【作者】 高秋菊；

【导师】 刘殿武；

【作者基本信息】 河北医科大学 ， 病原生物学， 2007， 博士

【摘要】 不同个体感染HCV和/或HBV后,临床表现复杂多样。约15%的感染者可自发清除病毒而免受疾病侵袭,约85%的感染者会转为慢性,并且在慢转者中约20%可进展为肝硬化,甚至原发性肝细胞癌。分析原因可能是病毒因素和宿主遗传因素综合作用的结果,但有报道认为即使是相同的病毒分离株在感染不同个体时其临床表现也有很大差异,因此宿主的遗传因素在丙型、乙型肝炎慢性化作用中起着重要作用。据报道,不同个体对HCV和/或HBV感染所发生的免疫反应不同,个体间某些细胞因子的基因多态性可能导致机体免疫功能状态的差异,因而决定着HCV/HBV感染的不同临床转归。辅助性T细胞(Th)是免疫调节的核心细胞,在IFN-γ的作用下,Th0细胞可分化为Th1细胞,Th1主要分泌IL-2、IFN-γ等,通过介导细胞免疫,用于宿主的抗病毒、细胞毒效应、NK细胞自然杀伤作用等;在IL-4作用下,Th0细胞分化为Th2细胞,Th2主要分泌IL-4、IL-10等,刺激B淋巴细胞分化增殖产生抗体。Th1和Th2细胞因子的功能互相拮抗,Th1和Th2细胞功能的失衡将严重扰乱体液免疫和细胞免疫,进而影响着HCV、HBV感染者的慢性化和不同的临床转归。目前,关于慢性HCV和/或HBV感染者体内Th1主要细胞因子IFN-γ、IL-2和Th2主要细胞因子IL-4、IL-10的表达水平报道尚不统一,与表达细胞因子水平相关的基因多态性研究尚处于起步阶段,并且结果报道各异。为此,本研究以不同感染类型:慢性HCV重叠HBV感染、单纯HCV感染、单纯HBV感染和健康对照及不同临床转归:轻型、中重型肝炎、肝硬化和健康对照为研究对象,探讨IFN-γ+T874A、IL-2-T330G、IL-4-C589T、IL-10-G1082A、IL-10-A592C的基因多态性与不同感染类型、临床转归、病毒复制和肝功异常的关系及各细胞因子基因多态性对临床转归有无交互作用,探讨HCV和/或HBV感染者不同临床转归的免疫遗传机制。第一部分ELISA联合RT-n PCR检测慢性HCV感染者的结果分析目的:对比酶联免疫吸附试验(ELISA)和逆转录-套式-聚合酶链反应(RT-n PCR)检测慢性丙型肝炎病毒感染者的抗-HCV和HCV RNA结果,为临床检测和血站筛选健康献血员提供参考依据。方法:2005年5月对河北赵县某农村单采血浆还输血细胞HCV感染者133人及健康对照52人共185人采集空腹静脉血。该感染人群于1990年前单采血浆还输血细胞时感染、1993年经现场调查、血清生化指标及病原学标志检查确诊为HCV感染者、2002年追踪调查仍明确诊断者。用ELISA法检测抗-HCV,用RT-n PCR检测HCV RNA。结果:①185份血清标本中,抗-HCV和HCV RNA均阳性者92份,占49.73%;抗-HCV阴性、HCV-RNA阳性18份,占9.73%;抗-HCV阳性、HCV-RNA阴性22份,占11.89%,两者均阴性53份,占28.65%。两种方法检测结果一致符合率为78.38%((92+53)/185),Kappa=0.548(u=8.65, P<0.01),另外用配对卡方检验两种方法不一致率亦无差别(χ2paried=0.40,P>0.05),均说明两种检测方法高度一致;②ELISA法检测慢性HCV感染者的灵敏度为82.71%,特异度为92.31%,漏诊率为17.29%;RT-n PCR检测的灵敏度为81.20%,特异度为96.15%,漏诊率为18.80%,两种方法检测灵敏度无差别(χ2=0.10,P>0.05);③并联试验检测灵敏度为96.75%,特异度为88.76%,其灵敏度明显高于单一ELISA法时的82.71%(χ2=9.62,P<0.01),也明显高于串联试验67.16%的灵敏度(χ2=29.39,P=0.00)。结论:单独用ELISA法检测抗-HCV约有17%的漏检率,同时用RT-n PCR检测HCV RNA,可明显提高HCV感染者的阳性检出率。第二部分慢性HCV和/或HBV感染者Th1主要细胞因子基因多态性研究目的:研究Th1主要细胞因子基因IFN-γ+874和IL-2-330单核苷酸多态性(SNP)与单纯HCV、HBV感染及HCV重叠HBV感染、临床转归等的关系,探讨HCV/HBV感染者慢性化和临床转归的免疫遗传机制。方法:用序列特异性引物-聚合酶链反应(PCR-SSP)和限制性内切酶片段长度多态性-聚合酶链反应(RFLP-PCR)技术检测了79例慢性HCV重叠HBV感染、55例单纯HCV感染、69例单纯HBV感染和74例健康对照者的IFN-γ+874 SNP和IL-2-330SNP,Beckman LX-20生化分析仪检测肝功,RT-n PCR检测HCV RNA。分析了IFN-γ+874和IL-2-330基因型和等位基因频率与慢性HBV/HCV感染、临床转归、ALT异常和HCV RNA表达等的关系。结果:①不同感染类型与IFN-γ+874基因型频率有统计学关联(χ2=16.15, P=0.01),单纯HCV、HBV感染和HCV重叠HBV感染者AA基因型频率明显高于对照组,TA基因型频率明显低于对照组,携带AA基因型个体感染HCV、HBV和HCV重叠HBV感染的相对危险度(OR)及95%可信限(95%CI)分别是TA基因型的3.22(1.43~7.25)、2.70(1.24~5.92)、4.02倍(1.88~8.55);T、A等位基因频率与感染类型尚未发现统计学关联(χ2=4.87, P=0.18); HCV和/或HBV感染类型间基因型、等位基因频率差异均不明显(P>0.05)。不同感染类型IL-2-330基因型存在显著差异(χ2=14.24, P=0.03),单纯HCV、HBV感染和HCV重叠HBV感染者TT基因型频率明显高于对照组,GG基因型频率明显低于对照组,携带TT基因型个体感染HCV、HBV和HBV重叠HCV感染的OR及95%CI分别是GG基因型的3.46(1.17~10.02)、7.14(2.13~23.81)、2.93倍(1.15~7.46);T、G等位基因频率在感染类型间差异有统计学意义(χ2=12.33, P=0.01),携带T基因个体感染HCV、HBV、HCV重叠HBV的OR值和95%CI分别是G基因的1.82(1.09~3.03)、1.73(1.10~2.73)、2.26倍(1.39~3.69);HCV和/或HBV各感染类型间基因型和等位基因频率差异均不显著(P>0.05)。②不同临床转归类型IFN-γ+874基因型频率之间差异有统计学意义(χ2=14.78,P=0.02),轻型、中重型肝炎和肝硬化组AA基因型频率明显高于对照组,而TA基因型频率明显低于对照组;携带AA基因型个体进展为轻型、中重型肝炎、肝硬化的OR及95%CI分别是TA基因型的3.09(1.51~6.33 )、3.85(1.70~8.70)、3.14倍(1.08~9.17);T、A等位基因频率疾病转归尚未发现统计学关联(χ2=5.68, P=0.13)。不同疾病转归类型IL-2-330基因型频率差异存在显著的统计学意义(χ2=13.46,P=0.04),轻型、中重型肝炎和肝硬化组TT基因型频率明显高于对照组,GG基因型频率明显低于对照组;携带TT基因型个体进展为轻型、中重型肝炎、肝硬化的OR值及95%CI分别是GG基因型的3.42(1.45~8.13 )、3.29(1.10~9.80)、11.11倍(1.32~90.91);GG基因型频率与临床进展呈明显负相关(γ=-0.92,P<0.05);T、G等位基因频率与疾病转归有统计关联(χ2=11.69, P=0.01),携带T基因个体进展为轻型肝炎、中重肝炎和肝硬化的OR及95%CI分别是G基因的1.83(1.20~2.80)、1.70(1.02~2.82)、2.75倍(1.32~5.63)。