江苏省海门市肝癌高发地区人群乙型肝炎病毒进化分析

【作者】 王静霓；

【导师】 姜庆五； 郑英杰；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2010， 硕士

【摘要】 江苏省海门市地处长江三角洲地区,是原发性肝细胞癌的高发市。HBV感染是海门市肝癌高发的最重要危险因素,应在肝癌防治决策时予以充分重视。在同一生存环境条件下,HBV感染可促使肝癌发病年龄提前。乙型肝炎病毒是一种DNA病毒,有四个重叠的开放读码框架(S、C、P和X)。S基因的变异可改变HBsAg的空间结构和免疫原性,导致其抗原性和蛋白表达水平下降。C基因变异将影响HBV病毒载量及HCC进程,其主要突变是nt 1896 G→A,使HBeAg合成终止。HBV基本核心启动子中最重要、最常见的基因突变是nt 1762 A→T、nt 1764 G→A双突变,既增强HBV复制力,又降低HBeAg合成,还增加肝细胞凋亡,从而加重患者病情。P基因变异以YMDD基序的变异(aa M552V或aa M552I,生成YVDD或YIDD)最为重要,YMDD变异伴“a”决定簇内变异时可使HBV的复制能力明显增强。目前发现的HBV已有A-J共10种基因型,我国主要流行的是B和C基因型。HBV基因型与感染的预后有关,与B基因型相比,C基因型HBV与肝癌的发病更加密切,也会导致更严重的肝纤维化结局。而不同基因型混合感染可引起携带者肝炎复燃。本研究采用多阶段随机抽样,在充分考虑抽样可行性和科学性的基础上对海门市一般人群进行乙肝血清学调查,包括一般情况,肝炎患病史,乙肝病毒暴露史,乙肝疫苗免疫接种史等。采用ELISA检测方法定性检测HBsAg及半定量检测抗-HBs,对HBsAg阳性血清抽提DNA,并用巢氏PCR扩增HBV S基因及LA-PCR扩增HBV DNA,从而掌握当地HBV的流行规律、变异及主要基因型。本项目调查对象共计5,624人,HBsAg阳性率为6.035%。不同性别人群间的HBsAg阳性率不存在统计学差异。<30岁人群HBsAg阳性率明显低于≥30岁人群HBsAg阳性率,10岁以下儿童HBsAg阳性率为0%。文化程度越高,HBsAg阳性率越低。医护人员的HBsAg阳性率最低,农民的HBsAg阳性率最高。长期一起生活的直系血亲中有乙肝患者人群的HBsAg阳性率远高于直系血亲中没有乙肝患者人群的HBsAg阳性率。做过手术和做过内窥镜人群的HBsAg阳性率分别高于没有做过的人群的HBsAg阳性率。献血人群的HBsAg阳性率低于没有献血人群的HBsAg阳性率。不同出生地点调查对象的HBsAg阳性率存在统计学差异,表现为：出生于县级以上医院者<出生于乡镇级医院者<出生于家中者。Logistic回归分析显示,乙肝感染相关因素中,长期一起生活的亲人中患有乙肝、做过内窥镜是调查对象乙肝感染的危险因素,而献血是调查对象乙肝感染的保护因素。后者可能是健康工人偏倚所致。抗-HBs滴度＜10IU/ml者占43.334%；抗-HBs滴度＞10IU/ml人群中,抗-HBs滴度越高所占构成比越高。HBsAg阴性人群与一般人群的抗-HBs滴度分布比例相似；HBsAg阳性人群中,抗-HBs滴度＜10 IU/ml者所占比例为90.845%,抗-HBs滴度位于(10,160]IU/ml者所占比例均低于1.5%,但是抗-HBs滴度>160IU/ml者所占比例为5.282%。不同性别人群间抗-HBs滴度的分布比例基本相同。年龄越低,抗-HBs滴度＜10IU/ml者占该年龄段人群比例越低。抗-HBs滴度＞10IU/ml人群中,小于10岁人群里位于(40,80]IU/ml者所占比例最低；[10,60)岁人群抗-HBs滴度分布与一般人群抗-HBs滴度分布相似；60岁以上人群各个抗-HBs滴度人群所占比例基本相同。不同文化程度和职业人群的抗-HBs滴度分布近似于该年龄段人群的抗-HBs滴度分布。是否做过内窥镜(做过内窥镜者的抗-HBs滴度较低)和不同的出生地点(出生于县级以上医院>乡镇级医院>在家出生者的抗-HBs滴度)对人群的抗-HBs滴度分布比例有影响。Logistic回归分析显示,乙肝感染相关因素中,是否做过内窥镜是抗-HBs是否具有保护作用的危险因素。乙肝疫苗计划免疫前出生人群的HBsAg阳性率远高于乙肝疫苗计划免疫后出生人群的HBsAg阳性率,抗-HBs平均滴度则表现相反。曾经在村医或个体诊所做过牙科诊疗、与家人共用牙刷和在乡镇及乡镇级以上医院出生的人群中,乙肝疫苗计划免疫前出生人群HBsAg阳性率分别高于乙肝疫苗计划免疫后出生人群的HBsAg阳性率,抗-HBs平均滴度则表现相反。巢式PCR扩增HBV S基因检测阳性率为45.8%。B基因型占10.8%,C基因型占86.9%,B/C混合型占2.3%。C基因型HBV S基因(特别在亲水区(nt245-nt391))的变异度普遍高于B基因型。相同基因型的不同患病类型乙肝患者的HBVS基因之间并无明显分隔,而B基因型HBV S基因之间同源性更高。所有可能导致HBV的抗原性和免疫应答发生改变的样本均为C基因型,占5.38%,平均年龄为46.71±1.17岁,抗-HBs均小于10 IU/ml。海门市乙肝HBsAg主要为adrq+血清型(82.3%),其次为adw2血清型(16.2%)。LA-PCR扩增HBV DNA检测阳性率为15.4%,DNA在1814 nt-2452 nt区域相对比较保守。4例样本pre-C基因发生nt 1896 G→A单个碱基替换。B基因型HBV BCP并未发生双重突变(nt 1762 A→T和nt 1764 G→A); C基因型HBVBCP中,发生双重突变(nt 1762 A→T和nt 1764 G→A)的占全部C基因型的47.06%，仅发生nt 1762 A→T突变者占5.88%,仅发生nt 1764 G→A突变者占11.76%。海门市与黑龙江和上海的HBV平均进化距离差距最小,其次为北京、安徽、启东、台湾和西藏。海门市HBV平均进化距离与中国其他地区HBV平均进化距离的差距主要出于基因型分布的差异所致。就C基因型HBV DNA而言,海门株之间的进化距离比较大,也大于某些地区株与海门株之间的进化距离。海门株与本地区及其他各个地区株之间的pre-C/C基因、C基因和X基因进化距离普遍小于同样地区之间的HBV DNA进化距离,而P基因的进化距离则普遍大于同样地区之间的HBV DNA进化距离。与其他地区相比,海门株之间的pre-S1/pre-S2区域序列变异程度较大,同源性较低。尽管海门市为HCC高发区,但是从海门市一般人群中筛选出的HBV DNA与其他地区的HCC患者的HBV DNA之间的同源性并不高,反而与北京等地区的慢性HBV患者的HBV DNA之间的同源性较高。经过近二十年的乙肝疫苗计划免疫工作,海门市HBV的预防工作卓有成效,HBV感染情况已经得到明显好转。青少年的HBV感染率并未随着年龄的增长而有所上升；而随着当地成年人年龄的增长,乙肝患者体内HBV可能会被清除,或是由于HBV S基因变异所致的HBsAg假阴性。海门市HBV存在家庭内或家族聚集感染,可能是垂直传播,遗传因素,水平传播综合作用的结果；从病毒学方面看,HBV家族聚集性感染和非家族聚集感染临床类型差异不大。海门市HBV也通过内窥镜检查进行传播。仅出生地点的不同对HBV感染有影响,说明出生时所接受到的医疗水平程度对HBV感染有影响。乙肝疫苗计划免疫对于中低教育程度和中低收入家庭来说,是预防HBV感染的主要途径。乡镇及乡镇以下级别医院的牙科诊疗可能是海门市HBV社会传播的途径之一,家庭内共用牙刷可能也是海门市HBV家庭内传播的途径之一综上所述,海门市一般人群HBV感染的主要影响因素为：年龄、受教育程度、直系血亲中是否有人患有乙肝、是否做过内窥镜、出生地点和乙肝疫苗计划免疫。年龄、受教育程度和出生地点主要影响乙肝疫苗的免疫接种,而海门市HBV感染主要来源于垂直传播途径和通过内窥镜、牙科诊疗和家庭内共用牙刷等水平传播途径。乙肝疫苗的及时接种是阻止海门市HBV传播的最有效的方法。海门市HBV S基因已经出现了能改变HBV抗原性和免疫原性的变异,应当密切关注以防止乙肝疫苗失效。海门市作为HCC高发区,当地HBV DNA与其他非HCC高发区HBV DNA(无论是否来源于HCC患者)的差异并不显著,X基因的差异甚至更小,提示尽管血清HBV病毒水平是HCC发生独立的预期因素,且慢性乙型肝炎炎症应答过程中逐渐累积的基因改变是HCC发生的最重要因素,但HBV的变异程度并不是海门市HCC高发的直接原因,而可能是HCC其它直接危险因素的促进因素。本研究受实验条件所限制,并未测量HBsAg阳性标本血清中的HBV DNA水平,可以在后续研究中进行近一步分析及探讨。更多还原

