后会造成广泛的疾病谱,包括无症状的携带者,潜伏性肝炎、急性肝炎、慢性肝炎、爆发性肝炎,甚至肝硬化、原发性肝细胞癌。而丙型肝炎病毒(HCV)感染后,大约20%-30%感染者会发生自发性清除(Spontaneous Clearance),但70%-80%将会持续感染。在抗病毒治疗过程中,不同个体对干扰素治疗的应答有很大差异,约70%的病人经长效干扰素联合利巴韦林治疗一年后,可获得持久的病毒学应答(Sustained Virologic Response, SVR);而剩余30%患者对该治疗表现为无应答(No Response)。这两种肝炎病毒感染后,疾病谱多样化,严重程度各有不同,对治疗的反应也大相径庭。大家普遍认为的病毒、环境、种族和患者的临床表现可能与疾病谱的多样化有关系,但尚不能完全解释疾病自然转归不同的真正原因。目前研究表明,宿主基因的变异是最重要的影响因素。尤其是近年来,国内外关于宿主遗传基因多态性与HCV感染转归的研究成果不断涌现,相继报道了诸如人类白细胞抗原、炎症趋化因子类,肿瘤坏死因子α(TNF-α)白介素和干扰素刺激基因等多个基因的多态性与HCV、HBV感染转归相关,都证实了宿主遗传基因变异可能导致机体免疫功能状态的差异,进而影响到HBV、HCV感染的不同临床结局。IL28B基因编码IFN-λ3,为Ⅲ型干扰素位于19号染色体上,包含6个外显子,属于IFN-λs家族。近期在非洲、欧洲人群中进行的全基因组关联分析(Genome-wide Association Study, GWAS)鉴定发现编码IFNλ3的IL28B基因附近有一些单核苷酸多态位点与HCV病毒自发清除能力及对干扰素的应答有关,所以我们推测IL28B基因的变异及其表达可能与HCV、HBV的感染密切相关。本文从宿主遗传易感性角度,借助国际最新关于慢性丙型肝炎宿主基因多态性的研究结果的启示,分别对丙肝感染者和乙肝感染者进行了IL28B rs12979860基因多态性及其表达水平的研究,同时对一部分丙肝患者进行了抗病毒治疗。收集我省汉族人口丙肝人群血样共896例(其中235例慢性丙肝患者参加抗病毒治疗),乙肝人群156例,所选人群符合Hardy-Weinberg平衡,通过焦磷酸测序法,分别对上述人群的IL28B SNP(rsl2979860)多态性位点(C/T)进行检测,应用ELISA试剂盒检测相关组个体血清中IL28B含量；应用Real Time PCR检测外周血单个核细胞(PBMC)中IL28B mRNA的表达情况。同时分析并评价不同基因型人群抗病毒治疗的应答情况。结果发现,在丙肝人群研究中,中国健康人群中rs12979860 CC基因型频率为90.1%。自发清除组的C等位基因频率(rs12979860)高于于慢性感染组患者(P=0.04)。多变量Logistic回归分析,调整了性别和HBV共感染后,rs12979860基因多态性仍然是HCV自发清除的独立影响因素。在携带CC基因型患者中,HCV的自发清除率是T/C和T/T基因型患者的2倍(OR=2.13,P=0.04)。感染了HCV基因型1b型患者中,C/C基因型患者中HCV载量明显高于TC/TT基因型患者(P=0.014)。与C/C基因型患者相比,T/C或T/T基因型患者的转氨酶水平增高,差异显著。我们检测了HCV感染后不同临床转归的患者和健康人的IL28B血清水平以及其mRNA的表达水平,发现慢性HCV感染的患者中IL28B血清学和mRNA水平明显低于自发清除组和健康对照组人群。rs12979860 C/C基因型人群IL28B血清水平和IL28的mRNA表达水平都比携带T等位基因的人群稍高。在中国慢性丙肝患者的抗病毒治疗过程中,IL28B CC基因型的患者更可能获得病毒学应答,尤其是cEVR,多因素分析亦证实IL28B基因多态性是丙肝患者治疗成功与否的一项重要的预测因子。在乙肝人群的研究中,我们共检测了156名HBV感染者和健康对照者,检测各组间IL28B血清学水平和IL28mRNA的表达水平,发现HBV携带组和健康对照组人群的IL28B无论是血清学还是mRNA的表达都明显高于其它各组(肝炎、肝硬化和肝癌组)(所有的P<0.05)。IL28B血清学水平在C/C基因型中的表达高于T/C基因型人群(P<0.05)。而在肝硬化+肝癌患者中,高转氨酶(ALT/AST)患者的IL28B无论在血清学还是mRNA水平都明显高于低转氨酶水平的患者(P<0.05)总之,我们的结果表明,在中国人群中,IL28B上游的rs12979860 SNP及IL28B的表达不仅与HCV感染后的临床转归有关。而且也与HBV感染后的慢性化及疾病进展有关,同时也对慢性丙肝患者的抗病毒应答与否发挥重要作用。进一步研究也表明携带IL28B保护性等位基因能够引起IL28B表达水平的升高,IL28B在mRNA水平和血清学水平的表达两方面都证实,宿主IL28B基因多态性可能直接影响了IL28B的表达,而IL28B的表达水平及其功能变化则对HCV和HBV感染后的临床转归的进程中发挥重大作用,同时机体转氨酶水平增高,免疫应答反应增强的患者中,可能IL28B也积极的参与其中。可见,无论在HCV还是HBV感染的患者人群中,IL28B SNP位点变异及其表达水平改变可能是控制HCV、HBV复制的有益因素,IL28B基因多态性及其表达与疾病进展过程密不可分。更多还原

【Abstract】 Hepatitis B and C are two infectious diseases in worldwide,which are the main reason of acute and chronic liver disease. HBV infection can cause a wide range of disease spectrum, including asymptomatic carriers, latent hepatitis, acute hepatitis, chronic hepatitis, fulminant hepatitis, and even cirrhosis, primary hepatocellular carcinoma. The hepatitis C virus (HCV) infection, approximately 20% -30% will clear the virus through spontaneous clearance, but 70% -80% will be persistent infection. In the course of antiviral treatment, the response to interferon therapy of HCV infection patients are very different, after Peg-interferon and ribavirin treatment, about 70% of the patients receive sustained virological response (SVR); while the remaining 30% of the patients showed no response to the treatment (No Response).After infected by both hepatitis viruses, there are diversity, different severity of disease, and different response to therapy. We have a common view that the virus, environment, race and clinical manifestations of patients may be related with diversity of disease, but it can not fully explain the real reason of the natural outcome of the disease. Present study shows that the host gene mutation is the most important factor. Especially in recent years, the results of research on host genetic polymorphisms and outcome of HCV infection continue to emerge, which have been reported, such as human leukocyte antigen, inflammatory chemokines, tumor necrosis factorα(TNF-α),interleukin and interferon stimulated genes,that gene polymorphisms related to HCV, HBV infection outcomes.It have confirmed the host genetic variation may lead to differences in the body immune function, further influencing different clinical outcomes of HBV, HCV infection.IL28B gene located on chromosome 19 interferon, consists of 6 exons, encoding IFN-λ3, as typeⅢ,are IFN-λs family. Recently Genome-wide Association Study(GWAS) of Africa, Europe population show that some single nucleotide polymorphisms near the IL28B coding IFNλ3 is related with spontaneous clearance capabilities of HCV viral and Response to interferon, so we speculated