③IFN-γ+874基因型和等位基因频率与HCV病毒复制未发现统计学关联(χ2=2.36,P=0.31;χ2=1.92,P=0.17)。IL-2-330基因型和等位基因频率与HCV病毒复制亦无统计学关联(χ2=0.83,P=0.66;χ2=1.97,P=0.66);④IFN-γ+874基因型和等位基因频率与ALT异常未发现统计学关联(χ2=0.23,P=0.89;χ2=0.12,P=0.74)。IL-2-330基因型频率和等位基因频率与ALT水平亦无统计学关联(χ2=1.35,P=0.51;χ2=0.24,P=0.62)。结论:Th1主要细胞因子IFN-γ+874和IL-2-330基因多态性与HCV/HBV感染者慢性化及临床转归有明显的统计学关联,与病毒复制和肝功损伤无明显关联。IFN-γ+874AA基因型、IL-2-330 TT基因型和T等位基因可能是HCV和/或HBV感染及其临床转归的危险基因(型),IFN-γ+874 TA、IL-2-330 GG基因型和T等位基因可能为其保护基因(型)。第三部分慢性HCV和/或HBV感染者Th2主要细胞因子基因多态性研究目的:研究Th2主要细胞因子IL-4-589、IL-10-1082和IL-10-592基因多态性与单纯HCV、HBV感染及HCV重叠HBVV感染、临床转归等的关系,探讨HBV和/或HCV慢性感染者和不同临床转归的遗传易感机制。方法:用SSP-PCR和RFLP-PCR技术检测79例HCV重叠HBV感染、55例单纯HCV感染、69例单纯HBV感染和74例健康对照者的IL-4-589 SNP、IL-10-1082和IL-10-592;用RT-n PCR方法检测血清HCV RNA;用Beckman LX-20全自动生化分析仪检测肝功能。结果:①IL-4-589位点基因型和等位基因频率与不同感染类型无明显统计关联(χ2=4.41,P=0.62;χ2=0.26,P=0.97)。不同感染类型与IL-10-1082位点基因型频率有统计学关联(χ2=13.05, P=0.04),HBV和/或HCV感染者AA基因型频率明显高于对照组,而AG基因型频率明显低于对照组;携带AA基因型个体感染HCV、HBV、HCV重叠HBV的危险是AG基因型的2.34(1.07~5.10)、2.29 (1.10~4.78)、2.56倍(1.25~5.21);HCV和/或HBV各感染类型间基因型差异均不显著(P>0.05)。IL-10-1082等位基因频率与不同感染类型间无明显的统计关联(χ2=4.65,P=0.20)。IL-10-592位点基因型和等位基因频率与不同感染类型无明显统计关联(χ2=2.83,P=0.83;χ2=1.10,P=0.78)。②IL-4-589位点基因型和等位基因频率与临床转归类型无明显统计关联(χ2=4.58,P=0.60;χ2=2.46,P=0.48)。IL-10-1082位点基因型频率与不同临床转归类型有统计学关联(χ2=11.40, P=0.07),轻型、中重型肝炎和肝硬化组AA基因型频率明显高于健康对照组,而AG基因型频率明显低于健康对照;携带AA基因型个体进展为轻型、中重型肝炎和肝硬化的风险是AG基因型的2.03(1.03~3.98)、2.70(1.24~5.88)和4.26倍(1.59~ 11.36) ; IL-10-1082 AA基因型频率与临床转归呈明显正相关(γ=0.99,P<0.01),AG基因型频率与临床转归呈明显负相关(γ=-0.99,P<0.01);IL-10-1082等位基因频率与临床转归未见统计学关联(χ2=5.85, P=0.12);IL-10-592位点基因型和等位基因频率与临床转归无明显统计关联(χ2=2.50,P=0.87;χ2=0.92,P=0.82)。③IL-4-589位点基因型频率和等位基因与体内HCV病毒复制无统计学关联(χ2=1.53,P=0.47;χ2=1.24 P=0.27)。IL-10-1082位点基因型和等位基因频率与HCV病毒复制有统计学关联(χ2=12.27,P=0.00;χ2=5.36, P=0.02);病毒复制组AA基因型和A等位基因频率明显高于无病毒复制组,而AG基因型和G基因频率明显低于无病毒复制组(OR=3.36(1.67~6.76)、1.67倍(1.08~2.57));IL-10-592位点基因型频率和等位基因与体内有无HCV病毒复制无统计学关联(χ2=2.10,P=0.35;χ2=1.88,P=0.17)。④IL-4-589位点基因型和等位基因频率在ALT<80 U/L和≥80U/L间差异有明显的统计学意义(χ2=12.46,P=0.00;χ2=9.97, P=0.00),ALT≥80U/L组CT和CC基因型频率明显高于肝功<80U/L组,ALT≥80U/L组TT基因型频率明显低于肝功<80U/L组,携带CT和CC基因型个体肝功损伤的OR值和95%CI是TT基因型的3.43(1.47~8.00)、8.25倍(1.73~39.22);ALT≥80U/L组C等位基因频率明显高于肝功<80U/L组,T等基因频率明显低于肝功<80U/L组,携带C等位基因的个体其肝功异常的风险是T等位基因的2.31倍(1.36~3.93)。IL-10-1082位点基因型和等位基因频率与ALT水平无明显统计学关联(χ2=3.25,P=0.20;χ2=1.79, P=0.18)。IL-10-592位点基因型频率在ALT<80 U/L和≥80U/L间差异有显著性(χ2=6.32,P=0.04),ALT≥80U/L组AC基因型频率明显高于肝功<80U/L组,AC基因型个体ALT异常风险是AA基因型的2.83倍(1.26~6.37),而等位基因频率与肝功异常无明显统计关联(χ2=2.30,P=0.13)。结论:Th2主要细胞因子IL-10-1082基因型与HCV和/或HBV感染类型、不同临床转归、病毒复制有明显的统计学关联,AA基因型可能是其危险基因型,AG基因型可能是其保护基因型;IL-10-592和IL-4-589位点多态性与HBV和/或HCV感染者的肝功损伤有一定联系,IL-10-592AC基因型、IL-4-589 CT、CC基因型和C等位基因可能是其危险基因(型),IL-10-592AA基因型、IL-4-589 TT基因型和T等位基因可能是其保护基因(型)。第四部分Th1、Th2细胞因子基因多态性在慢性HCV和/或HBV感染者临床转归中的交互作用研究目的:探讨Th1、Th2细胞因子基因多态性在慢性HCV和/或HBV感染者临床转归中的交互作用。方法:用多因子降维法(MDR)分析Th1、Th2细胞因子基因多态性在慢性HCV和/或HBV感染者及临床转归中有无交互作用,患病风险用OR及95% CI评价,交互作用大小用交互作用指数(S)、交互作用归因比(API)、交互作用超额相对危险度(RERI)及95%可信限(95%CI)评价。结果:①MDR分析Th1主要细胞因子基因多态性,发现IFN-γ+874、IL-2-330位点对慢性HCV/HBV感染者临床转归有明显的交互作用(P=0.00)。IFN-γ+874 AA和IL-2-330 TT基因型共存时,HCV/HBV感染者进展为轻型.中重型肝炎和肝硬化的OR值分别为10.1(2.8~ 36.0)、10.6(2.9~38.8)和13.6(4.9~66.8),分别是IFN-γ+874 AA基因型单独暴露的6.8、4.1和8.1倍,是IL-2-330 TT基因型单独暴露时的8.4、8.1和6.6倍;S分别为13.04.5.01和7.22;RERI分别为8.43(2.61~27.23)、7.65(2.50~23.42)和10.83(2.53~46.32);API分别为83.22%.72.44%和79.81%。