【Abstract】 Haimen City, Jiangsu Province, located in the Yangtze River Delta, is the high incidence city of primary hepatocellular carcinoma (HCC). HBV infection, the most important risk factors and promotion condition in younger while living in the same environmental, should be full attention to decision-making in the HCC control.As a DNA virus, there are four overlapping open reading frames (S, C, P and X) in Hepatitis B virus. S gene mutation changes the HBsAg spatial structure and immunogenicity, leading to low levels of antigenicity and protein. C gene mutation, mainly nt 1896 G→A for termination of HBeAg synthesis, will affect viral load and the process of HCC. The most important and common mutation in Basic core promoter is nt 1762 A→T, nt 1764 G→A double mutation,which not only enhanced HBV replication ability, but also reduce HBeAg synthesis as well as increased hepatocyte apoptosis, thereby increasing the patient condition. YMDD mutation (aa M552V or aa M552Ⅰ, generate YVDD or YIDD) is the most important mutation in P gene. HBV replication is obviously enhanced due to YMDD mutation with "a" determinant.Currently there are 10 HBV genotypes in the world. The popular genotypes in China are B and C. HBV genotype is connected with prognosis of HBV infection. Compared with genotype B, genotype C is more closely with HCC, and will lead to more severe liver fibrosis. The mixed infection of different genotypes of HBV can cause resurgence.We carried out the hepatitis B serological surveys in the general population of Haimen City with multi-stage random sampling, including general conditions, history of liver diseases, exposure history of hepatitis B virus, and vaccination history for hepatitis B. After HBsAg qualitative detection and semi-quantitative detection of anti-HBs by ELISA, extraction of HBV DNA, Nested PCR amplification of HBV S gene and the LA-PCR amplification of HBV DNA, we grasp the prevalence, the variation and the major genotype of HBV in Haimen.HBsAg positive rate was 6.035% in the totally investigatory population of 5,624. The HBsAg-positive rate between two gender is not statistics different.<30 HBsAg-positive rate in population at aged<30 years was significantly lower than aged≥30 years of age, and HBsAg-positive rate of children under the age of 10 was 0. The higher educational level, HBsAg-positive rate is lower. The lowest rate of HBsAg-positive was in health care workers, but the highest was in peasants. The HBsAg-positive rate in the groups of consanguinities who were hepatitis B patients was significantly higher than the groups of consanguinities without hepatitis B virus. The HBsAg-positive rate in surgery and endoscopy groups was significantly higher than the groups without surgery and endoscopy. The HBsAg positive rate among blood donors is lower than the non-blood donors. The HBsAg-positive rate among the different place of birth was significantly different, as follows: who was born in hospitals above county level< who was born in the township-level hospitals<who was born at home. logistic regression analysis showed