發(fā)布時(shí)間：2018-05-26 18:25

  本文選題：丙型肝炎病毒 + 基因型　； 參考：《南京醫(yī)科大學(xué)》2014年碩士論文

【摘要】：丙型肝炎病毒(Hepatitis C Virus, HCV)感染呈全球流行,據(jù)世界衛(wèi)生組織(WHO)估計(jì),全世界約有3%的人口(即1.7億)感染HCV。目前,中國(guó)自然人群感染率約在0.9%-5.1%,平均約為3.2%,且呈增長(zhǎng)趨勢(shì)。HCV感染所致的丙型肝炎慢性化率高達(dá)75%～85%,可導(dǎo)致肝臟慢性炎癥壞死和纖維化,部分患者可發(fā)展為肝硬化甚至肝細(xì)胞癌,對(duì)人類的健康和生命構(gòu)成了嚴(yán)重威脅。HCV為單股正鏈RNA病毒且屬于黃病毒家族成員。其基因組全長(zhǎng)約9.5kb,分為5’非編碼區(qū)(5'untranslated region,5'UTR)、編碼區(qū)以及3’非編碼區(qū)(3'UTR),其中5'UTR區(qū)、編碼區(qū)中的核心區(qū)最保守,E1區(qū)、E2區(qū)最易變異。HCV高度變異導(dǎo)致其基因型和亞型的在全球呈區(qū)域性分布。我國(guó)大部分地區(qū)流行的HCV型別為1b型,其次為2a型,在南方城市1b型感染率占90%以上,從南向北2a型逐漸增多。HCV不同基因型、亞型與HCV感染者臨床癥狀的嚴(yán)重程度、疾病的轉(zhuǎn)歸以及HCV的感染途徑、地區(qū)分布規(guī)律等具有高度的相關(guān)性。此外,HCV基因型也是影響丙型肝炎患者干擾素治療效果的重要因素之一。 HCV感染時(shí),體液免疫在抗病毒感染中作用較小,而CD4+T淋巴細(xì)胞反應(yīng)起很重要的作用。人類白細(xì)胞抗原(]human leukocyte antigen, HLA) Ⅱ類抗原是由人類主要組織相容性復(fù)合體(najor histocompatibility complex, MHC)中II類基因編碼的HLA蛋白分子,主要分布于B淋巴細(xì)胞、巨噬細(xì)胞和活化的T淋巴細(xì)胞表面,其功能是將外源性抗原肽遞呈給CD4+T淋巴細(xì)胞。因此,HLA-Ⅱ類基因的高度多態(tài)性是決定宿主免疫應(yīng)答能力的最重要的遺傳因素。HLA-Ⅱ類抗原基因主要包括HLA-DR、DQ和DP三個(gè)亞區(qū),每個(gè)亞區(qū)的基因結(jié)構(gòu)都呈高度多態(tài)性。HLA-DP分子是MHCⅡ類抗原,為人類的主要組織相容性復(fù)合體中一種蛋白質(zhì)/肽抗原受體,HLA-DP在抗原提呈和免疫應(yīng)答中發(fā)揮著重要作用,HLA-DP分子在宿主清除病毒過程中發(fā)揮重要的作用,提示HLA-DP基因型可能在HCV感染的慢性化過程中扮演重要角色。 本研究擬以HLA-DP基因多態(tài)性為切入點(diǎn),進(jìn)一步采用病例-對(duì)照研究在中國(guó)HCV高危人群中,探討不同感染途徑丙型肝炎患者HCV基因型的分布和HLA-DP基因多態(tài)性與HCV感染及其轉(zhuǎn)歸的關(guān)系。 第一部分 江蘇地區(qū)不同感染途徑丙型肝炎患者HCV基因分型的研究 丙型肝炎病毒(Hepatitis C Virus, HCV)為單股正鏈RNA,其基因組分為5’非編碼區(qū)(5'untranslated region,5'UTR)、編碼區(qū)以及3’非編碼區(qū)(3UTR),其中5'UTR區(qū)、編碼區(qū)中的核心區(qū)最保守,E1區(qū)、E2區(qū)最易變異。HCV高度變異導(dǎo)致其基因型和亞型在全球呈區(qū)域性分布。HCV基因型對(duì)HCV感染的流行病學(xué)研究具有重要意義。此外,HCV基因型也是影響丙型肝炎患者干擾素治療效果的重要因素之一。為此,本研究于2006年5月——2009年12月在江蘇省收集505例不同感染途徑的丙型肝炎患者,并對(duì)其進(jìn)行HCV常見基因型分型檢測(cè),探討江蘇省HCV基因型在不同感染途徑人群中的分布情況,為丙型肝炎的臨床治療提供科學(xué)依據(jù)。 [目的]探討江蘇地區(qū)不同感染途徑丙型肝炎患者病毒(HCV)基因型的分布及其與感染途徑、肝功能的關(guān)系,為丙型肝炎的治療策略和措施提供理論依據(jù)。 [方法]按照Simmonds分型方法對(duì)505例不同感染途徑的丙型肝炎患者進(jìn)行HCV基因型分型檢測(cè)并分析年齡、性別、肝功能以及感染途徑與HCV基因型的關(guān)系。 [結(jié)果] 1.HCV基因分型檢測(cè)結(jié)果505例HCVRNA陽(yáng)性標(biāo)本中,1a型占1.6%,1b型占68.9%,2型占4.8%,3型占7.9%,其它分型占16.8%。 