②MDR分析Th2主要细胞因子基因多态性,发现IL-10-592和IL-4-589位点对慢性HCV和HBV感染者的肝脏炎症性损伤有明显的交互作用(P=0.00)。单独IL-10-592 AC基因型对慢性HCV/HBV感染者肝脏炎症性损伤无明显作用(OR=1.0),但IL-4-589 CC或CT和IL-10-592 AC共存时OR为5.5(2.6~11.5);S=7.10,RERI=3.70(2.11~6.49);API=67.64%。③MDR分析Th1、Th2主要细胞因子基因多态性交互作用,发现IFN-γ+874、IL-2-330和IL-10-1082三个位点对慢性HCV和HBV感染者的临床转归有明显交互作用(P=0.01)。若只考虑三种基因型的单独效应,对HCV/HBV感染者临床转归的危险性从大到小依次是IL-10-1082 AA基因型(OR=2.7,4.3,7.1)、IFN-γ+874 AA基因型(OR=2.0,3.9,2.4)和IL-2-330 TT基因型(OR=1.2,1.3,2.8),并且IL-10-1082 AA基因型与临床进展严重程度正相关(γ=0.99,P<0.05)。若考虑其中两种基因型的联合作用效应,IFN-γ+874 AA和IL-2-330 TT基因型共存时,感染者不同临床转归类型对应的OR值分别为7.5(2.0~28.4)、9.6(2.6~35.6)和10.7(1.4~ 80.2)(与单独分析IFN-γ+874 AA和IL-2-330 TT基因型的OR值不等,是因为存在IL-10-1082 AA基因型的交互作用),分别是IFN-γ+874 AA单基因型暴露的3.8、2.5和4.5倍,是IL-2-330 TT单基因型暴露的6.3、7.4和3.8倍;S分别为5.3、2.7和3.0,RERI分别为5.3(1.1~24.5)、5.41(1.8~16.2)和6.5(1.3~31.5),API分别为70.3%、56.4%、60.5%。IFN-γ+874 AA和IL-10-1082 AA共存时,感染者不同临床转归类型对应的OR值分别为1.6(1.0~2.5)、4.0(1.6~ 9.9)、5.3(1.5~ 19.1),均小于IL-10-1082 AA基因型单独作用的OR值(2.7、4.3、7.1);S分别为0.2、0.5、0.6,RERI分别为-2.1(-1.1~-4.1)、-3.2(-1.5~-6.8)、-3.2 (-1.3~ -7.6),API分别为-130.0%、-79.5%、-59.1%。IL-2-330 TT和IL-10-1082 AA基因型共存时,感染者不同临床转归类型对应的OR值分别为3.0(1.3~7.2)、4.5(1.6~12.3)和8.5(2.0~35.7),与IL-10-1082 AA基因型单独暴露时的OR值(2.7、4.3、7.1)相差不大;S分别为1.1、1.0、1.0,RERI分别为0.1、-0.1、-0.4,API分别为3.7%、0.0%、-0.4%。当IFN-γ+874 AA、IL-2-330 TT和IL-10-1082 AA三种基因型共存时,感染者不同临床转归类型对应的OR值分别为9.6(2.9~31.8)、12.8 ( 2.0~80.2 )和32.0(5.1~200.9),是IFN-γ+874 AA、IL-2-330 TT共存时OR值的1.3、1.3和3.0倍;S分别为1.1、1.0、2.0,API为4.2%、-0.8%和47.5%,RERI为0.4(0.2~0.7)、-0.1(0.0~0.5)和15.2(3.6~64.3)。结论:Th1主要细胞因子IFN-γ+874 AA、IL-2-330 TT基因型对慢性HCV/HBV感染者不良临床转归有明显的协同作用;Th2主要细胞因子IL-4-589 CC或CT和IL-10-592 AC基因型对慢性HCV/HBV感染者肝脏炎症性损伤有明显的协同作用;Th1主要细胞因子基因型IFN-γ+874 AA、IL-2-330 TT和Th2主要细胞因子基因型IL-10-1082 AA对慢性HCV/HBV感染者进展为轻型、中重型肝炎交互作用不明显,对进展为肝硬化有协同作用。更多还原

【Abstract】 Infection with the hepatitis C virus (HCV) and/or hepatitis B virus (HBV) is characterized by a broad spectrum of possible outcomes. Infection is self-limited in a very minority, while the majority of infections develop persistent infection. Among those individuals with persistent HCV and/or HBV infection, the majority develops chronic hepatitis, some progress to fibrosis and even primary hepatocellular carcinoma. The variation may be attributable to viral and host genetic background. However it has been reported that the natural outcome in HCV or HBV infection varies among individuals though infected by the same viral. So there is strong evidence in HCV/HBV infection that host genetic factors play a major role in determining the outcome of infection.The immunity responses to HCV and HBV infection vary among individuals to a far extent. The single nucleotide polymorphism (SNP) in some cytokines gene greatly influences the clinical outcome of individuals with HCV and HBV infection. Helper T cell (Th) plays a key role in mediated immunity. Th1 cell is produced by the origin of Th in the stimulation of interferon-gamma and Th1 secrete cytokines such as interleukin 2, interferon-gamma, and so on. Th1 cytokines participate in cell-mediated immunity for controlling such intracellular pathogens as viruses, cytotoxic T lymphocyte (CTL) proliferation and activation, and natural killer