【作者】 刘翠萍；

【导师】 余祖江；

【作者基本信息】 郑州大学 ， 内科学， 2011， 硕士

【摘要】 背景丙型肝炎(hepatitis C)是一全球健康问题,是由丙型肝炎病毒(hepatitis C virus, HCV)引起的肝脏慢性炎症,HCV是导致肝硬化和肝细胞癌的主要病原体之一。据世界卫生组织(The World Health Organization WHO)统计,全球丙型肝炎病毒的感染率约为3%,即约有1.8亿人感染HCV,每年新发感染者增加约300-400万,在中国血清感染率约为3.2%,其中HCV抗体阳性者达4000万。HCV主要通过血源性途径进行传播,包括输血、性接触、纹身和吸毒等方式。急性HCV感染者中只有20%-30%的患者可发生自发病毒清除,70%-80%的患者发展为慢性感染者,其中30%可进展为终末期肝病,包括肝硬化、肝癌等。目前以干扰素(interferon, IFN)为基础的抗病毒治疗,是丙型肝炎病毒感染唯-的有效治疗手段,最理想的抗病毒治疗方案为聚乙二醇干扰素(pegylated interferon, PEG-IFN)联合利巴韦林(ribavirin, RBV),作为抗HCV治疗的“标准方案”,但并不是所有患者都能达到持续病毒学应答。在抗病毒治疗过程中,一些病毒因素,如基因1型、高基线病毒载量、治疗期间的病毒动力学、干扰素敏感决定区域的氨基酸类型,及这些因素与治疗结果有着显著的联系。对HCV1型的患者治疗12月后,只有42%-52%的患者可获得持续病毒学应答(sustained virological response, SVR), HCV 2/3型患者中也有5%的长期处于无病毒学应答(null virological response, NVR);其中干扰素的不良反应可以导致10%-14%的患者被迫减量或提前停药。近年来,在非洲、欧洲、东亚人群中进行的全基因组关联分析(Genome-wide Association Study, GWAS)研究发现编码IFNλ3的IL28B基因附近有一些单核苷酸的多态位点与HCV病毒自发清除能力及对干扰素抗病毒治疗应答有关,当这些SNP的等位基因发生变异后,患者对丙型肝炎病毒的自发清除能力和对干扰素应答能力发生明显变化。SNP rs8099917位于19号染色体上编码IFNλ3的IL28B下游约8kb位点处。在欧洲人群中,Suppiah等和Rauch等都论证了IL28BSNP rs8099917与治疗应答有效和治疗失败相关。Tanaka等对日本患者也鉴定了IL28B SNP rs8099917与SVR相关,这比在欧洲人群中更具有深刻的影响,同时他们也发现SNP rs12980275与无病毒学应答关系密切。rs8099917基因型为T/T的患者,其发生病毒自发清除的比率和发生持续病毒学应答(SVR)百分比明显高于G/G基因型患者。rs12980275基因型为A/A的患者的持续病毒学应答百分比也明显高于G/G基因型患者。目的目前国内对IL28B等位基因与干扰素应答关系鲜见报道,因此,对我院慢性丙型肝炎患者对干扰素治疗应答情况和IL28B基因多态性进行回顾性分析研究,比较应答组(SVR)与无应答组(NVR)的IL28B基因变异频率,为早期预测干扰素疗效提供理论依据。方法1.病例收集：入选病例均为慢性丙型肝炎诊断符合2004年3月中华医学会肝病学分会制定的《丙型肝炎防治指南》,基因型均为HCV 1型患者,且均接受标准干扰素联合利巴韦林抗病毒治疗一年,半年后检测HCV-RNA是否阳性。2.实验方法：收集这些患者经EDTA处理过的外周血液,运用分子生物学方法提取基因组DNA,PCR扩增后直接测序,对干扰素治疗应答与无应答的慢性丙型肝炎患者的SNPs rs8099917和rs12980275位点进行检测。3.统计学分析：应用SPSS 17.0统计软件进行数据分析,对干扰素抗病毒治疗应答与不应答的慢性丙型肝炎患者的IL28B基因多态性rs12980275和rs8099917位点的基因表型及等位基因频率进行分析统计,采用Pearsonx2和关联性检验进行等位基因单因素分析和关联分析,比较应答组和不应答组患者之间的频率差异,以比值比(odds ratio, OR)及其95%可信区间(confidential interval,CI)表示相对风险度,以R表示关联性。P值双侧,a=0.05,当P<0.05时认为差异有统计学意义。R>0为正相关,R<0为负相关。结果1.基因型频率：在63名SVR患者中,rs12980275基因型A/A 55人,占87%,A/G8人,占13%,在34名NVR患者中,rsl2980275基因型A/A 23人,占68%,A/G11人,占32%。在63名SVR患者中,rs8099917基因型T/T 55人,占87%,T/G 8人,占13%,在34名NVR患者中,rs8099917基因型T/T 24人,占71%,T/G 10人,占29%。在97名丙肝患者中rs12980275和rs8099917均未观测到G/G基因型。2.等位基因频率：在63名SVR患者中,rs12980275 A等位基因频率为93.7%,G等位基因频率为6.3%,在34名NVR患者中,rs12980275 A等位基因频率为83.8%,G等位基因频率为16.2%。在63名SVR患者中,rs8099917 T等位基因频率为93.7%,G等位基因频率为6.3%,在34名NVR患者中,rs8099917 T等位基因频率为85.3%,G等位基因频率为14.7%。3.对SVR组和NVR组的rs12980275基因型比较,OR=3.288,95%CI=[1.171-9.234],利用Pearson’s检验χ2=5.416,P=0.020<0.05,二者之间有显著差异,Pearson’s R=0.236>0,二者呈正相关。对SVR组和NVR组的rs8099917基因型比较,OR=2.865,95%CI=[1.006-8.154],利用Pearson’s检验χ2=4.081,P=0.04<30.05,二者之间有显著差异,Pearson’s R=0.205>0,二者呈正相关。对SVR和NVR的rs12980275等位基因频率比较,OR=2.846,95%CI=[1.086-7.464],利用Pearson’s检验χ2=4.828,P=0.028<0.05,二者之间有显著差异,Pearson’s R=0.158>0,二者呈正相关。对SVR和NVR的rs8099917等位基因频率比较,OR=2.543,95%CI=[0.953-6.785],利用Pearson’s检验χ2=3.664,P=0.056>0.05,二者之间无显著差异,Pearson’s R=0.137>0,二者呈正相关。结论1.通过对我院治疗的CHC患者IL28B基因多态性分析,SVR患者中SNPrs12980275位点的A等位基因频率为93.7%,G等位基因频率为6.3%,在NVR患者中分别为83.8%,16.2%,二者之间有显著差异。SVR患者中SNP rs8099917位点的T等位基因频率为93.7%,G等位基因频率为6.3%,在NVR患者中分别为85.3%,14.7%,二者之间无显著差异。rs12980275和rs8099917的基因型和等位基因频率在SVR、NVR组中的相关性分析Pearson’s R均大于0,相关性均呈正相关。2.rs12980275位点基因型主要为AA、AG、GG, rs12980275位点基因型主要为TT、TG、GG,而在我们的研究中rs12980275只检测到AA、AG,rs8099917只检测到TT、TG,二者均未发现GG型,rs12980275和rs8099917的GG型所在比例极低。3.rs12980275和rs8099917多态性可有效地预测干扰素抗病毒治疗应答效果。更多还原