that the mutation and expression of IL28B gene may be associated closely with HCV, HBV infection.From the perspective of host genetic susceptibility, with the enlightenment from the latest international host of chronic hepatitis C gene polymorphism results, We studied the L28B rs12979860 polymorphism and its expression level in HBV and HCV patients respectively, meanwhile, some chronic HCV infection patients accepted antviral therapy. We collected blood samples of Jilin province among the Han population, including 896 subjects of hepatitis C (among of them,235 chronic HCV infection patients accepted treatment),156 subjects of hepatitis B.the selected population accorded with Hardy-Weinberg equilibrium, these IL28B SNP (rs12979860) polymorphism (C/T) were detected respectively through the pyrosequencing method. ELISA test was used for IL28B serum concentration; the Real Time PCR was applicated for mRNA expression of IL28 of peripheral blood mononuclear cells (PBMC). At the same time, we also analysised the response status of the 235 HCV infection patients after treatment.It was found that among populations of hepatitis C, the genotype frequency of rs12979860 CC of Chinese healthy population was 90.1%. C allele frequency (rs12979860) in spontaneous clearance group was higher than persistent infection patients (P=0.04). Maultivariate logistic regression analysis found, after adjusted gender and HBV infection, rs12979860 polymorphism is still an independent factor for HCV spontaneous clearance. Among patients carrying the C/C genotype, the rate of HCV spontaneous clearance are 2 times to T/C and T/T genotype patients (OR=2.13, P=0.04).Patients infected HCV genotype lb, HCV load of patients with C/C genotype was significantly higher than T/C or T/T genotype (P=0.014). Comparing with C/C genotype patients, the patients with T/C or T/T genotype had higher ALT level, and the difference was significant. We detected the serum and mRNA expression levels of IL28B and found that in the chronic HCV infection patients, serology and IL28B mRNA levels were significantly lower than the spontaneous group and the healthy control group. In IL28B rs12979860 C/C genotype group, serum levels and mRNA expression levels of IL28B were slightly higher than the population carrying T allele.In the course of IFN/RBV treatment for Chinese chronic HCV infection patients, the patients carrying C/C genotype of IL28B could produced higher virologic response rate, especially cEVR, and multivariate logistic regression analysis also confirmed that the IL28B polymorphism is an important predictor for chronic hepatitis C patients treatment.In the population infected hepatitis B, we detected a total of 156 HBV infected patients and healthy controls, testing serum levels and mRNA expression levels of IL28B in each groups, which found serum IL28B or mRNA expression of both groups(HBV carrier and healthy groups) were significantly higher than other groups (hepatitis, cirrhosis and liver cancer groups) (all P<0.05). Serum levels of IL28B of C/C genotype was higher than T/C genotype subjects (P<0.05). In the cirrhosis + hepatocellular carcinoma (HCC) patients, serum or IL28B mRNA levels with high aminotransferase (ALT/AST) of patient are significantly higher than patients with low transaminase levels (P<0.05)In summary, our research show that in the Chinese population, the rs12979860 SNP located IL28B upstream and the expression of IL28B is not only related with clinical outcomes of HCV infection and the response of antivirologic treatment, but also with the chronic HBV infection and disease progression. Further studies have shown that carrying the protective allele of IL28B can cause elevated expression levels of IL28B. both the mRNA level and serum levels of IL28B confirmed that host gene IL28B polymorphisms may directly affect its expression, whereas expression and function of IL28B may play a major role on the clinical outcome of the process in the HCV and HBV infection, In patients with high transaminase level and strong immune responses, IL28B may also actively participate in it. So we believe that in patients both with HCV or HBV infection of the Chinese population, IL28B SNP variations and the change of the expression level may be a beneficial factor to control HCV, HBV replication. Gene polymorphism of IL28B and its expression are inseparable from disease progression.更多还原