cells (NK) activation. Th2 cell is produced by the origin of Th with the stimulation of interleukin 4. The major lymphokines secreted by Th2 cell are interleukin 4, interleukin 10 and so on. Th2 cytokines are essential for antibody-mediated immunity by proliferating and activating the B lymphocell. The cytokines from Th1 cells inhibit Th2 cells, and vice versa. The imbalance of Th1 and Th2 heavily disturbed the balance between the cell-mediated immunity and antibody-mediated immunity, which may be the key to the clinical outcome of chronic HCV and/or HBV infection.There is compelling evidence that cytokines play a significant role in chronic HCV and HBV disease pathogenesis. However, contradictory reports exist as to the exact effect of the cytokines promoter polymorphisms on the natural outcome of HCV and HBV infection. In order to explore the possible association of the cytokines gene polymorphism with the susceptibility of chronic HCV/HBV infection and the outcome of these infections, the IFN-γ+874, IL-2-330, IL-4-589, IL-10-1082, IL-10-592 gene polymorphisms were studied in subjects of mild hepatitis, moderate and severe hepatitis, and cirrhosis infected with HCV, HBV, HCV co-infected with HBV. These results would explore the immune genetic mechanisms of the clinical outcome of HCV and HBV infection.Part 1 Result comparison of anti-HCV detected by ELISA and HCV RNA detected by RT-n PCR in chronic hepatitis C virus infectionObjective: In order to provide the basis for the clinical test and the blood station screening the health donator, the results of anti-HCV tested by enzyme-linked-immuno-absorbed assay (ELISA) and HCV RNA tested by Reverse-Transcript–nested-Polymerase-Chain-Reaction (RT-n PCR) were compared in the chronic HCV infection.Methods: Venous blood samples of 133 chronic HCV infections, which were infected with HCV nearly in 1990 through plasma donator and diagnosed in 1993 in a rural area of Zhao County of Hebei Province, and 52 health controls were collected in May 2005. The antibody to HCV was tested by ELISA method and HCV RNA was tested by RT-n PCR method.Results:①In 185 cases, the rate of both anti-HCV and HCV RNA positive was 49.73% (92/185). The rate of anti-HCV negative but HCV RNA positive was 9.73% (18/185). The rate of anti-HCV positive but HCV RNA negative was 11.89% (22/185). The rate of both anti-HCV and HCV RNA negative was 28.65% (53/185). The result-agreement rate of ELISA and RT-n PCR testing methods was 78.38% ((92+53)/185). Kappa value equals 0.548 (u=8.65, P<0.01). The disagreement rate was not obviously different between ELISA and RT-n PCR testing methods (χ2 paired =0.40, P>0.05), which illustrated high agreement between the ELISA and RT-nPCR methods.②In the diagnosis of chronic HCV infection, the sensitivity of anti-HCV tested by ELISA was 82.71%, the specificity was 92.31%, and the omitting rate was 17.29%. The sensitivity of HCV RNA tested by RT-n PCR was 81.20%, and the specificity was 96.15%, and the omitting rate was 18.80%. The sensitivity was not obviously different between ELISA and RT-n PCR (χ2=0.10,P>0.05).