【Abstract】 BackgroundHepatitis C is a global health problem and a chronic inflammation of the liver caused by hepatitis c virus. HCV is one of the main pathogens cause liver cirrhosis and hepatocellular carcinoma. The World Health Organization estimated, there were about 3% of the world population, i.e., more than 180 million people, currently infected, with 3-4 million new cases appearing each year. HCV infection affects more than 4 million people in China, about 3.2% serum infection rates. The primary source of HCV transmission is infected blood or blood products, unsafe injection, exposure to an infected sexual partner or having multiple sexual partners, tattooing and so on. Only 20%-30% of acute HCV-infected individuals recover spontaneously, with the remaining 70%-80% going on to develop chronic infection and 30 percent of them may progress to cirrhosis and hepatocellular carcinoma. Currently, the only effective treatments of hepatitis c virus infection is based in interferon treatment, the standard of care in patients with chronic hepatitis C, a combination of PEG-IFN-αwith ribavirin, does not produce SVR in all patients treated. In antiviral treatment process, several viral factors, such as genotype 1, high baseline viral load, viral kinetics during treatment, and amino acid pattern in the interferon sensitivity-determining region, these factors are significantly associated with the treatment outcome. However, only 42-52% of patients with hepatitis C genotype 1 on 48-week-long treatment can achieve sustained virologic response, and 5% of patients with genotype 2 or 3 have a null response to PEG-IFN-α/RBV, and 10-14% of patients require premature withdrawal from interferon-based therapy due to the side effects.In recent years, in Africa, Europe and East Asia cohorts, using Genome-wide Association Study identified Single Nucleotide Polymorphism in the IL-28B (encode IFNλ3) region as associated with spontaneous clearance of HCV and an effect on treatment response of interferon. Genetic variance could influence the spontaneous clearance of HCV and response to interferon of patients with hepatitis c. A single nucleotide polymorphism (rs8099917) located 8kb upstream of IL28B gene which located on chromosome 19 and encode IFN-λ3. Suppiah et al. and Rauch et al. demonstrated IL-28B polymorphisms in European cohorts, which were associated with an effect on treatment response and on treatment failure, respectively. Tanaka et al. identified IL-28B SNP rs8099917 to be associated with SVR in Japanese patients, which is a more profound effect than in European cohorts. They also found that SNP rs12980275 was associated with treatment response. The T/T genotype of rs8099917 can achieve SVR more possibly than G/G genotype. The A/A genotype of rs8099917 can also achieve SVR more possibly than G/G genotype.ObjectiveAt present, the relationship of IL28 alleles and interferon response is rare reported in our country. Therefore, we can retrospective study the condition of treatment response of interferon and IL28B gene polymorphism of chronic hepatitis c in our hospital, and compare with IL28B gene variants frequency of sustained virological response group and null virological response. To early predict curative effect of interferon provide theoretical basis. Methods1. Cases collected:Selected cases were all chronic hepatitis c patients, the diagnostic standard meets the criteria drawn up by Hepatology branch of Chinese medical association on March 2004, genotype is all HCV 1 type, and they are all accept antiretroviral treatment with a combination of interferon IFN and ribavirin (RBV) for one year, six months