2.不同特征人群HCV基因型分布男性與女性的HCV基因型分布差異無(wú)統(tǒng)計(jì)學(xué)意義(χ2=4.94,P=0.08)；不同年齡人群中的HCV基因型分布差異亦無(wú)統(tǒng)計(jì)學(xué)意義(χ2=1.61,P=0.81)；不同HCV基因型人群的ALT.AST水平均無(wú)統(tǒng)計(jì)學(xué)差異(PALT＝0.64,PAsT=0.28)。 3.不同感染途徑患者HCV基因型分布情況不同感染途徑人群的HCV基因型分布存在統(tǒng)計(jì)學(xué)差異(χ2=73.35,P0.01)。 [結(jié)論]江蘇地區(qū)丙型肝炎患者HCV基因型以1b型為主,1a型HCV在中國(guó)大陸地區(qū)流行程度有擴(kuò)大趨勢(shì),HCV基因型與其感染途徑有關(guān)。 第二部分 HLA-DP基因多態(tài)性與HCV感染轉(zhuǎn)歸的關(guān)系 丙型肝炎病毒(hepatitis C virus, HCV)是單鏈RNA病毒,人體感染HCV后,遺傳因素參與了機(jī)體免疫,可能影響疾病的易感性及病程的進(jìn)展。HLA是迄今所知最復(fù)雜、多態(tài)性最高的免疫相關(guān)遺傳系統(tǒng),在人類的免疫應(yīng)答反應(yīng)中起著十分重要的作用。HLA-Ⅱ類基因分為經(jīng)典HLA-DP-DQ、-DR和非經(jīng)典的HLA-DM.-DO兩類。HLA-DP由組成2個(gè)亞基,DPα和DPp的抗原。α鏈和β鏈的遠(yuǎn)膜區(qū)有一個(gè)抗原結(jié)合槽(antigen binding cleft),因此可以推鋇HLA-DP基因的多態(tài)性可導(dǎo)致了抗原結(jié)合槽的構(gòu)象、結(jié)合及遞呈抗原肽給T細(xì)胞的效率不同。從而影響著免疫反應(yīng)的強(qiáng)弱。目前國(guó)內(nèi)外大量研究表明HLA-DP基因區(qū)域的某些多態(tài)性位點(diǎn)與個(gè)體更易導(dǎo)致感染HCV慢性化。然而,未發(fā)現(xiàn)有關(guān)于HLA-DP基因多態(tài)性與HCV感染轉(zhuǎn)歸的研究出現(xiàn)。 本研究探討HLA-DP基因多態(tài)性與HCV感染轉(zhuǎn)歸的相關(guān)性,為研究HCV感染的發(fā)生機(jī)制和臨床治療提供新的線索。 [目的]探討HLA-DP基因多態(tài)性與HCV感染轉(zhuǎn)歸的關(guān)系。 [方法]采用病例-對(duì)照研究設(shè)計(jì),收集1653例HCV高危人群,其中包括401例HCV持續(xù)感染者(HCVRNA陽(yáng)性),268例HCV自限清除者(抗-HCV抗體陽(yáng)性,HCVRNA陰性)以及984例健康對(duì)照者(抗-HCV抗體陰性),采用TaqMan-MGB探針技術(shù)檢測(cè)HLA-DP單核苷酸多態(tài)性,比較不同基因型與HCV感染慢性化關(guān)系。 [結(jié)果] 1.一般情況比較 本研究有研究對(duì)象共1653人,分別為對(duì)照組984人、自限清除組268人和持續(xù)感染組401人。三組的性別和年齡構(gòu)成差異均無(wú)統(tǒng)計(jì)學(xué)意義(P性別=0.24,P年齡=0.07),但是三組之間的谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)水平以及高危人群種類分布差異均有統(tǒng)計(jì)學(xué)意義。 2. HLA-DP基因多態(tài)性與HCV感染轉(zhuǎn)歸的關(guān)系 將HCV感染組(自限清除組+持續(xù)感染組)與對(duì)照組進(jìn)行比較,采用多因素Logistic回歸分析,對(duì)性別、年齡和高危人群種類因素進(jìn)行調(diào)整,估計(jì)HLA-DP突變基因型對(duì)HCV易感性的影響。rs3077位點(diǎn)分析結(jié)果顯示：雜合基因型CT和突變型TT在感染組中的頻率均高于對(duì)照組(調(diào)整OR和95%C1分別為1.31,1.05-1.64和1.93,1.36-2.73),提示雜合基因型CT和突變型TT可增加個(gè)體對(duì)HCV的易感性。rs2395309位點(diǎn)分析結(jié)果顯示：雜合基因型GA和突變型AA在感染組中的頻率均高于對(duì)照組(調(diào)整OR和95%C1分別為1.29,1.03-1.61和1.89,1.34-2.68),提示雜合基因型GA和突變型AA可增加個(gè)體對(duì)HCV的易感性。