③The sensitivity tested by ELISA parallel with RT-n PCR was 96.75%, which was obviously higher than that of single ELISA (82.71%) (χ2=9.62,P=0.00) and ELISA series with RT-n PCR(χ2=29.39, P=0.00).Conclusion: The false negative rate was nearly 17% when anti-HCV was used to detect HCV infection. The positive testing rate of HCV infection was increased remarkably when ELISA and RT-n PCR were used simultaneously. Part 2 The study of Th1 cytokine SNP in chronic HCV and/or HBV infectionObjective: To explore the possible association of the polymorphisms in interferon-gamma gene intron 1 at position +874 (IFN-γ+874), interleukin-2 gene at position–330 (IL-2-330) with the susceptibility of HCV and/or HBV infection and the outcome of these infections.Methods: IFN-γ+874 gene and IL-2-330 gene SNP were detected in 277 subjects including 79 chronic HCV co-HBV infection, 55 individuals only with HCV infection, 69 individuals only with HBV infection and 74 healthy controls, by sequence specific primers-PCR (SSP-PCR) and by restricted fragment long polymorphism-PCR (RFLP-PCR). Hepatocellular injury as suggested by alanine aminotransferase (ALT) was detected by BeckmanLX-20. The presence or absence of viral particles in serum was determined by RT-nPCR. The possible association of the polymorphism of IFN-γ+874, IL-2-330 with the susceptibility of HCV and/or HBV infection and the outcome of these infections were analyzed. Results:①IFN-γ+874 AA genotype frequency in individuals with chronic HCV, HBV, HCV co-HBV infection was significant higher than that in controls(χ2=16.15,P=0.01). Odds Ratio (OR) and 95% Confidence Interval (95% CI) of IFN-γ+874 AA genotype in chronic infection with HCV, HBV, HCV co- HBV were 3.22 (1.43~7.25), 2.70 (1.24~5.92) and 4.02 (1.88~8.55) compared with TA genotype. No significant differences were found among the HCV, HBV, HCV and HBV infections (P>0.05). There were no significant association of IFN-γ+874 A/T allele frequency with HCV/HBV infections (χ2=4.87, P=0.18); Chronic infection with HCV, HBV, HCV co-HBV was associated with higher IL-2-330 TT genotype frequency (χ2=14.24, P=0.03; OR (95%CI) = 3.46 (1.17~10.20), 7.14 (2.13~23.81), 2.93 (1.15~7.46)) and T allele frequency (χ2=12.33, P=0.01; OR (95%CI)=1.82 (1.09~3.03), 1.73 (1.10~ 2.73), 2.26 (1.39~3.69)). No significant differences were found among the HCV and / or HBV infections (P>0.05).②The outcome of mild chronic hepatitis (CH), moderate/severe CH and cirrhosis during HBV and/or HCV infection was associated with IFN-γ+874 AA genotype (χ2=14.78,P=0.02, OR=3.09 (1.51~6.33), 3.85 (1.70~8.70), 3.14 (1.08~9.17)); No significant relationships were found between IFN-γ+874 A/T allele frequency and the clinical outcome of HBV/HCV infection (χ2=5.68, P=0.13); The outcome of mild CH, moderate/severe CH and cirrhosis during HBV and/or HCV infection was associated with higher IL-2-330 TT genotype frequency (χ2=13.46, P=0.04; OR=3.42 (1.45~8.13), 3.29 (1.10~9.80), 11.11 (1.32~90.91)) and T allele frequency (χ2=11.69, p=0.01; OR=1.83 (1.20~2.80), 1.70 (1.02~2.82), 2.75 (1.32~5.63)), and the IL-2 (-330) GG genotype frequency showed negative correlation with the clinical progression (γ=-0.92, p<0.05).③There were no significant associations of IFN-γ+874 genotype / allele frequency with HCV duplication (χ2=2.36,P=0.31;χ2=1.92,P=0.17). No significant relationship was found between IL-2-330 genotype/allele frequency and HCV duplication (χ2=0.83,P=0.66;χ2=0.20,P=0.66).