latter test HCV-RNA.2. Experimental methods:Collecting the peripheral blood treated by EDTA of these patients, using the molecular biology methods extract human genomic DNA, after PCR directly sequencing. The test was done by sequencing for SNPs genotyping of rs8099917 and rs12980275 of sustained virological response and null virological response patients. The allele frequency between the patients with sustained virological response and null virological response were compared to assess the effect of SNP.3. Statistical analysis:SPSS 17.0 statistics software was used to analyze data. We analyze gene phenotype and allele frequency of the IL28B gene polymorphism rs12980275 and rs8099917 for patients of chronic hepatitis c of interferon antiviral treatment response and not response, and allele single factor analysis and correlation analysis are used Pearson X 2 and relevance test. Compare the frequency difference of response group and not response groups, use odds ratio and 95% confidential interval signify relative risk degrees, R signify relevance. P value bilateral,α= 0.05, regard P< 0.05 as statistical significance. R> 0 for positive correlation, R< 0 for negatively correlated.Results1. Genotype Frequency:In 63 SVR patients, rs12980275 genotypes distribution (A/A n=55,87%; A/G n=8,13%), in 34 NVR patients, rs12980275 genotypes distribution (A/A n=23,68%; A/G n=11,32%). There was significant difference between the two groups (OR=3.288,95%CI=[1.171-9.234], Pearson’s x 2=5.416, P=0.020<0.05), and positive correlation between the two groups (Pearson’s R=0.236>0). In 63 SVR patients, rs8099917 genotypes distribution (T/T n=55,87%; T/G n=8, 13%), in 34 NVR patients, rs8099917 genotypes distribution (T/T n=24,71%; T/G n=10,29%). There was significant difference between the two groups (OR=2.865, 95%CI=[1.006-8.154], Pearson’s x 2=4.081, P=0.043<0.05), and positive correlation between the two groups (Pearson’s R=0.205>0).Two genotypes (TT and TG of rs8099917, AA and AG of rs12980275) were identified in the study. No GG genotype was founded.2. The frequency of A allele of rs12980275 was 93.7% verse 83.8% and G allele frequency is 6.3% verse 16.2% in SVR patients and NVR patients, respectively. There was significant difference between the two groups (OR=2.846, 95%CI= [1.086-7.464], Pearson’s x2=4.828, P=0.028<0.05), and positive correlation between the two groups (Pearson’s R=0.158>0).The frequency of T allele of rs8099917 was 93.7% verse 85.3% and G allele frequency is 6.3% verse 14.7% in SVR patients and NVR patients, respectively. There was no significant difference between the two groups (OR=2.543, 95%CI=[0.953-6.785], Pearson’s X2=3.664, P=0.056>0.05), and positive correlation between the two groups (Pearson’s R=0.137>0).Conclusion1. There was significant difference between the A allele of rs12980275 (93.7% to 83.8%) and G allele (6.3% to 16.2%) in SVR patients and NVR patients.There was no significant difference between the T allele of rs8099917 (93.7% to 85.3%) and G allele (6.3% to 14.7%) in SVR patients and NVR patients.2. Genotype of rs12980275 mainly are AA, AG, GG, rs12980275 mainly are TT, TG, GG, but in our studies rs12980275 only detect AA, AG, rs8099917 only detect TT and TG, there were not found GG type in both rs8099917 and rs 12980275, and the proportion of GG type of rs8099917 and rs12980275 very low.3. Polymorphism of SNPs rs8099917 and rs12980275 can effectively predict interferon antiviral treatment response effect.更多还原