that the risk factors of hepatitis B infection include living with consanguinities who were hepatitis B patients and having endoscopy, but donating blood is a protective factor due to healthy worker bias.Whose Anti-HBs titers< 10 IU/ml were accounted for 43.334% in totally investigatory population; in anti-HBs titer of> 1OIU/ml population, however, the groups with higher anti-HBs titers is larger than the groups with lower anti-HBs titers. The distribution of anti-HBs titer in HBsAg negative population was same as the general population. In HBsAg-positive population, whose anti-HBs titers<10 IU/ml were accounted for 90.845%; whose anti-HBs titers in (10,160] IU/ml is lower than 1.5%; but whose anti-HBs titers> 160IU/mlwere accounted for 5.282%. The distribution of anti-HBs titer between two genders was similar. In anti-HBs titer<10 IU/ml population, the percentage of younger is gradully lower than elder. In anti-HBs titer> 10 IU/ml population, children below age of 10 whose located in (40,80] IU/ml was accounted for the lowest percentage; the distribution of anti-HBs titer in [10,60) years population was same as the general population; the percentage of different anti-HBs titer in people over the age of 60 was similar. The anti-HBs titer distribution in differently educational and occupational groups was similar to the age groups. Endoscopy done groups had lower anti-HBs titers. The distribution of anti-HBs titer can be impacted by different place of birth (born in or above the county level hospitals> born in township-level hospitals> born at home). Logistic regression analysis showed that endoscopy was the risk factors of anti-HBs protective effects.HBsAg-positive rate of population, especially in those who having done dental in the village doctor or individual clinics, sharing a toothbrush with family members and born in and above township level hospitals, who were born before the era of planned immunization was much higher than after the era of planned immunization, but the average anti-HBs titer was performance of the contrary. Amplified by nested PCR, test positive percentage for HBV S gene was 45.8%. B genotype was accounted for 10.8%, C genotype was accounted for 86.9%, and B/ C mixed genotype was accounted for 2.3%. The variability of genotype C HBV S gene, particularly in the hydrophilic region (nt 245-nt 391), was generally higher than genotype B. HBV S gene among different disease patients with the same genotype was no significant separation, and genotype B HBV S gene was higher homology. All that may lead to HBV antigens and immune response changes, accounting for 5.38%, were genotype C, which average age was 46.71±1.17 years and anti-HBs titer was less than 10 Ul/1. All Haimen’s HBV samples were mainly adrq+ HBsAg serotypes (accounted for 82.3%), followed by adw2 serotypes (accounted for 16.2%).Amplified by LA-PCR, test positive percentage for HBV DNA was 15.4%, and the nt 1814-nt 2452 region is relatively conservative.4 cases of pre-C gene occurred nt 1896 G→A single base substitutions. BCP double mutations did not occur (nt1762A→T and nt1764G→A) in genotype B; in genotype C, however, BCP double mutations (ntl762A→T and ntl764G→A) was accounted for 47.06%, where as only ntl762A→T mutation and only nt1764G→A mutation was accounted for 5.88% and 11.76%, respectively. The average evolutionary distance of HBV DNA among Haimen, Shanghai