④There were no significant associations of IFN-γ+874 genotype/allele, IL-2-330 genotype/allele frequency with abnormal ALT (χ2=0.23,P=0.89;χ2=0.12, P=0.74;χ2=1.35,P=0.51;χ2=0.24, P=0.62).Conclusion: These results suggest that polymorphisms in the IFN-γ+874, IL-2-330 appear to have some influence on the chronic HCV and/or HBV infection, and on the outcome of HCV and/or HBV infections. IFN-γ+874AA, IL-2-330 TT genotype and T allele are possible risk to the chronic HCV and/or HBV infection and to the outcome of HCV and/or HBV infection, however IFN-γ+874 TA, IL-2-330 GG genotype and G allele is possible protective to them.Part 3 The study of Th2 cytokine SNP in chronic HCV and/or HBV infectionObjective: To explore the possible associations of the polymorphism of interleukin-4 (IL-4) gene at position–589, interleukin-10 (IL-10) gene at position -1082, -592 with the susceptibility of hepatitis C and/or hepatitis B virus infection and the outcome of these infections.Methods: IL-4-589, IL-10-1082, IL-10-592 gene SNP were detected in 277 subjects including 79 HCV co-HBV infection, 55 individuals only with HCV infection, 69 individuals only with HBV infection and 74 health control, by SSP-PCR and RFLP-PCR. Hepato-cellular injury as suggested by ALT was detected by BeckmanLX-20. The presence or absence of viral particles in serum was determined by RT-nPCR. The possible associations of the polymorphisms of IL-4-589, IL-10-1082 and IL-10-592 with the susceptibility of HCV and/or HBV infection and the outcome of these infections were analyzed.Results:①Chronic infection with HCV, HBV, HCV co-HBV was not associated with IL-4-589 genotype and allele (χ2=4.41,P=0.62;χ2=0.26,P=0.97); Chronic infection with HCV, HBV, HCV and HBV was associated with higher IL-10-1082 AA genotype frequency (χ2=13.05, P=0.04; OR=2.34 (1.07~5.10), 2.29 (1.10~4.78), 2.56 (1.25~5.21)). However no significant differences were found among the HCV, HBV, HCV and HBV infection (P>0.05). There were no significant association of IL-10-1082 allele frequency among HCV, HBV and HCV co-HBV infections (χ2=4.65,P=0.20). There were no significant association of IL-10-592 genotype and allele frequency with chronic HBV and/or HCV infection(χ2=2.83,P=0.83;χ2=1.10,P=0.78);②The outcome of mild CH, moderate/severe CH and cirrhosis during HBV and/or HCV infection was associated with IL-10-1082 AA genotype frequency (χ2=11.40, p=0.07; OR=2.03 (1.03~3.98), 2.70 (1.24~5.88), 4.26 (1.59~11.36)), and the IL-10-1082 AA genotype frequency showed positive correlation with the clinical outcome (γ=-0.99, p<0.01), however IL-10-1082 AG genotype frequency showed negative correlation with the clinical outcome (γ=-0.99, p<0.01). There were no significant associations of IL-10-1082 allele frequency, IL-10-592 genotype and allele frequency with HCV/HBV infection and clinical outcome (χ2=5.85, P=0.12;χ2=2.50, P=0.87;χ2=0.92, P=0.82); The outcome of mild CH, moderate/severe CH and cirrhosis during HBV and/or HCV infection was not associated with IL-4-589 genotype and allele frequency (χ2=4.58, P=0.60;χ2=2.46, P=0.48).