3. HLA-DP基因多態(tài)性與HCV自限清除的關(guān)系 將HCV自限清除組與持續(xù)感染組進(jìn)行比較,采用多因素Logistic回歸分析,對(duì)性別、年齡和高危人群種類因素進(jìn)行調(diào)整,估計(jì)HLA-DP基因多態(tài)性與HCV感染自限清除的關(guān)系。rs9277535位點(diǎn)分析結(jié)果顯示：雜合基因型GA和突變型AA在持續(xù)性感染組中的頻率均高于自限清除組(調(diào)整OR和95%C1分別為1.54,1.05-2.28和1.62,1.01-2.60),提示雜合基因型GA和突變型AA可降低個(gè)體自限清除病毒的能力。 4.單倍型分析 以HLA-DP基因3個(gè)SNP位點(diǎn)(rs3077, rs9377535和rs2395309)中均成功進(jìn)行基因型檢測(cè)的樣本為研究對(duì)象,用PHASE2.0推斷可能的單倍型及其頻率。與最常見的單倍型CGG相比,含三個(gè)位點(diǎn)突變等位基因的單倍型TAA可以增加HCV的易感性(調(diào)整OR和95%CI為1.31,1.09-1.55)。 [結(jié)論]HLA-DP基因多態(tài)性可能與丙型肝炎易感性及慢性化有關(guān)。
[Abstract]:Hepatitis C Virus (HCV) infection is a global epidemic. According to WHO (WHO), about 3% of the population (or 170 million) of the world is infected with HCV.. The infection rate of natural population in China is about 0.9%-5.1%, with an average of about 3.2%, and the chronicity of hepatitis C caused by.HCV infection is up to 75% ~ 85%, which can lead to Chronic inflammatory necrosis and fibrosis of the liver, some patients can develop into liver cirrhosis and even hepatocellular carcinoma, which poses a serious threat to human health and life..HCV is a single strand of RNA virus and belongs to the family of the yellow virus. The total length of the genome is about 9.5kb, divided into 5 'non coding region (5'untranslated region, 5'UTR), coding region and 3' non. The coding area (3'UTR), in which the core area of the 5'UTR region and the coding region is the most conservative, the E1 region and the most variable.HCV height variation in the E2 region lead to the regional distribution of the genotype and subtype in the world. The prevalent HCV types in most areas of China are 1B type, followed by 2 A, the 1b type infection rate in southern city is over 90%, and the 2A type is gradually increasing from south to north to.H. CV different genotypes, subtypes and the severity of the clinical symptoms of HCV infected people, the prognosis of the disease and the route of HCV infection, regional distribution, and so on. In addition, the HCV genotype is also one of the important factors affecting the effect of interferon therapy in hepatitis C patients.