and Heilongjiang was the smallest gap, followed by Beijing, Anhui, Qidong, Taiwan and Tibet, which is mainly due to different genotype distribution.On the genotype C HBV DNA, the evolutionary distance among Haimen strains was relatively large, even much greater than the distance between Haimen and some areas’strains. The distance on the pre-C/C gene, C gene and X gene, in the opposite of P gene, in Haimen strains and between Haimen and other areas’ strains were lower than the HBV DNA among the same areas’ strains. Compared with other regions, the pre-S1/pre-S2 gene in Haimen strains had grester variation. Despite of the high HCC incidence in Haimen, the evolutionary distance on HBV DNA between Haimen strains from the general population and other areas’ strains from HCC patients is larger than those between Haimen and Beijing strains from HB patiants.After nearly two decades of hepatitis B vaccine program immunization, HBV prevention in Haimen City is effective and HBV infection has been significantly decreased. HBV infection rate of young people is increased with growing up; and in the local adult, HBV hepatitis B patients is likely to be removed with aging, or false negative HBsAg caused by HBV S gene mutation. There is HBV infection within the family or family gathering in Haimen, which may be the combined result of vertical transmission, genetic factors and horizontal transmission. But there is non-significant difference of clinical types between familial and non-familial aggregation in virology. Places of birth can influence on HBV infection, indicating that the level of medical received at birth can imapct on HBV infection. For the low education and income families, Hepatitis B vaccine immunization is the main way to prevent HBV infection. HBV is spread through not only some ways of social transmission, such as the endoscope and dental in and below township-level hospitals, but the ways of family transmission by sharing a toothbrush as well. In summary, the main factors of HBV infection in the general Haimen population include:age, educational level, consanguinities who were hepatitis B patients, endoscopy, place of birth and hepatitis B vaccine immunization. Age, educational level and place of birth mainly effect hepatitis B vaccine immunization, and Haimen HBV infection is mainly from vertical transmission and horizontal transmission by endoscopy, dental and sharing a toothbrush in family. Hepatitis B vaccine in the right time is the best way to prevent the spread of HBV in Haimen.The variation of HBV S gene in Haimen, which can changes in antigenicity and immunogenicity, should be closely watched to prevent hepatitis B vaccine failure. As the high-incidence HCC area, the distance of HBV DNA, as well as X gene, was not significant between Haimen and other low-incidence HCC areas, regardless whether from HCC patients, which suggesting that variation of HBV is not the direct cause of high-incidence HCC, but maybe the simulating factor to other direct factors in Haimen, although the viral load of serum HBV is independent expected factors and genetic changes during chronic hepatitis B inflammatory response is the most important factor in HCC occurred.Limited by the experimental conditions, this study did not measure HBV load in HBsAg positive samples, which need to be carried out taking a step forward in follow-up studies.更多还原