③There were obviously associations of IL-10-1082 higher AA genotype frequency and A allele frequency with HCV replication (χ2=12.27, P=0.00;χ2=5.36, P=0.02). The risk of subjects with AA genotype or A allele was 3.36 times (1.67~6.76), 1.67 times (1.08~2.57) than that with AG genotype and G allele; There were no significant associations of IL-10-592, IL-4-589 genotype/allele frequency with HCV replication (χ2=2.10, P=0.35;χ2=1.88, P=0.17;χ2=1.53, P=0.47;χ2=1.24, P=0.27).④There were no significant associations of IL-10-1082 genotype and allele frequency and IL-10-592 allele frequency with abnormal ALT (χ2=3.25, P=0.20;χ2=1.79, P=0.18;χ2=2.30, P=0.13); Abnormal ALT level was associated with higher IL-10-592 AC genotype (χ2=6.32, P=0.04; OR=2.83(1.26~6.37)); Abnormal serum ALT level was associated with IL-4-589 CC and CT genotype (χ2=12.46, P=0.00; OR=8.25 (1.74~ 39.22), 3.43 (1.47~8.00)) and the C allele (χ2=9.97, P=0.00, OR=2.31 (1.36~3.93)).Conclusion: These results suggest that polymorphism in the IL-10-1082 appears to have some influence on the chronic HCV and/or HBV infection, and on the outcome of HCV and/or HBV infections and HCV replication. IL-10-1082 AA genotype is possible risk to the chronic HCV, HBV and HCV co-HBV infection and the clinical outcome, however AG genotype is possible protective to them. IL-10-592 AC genotype frequency has influence on the liver damage, and IL-4-589 CT, CC genotype, and C allele are possible risk to the liver inflammatory injury, and TT genotype and T allele is possible protective to it.Part 4 The interaction of Th1 and Th2 cytokine SNP in the clinical outcome infected with chronic HCV and/or HBVObjective: To analyze the interaction of the Th1 and Th2 cytokine SNP in HCV/HBV infection and in the outcome of these infections. Methods: The interaction of the Th1 and Th2 cytokine SNP in HCV and/ or HBV infection and the outcome of these infections was analyzed by multifactor dimensionality reduction (MDR). The OR was used to evaluate the risk of clinical outcome during HCV/HBV infection. The synergy index (S), Attributable proportion of interaction (API), Relative excess risk of interaction (RERI) and 95% CI were used to evaluate the interaction degree.Results:①The interaction of IFN-γ+874 AA and IL-2-330 TT genotype was found in the clinical outcome of chronic HCV/HBV infection by MDR (P=0.00). When individuals carried with IFN-γ+874 AA genotype and IL-2-330 TT genotype, the OR and 95% CI for the HCV/HBV to develop to mild CH, moderate/severe CH and cirrhosis was 10.1(2.8~36.0), 10.6(2.9~38.8) and 13.6(4.9~66.8) respectively, and the OR in individuals with IFN-γ+874 AA and IL-2-330 TT was 6.8 times, 4.1 times and 6.6 times compared with individuals of only IFN-γ+874 AA, and was 8.4 times, 8.1 times and 6.6 times comparied with individuals of only. The S of chronic HCV/HBV infection developing to mild CH, moderate/severe CH and cirrhosis in IFN-γ+874 AA and IL-2-330 TT genotype was 13.04, 5.01 and 7.22 respectively. RERI and 95% CI were 8.43(2.61~27.23), 7.65(2.50~23.42) and 10.83(2.53~46.32). API was 83.22%, 72.44% and 79.81% respectively.②Interaction of IL-10-592 gene and IL-4-589 gene was found in the hepato-cellular injury of chronic HCV and / or HBV infection by MDR (P=0.00). When only with IL-10-592 AC, there was no significant effect on the hepato-cellular injury of chronic HCV/HBV infection (OR=1.0). However together with IL-4-589 CC or CT and IL-10-592 AC, the OR was 5.5 (2.6~11.5). It was found to have significant interaction to the hepato-cellular injury, which the S was 7.1, RERI was 3.7(2.11~6.49), and API was 67.6%.