In HCV infection, humoral immunity plays a small role in antiviral infection, and CD4+T lymphocyte reaction plays an important role. Human leukocyte antigen (]human leukocyte antigen, HLA) class II antigen is a HLA protein molecule encoded by the II class of human major histocompatibility complex (najor histocompatibility complex, MHC). Distributed in B lymphocyte, macrophage and activated T lymphocyte, its function is to send exogenous antigen peptide to CD4+T lymphocyte. Therefore, the high polymorphism of HLA- class II gene is the most important genetic factor determining host immune response,.HLA- class II anti primordia mainly includes three subregions of HLA-DR, DQ and DP, each subregion. The genetic structure of the region is highly polymorphic and.HLA-DP is a MHC class II antigen. It is a protein / peptide antigen receptor in the major histocompatibility complex of human beings. HLA-DP plays an important role in antigen presentation and immune response. HLA-DP molecules play an important role in the process of virus removal in the host, suggesting that the HLA-DP genotypes are available. It plays an important role in the chronicity of HCV infection.
In this study, we use the HLA-DP gene polymorphism as the breakthrough point to further use case control study to investigate the distribution of HCV genotypes and the relationship between the polymorphism of HLA-DP gene and the HCV infection and the outcome of the hepatitis C patients with different infection routes in the high risk population of HCV in China.
Part one
HCV genotyping of hepatitis C patients with different routes of infection in Jiangsu
Hepatitis C Virus (HCV) is a single strand positive chain RNA whose genome is divided into 5 'non coding region (5'untranslated region, 5'UTR), coding region and 3' non coding region (3UTR). The 5'UTR region, the core region of the coding region is the most conservative, E1 region, and the largest variation in the E2 region causes its genotypes and subtypes to be regional in the global region. The distribution of.HCV genotype is of great significance to the epidemiological study of HCV infection. In addition, the HCV genotype is one of the important factors affecting the effect of interferon therapy in hepatitis C patients. In this study, 505 cases of hepatitis C in different routes of infection were collected in Jiangsu province in May 2006 and December 2009, and the common HCV was common. Genotypic typing was used to investigate the distribution of HCV genotypes in Jiangsu among different routes of infection, so as to provide a scientific basis for clinical treatment of hepatitis C.
[Objective] to investigate the distribution of virus (HCV) genotype of hepatitis C virus (HCV) in different infection routes in Jiangsu and its relationship with the way of infection and liver function, and provide a theoretical basis for the treatment strategy and measures of hepatitis C.
[Methods] HCV genotyping was carried out in 505 hepatitis C patients with different infection routes according to the Simmonds typing method and the relationship between age, sex, liver function and the infection pathway and HCV genotype was analyzed.
[results]
The results of 1.HCV genotyping test showed that among 505 HCVRNA positive specimens, 1.6% were 1a, 68.9% were 1b, 2 were 4.8%, 3 were 7.9%, and the others were 16.8%..
2. there was no significant difference in the distribution of HCV genotypes between men and women in different HCV genotypes (x 2=4.94, P=0.08), and there was no statistical difference in the distribution of HCV genotypes in different age groups (x 2=1.61, P=0.81), and there was no statistical difference in the level of ALT.AST among the different HCV genotypes (PALT = 0.64, PAsT=0.28).
3. the distribution of HCV genotypes in patients with different routes of infection was statistically different from those in the infected group (HCV 2=73.35, P0.01).
[Conclusion] the HCV genotype of hepatitis C patients in Jiangsu area is mainly 1b type, and the prevalence of type 1 a HCV in the mainland of China has a tendency to expand, and the HCV genotype is related to the infection pathway.