③The interaction of IFN-γ+874 gene, IL-2-330 gene and IL-10-1082 gene was found in the clinical outcome of chronic HCV/HBV infection by MDR (P=0.01). When one SNP were analyzed, the IL-10-1082 AA genotype had the highest risk (OR=2.7, 4.3, 7.1), IFN-γ+874 AA genotype had the middle risk (OR=2.0, 3.9, 2.4), IL-2-330 TT had the lowest risk (OR=1.2, 1.3, 2.8) to the outcome of HCV/HBV infection. The IL-10-1082 AA genotype was positive correlation with the clinical progression (γ=0.99, P<0.05). When two SNP were analyzed at a time, IFN-γ+874 AA genotype and IL-2-330 TT genotype had a high risk for the HCV/HBV to develop to mild CH, moderate/severe CH and cirrhosis, OR=7.5(2.0~28.4), 9.6(2.6~35.6) and 10.7(1.4~ 80.2)), which were not equal to the OR=10.1(2.8~36.0), 10.6(2.9~38.8) and 13.6(4.9~66.8) of IFN-γ+874 AA genotype and IL-2-330 TT genotype was due to with or without considering the interaction of IL-10-1082 AA genotype. The risk of both IFN-γ+874 AA genotype and IL-2-330 TT genotype in mild CH, moderate/severe CH and cirrhosis was 3.8 times, 2.5 times and 4.5 times comparied with only IFN-γ+874 AA genotype, and was 6.3 times, 7.4 times and 3.8 times comparied with only IL-2-330 TT genotype. The S was 5.3, 2.7 and 3.0 respectively , the RERI was 5.25(1.1~24.5), 5.41(1.8~16.2) and 6.5(1.3~31.5), the API was 70.28%, 56.35%, 60.54%. When together with IFN-γ+874 AA genotype and IL-10-1082 AA genotype, the OR in mild CH, moderate/severe CH and cirrhosis was 1.6(1.0~2.5), 4.0(1.6~9.9) and 5.3(1.5~ 19.1) respectively, which was lower than that with only IL-10-1082 AA genotype. The S was 0.22, 0.49, 0.58 respectively, the RERI was -2.1(-1.1~-4.1), -3.2(-1.5~ -6.8), -3.15 (-1.3~ -7.6) respectively, the API was -130.00%, -79.50%, -59.10% respectively. When together with IL-2-330 TT genotype and IL-10-1082 AA genotype, the OR in mild CH, moderate/severe CH and cirrhosis was 3.0(1.3~7.2), 4.5(1.6~12.3), 8.5(2.0~35.7), which was similar to the OR of only with IL-10-1082 AA genotype. S was 1.06, 0.98, 0.95 respectively, RERI was 0.11, -0.07, -0.42, API was 3.68%, -0.02%, -0.42%. When three SNPs were analyzed at a time, OR in mild CH, moderate/severe CH and cirrhosis was 9.6(2.9~31.8), 12.8(2.0~80.2) and 32.0(5.1~200.9). The OR was the 1.3 times, 1.3 times and 3.0 times compared with IFN-γ+874 AA and IL-2-330 TT. S was 1.1, 1.0 and 2.0, API was 4.2%, -0.8% and 47.5%, RERI was 0.4(0.2~0.7), -0.1(0.0~0.5) and 15.2(3.6~64.3) respectively.Conclusion: These results suggest that subjects with IFN-γ+874 AA genotype and IL-2-330 TT genotype had higher risk for the clinical outcome of chronic HCV/HBV infection, the subjects with IL-10-592 AC genotype and IL-4-589 CC or CT genotype had a higher risk for the hepato-cellular injury of chronic HCV/HBV infection. IFN-γ+874 AA and IL-2-330 TT together with IL-10-1082 AA had no significant interaction for HCV/HBV infection to the mild CH, moderate and severe CH, however had positive interaction for HCV/HBV infection develop to cirrhosis.更多还原