The second part
The relationship between the polymorphism of HLA-DP gene and the outcome of HCV infection
Hepatitis C virus (hepatitis C virus, HCV) is a single chain RNA virus. After human infection of HCV, genetic factors participate in the immune system, which may affect the susceptibility and progress of the disease..HLA is the most complex and polymorphic immune related genetic system known to date, which plays a very important role in the response of human immune response.HLA-. Class II genes are divided into classical HLA-DP-DQ, -DR and non classical HLA-DM.-DO two.HLA-DP, which consist of 2 subunits, DP alpha and DPp antigens. The alpha and beta chains have an antigen binding slot (antigen binding cleft). Therefore, the polymorphism of the barium HLA-DP gene can lead to the conformation of antigenic grooves, binding and presenting antigen peptides to T fines. At present, a large number of studies at home and abroad have shown that some polymorphic loci and individuals in the HLA-DP gene region are more likely to lead to the chronicity of HCV. However, there is no discovery of the HLA-DP gene polymorphism and the HCV infection.
The aim of this study was to investigate the association between HLA-DP gene polymorphism and the outcome of HCV infection, and to provide new clues for studying the pathogenesis and clinical treatment of HCV infection.
[Objective] to explore the relationship between HLA-DP gene polymorphism and the prognosis of HCV infection.
[Methods] a case control study was used to collect 1653 high risk groups of HCV, including 401 cases of HCV persistent infection (HCVRNA positive), 268 HCV self limiting scavengers (anti -HCV antibody positive, HCVRNA negative) and 984 healthy controls (anti -HCV negative). The HLA-DP single nucleotide polymorphisms were detected by TaqMan-MGB probe technique. The chronicity of the genotype and HCV infection.
[results]
1. general situation comparison
There were 1653 subjects in this study, 984 in the control group, 268 in the self clearance group and 401 in the continuous infection group. There was no significant difference in gender and age between the three groups (P sex =0.24, P age =0.07), but the level of alanine transaminase (ALT) and gluten transaminase (AST) between the three groups and the differences in the distribution of high risk groups There were statistical significance.
Relationship between 2. HLA-DP gene polymorphism and HCV infection outcome
The HCV infection group (self limited clearance group + persistent infection group) was compared with the control group. The multiple factor Logistic regression analysis was used to adjust the factors of sex, age and high risk population, and the effect of HLA-DP mutation genotype on the susceptibility of HCV was estimated. The results of.Rs3077 site analysis showed that the heterozygous genotype CT and the mutant TT were in the infected group. The frequency was higher than that of the control group (adjusted OR and 95%C1 respectively 1.31,1.05-1.64 and 1.93,1.36-2.73), suggesting that heterozygous genotype CT and mutant TT could increase the susceptibility of individual to HCV, which showed that the frequency of heterozygous genotype GA and mutant AA in infected group was higher than that of the control group (OR and 95%C1) were higher than those of the control group. 1.61 and 1.89,1.34-2.68), suggesting that heterozygous genotype GA and mutant AA can increase the susceptibility of individuals to HCV, and the relationship between.3. HLA-DP polymorphism and HCV self limited clearance.
The HCV self limited clearance group was compared with the continuous infection group. The multiple factor Logistic regression analysis was used to adjust the sex, age and high risk population, and the relationship between the HLA-DP gene polymorphism and the HCV infection self limiting clearance was estimated. The results of the.Rs9277535 site analysis showed that the heterozygous genotype GA and the mutant AA were in the persistent infection group. The frequency was higher than that of the self limited scavenging group (adjusting OR and 95%C1 for 1.54,1.05-2.28 and 1.62,1.01-2.60 respectively), suggesting that heterozygous genotype GA and mutant AA could reduce the ability of individual self limiting virus clearance.
4. haplotype analysis
The samples of the 3 SNP loci (rs3077, rs9377535 and rs2395309) of the HLA-DP gene (rs3077, rs9377535 and rs2395309) were studied. The possible haplotype and its frequency were deduced by PHASE2.0. The haplotype TAA containing three loci mutation alleles could increase the susceptibility to HCV (adjusted OR and 95%CI 1.31, compared with the most common haplotype CGG. " 1.09-1.55).
[conclusion]HLA-DP gene polymorphism may be associated with susceptibility to hepatitis C and chronicity.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.63

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