慢性乙型肝炎患者抗病毒治疗疗效相关的遗传因素研究

发布时间：2018-01-15 15:37

本文关键词：慢性乙型肝炎患者抗病毒治疗疗效相关的遗传因素研究　出处：《安徽医科大学》2013年博士论文　论文类型：学位论文

【摘要】：背景慢性乙型肝炎病毒(HBV)感染后的经典三步曲即慢性乙型肝炎(CHB)、肝硬化和原发性肝癌。抗病毒是慢性乙型肝炎的主要治疗方法，包括干扰素(IFN-ot)和核苷(酸)类似物，以清除病毒、延迟肝硬化、肝癌的进展以及提高生存率为长期治疗目标。长期以来，肝功能、乙肝五项和乙型肝炎病毒载量(HBVDNA)等实验室指标一直是评估乙肝患者病情的经典标志。随着科技的进步，许多与CHB患者抗病毒疗效相关的遗传背景标志物开始出现。本课题组前期采用前瞻性研究发现雌激素受体1(ESR1)单核苷酸多态性(SNP)与IFN-a初治患者早期病毒学应答相关。除了本课题组之外，尚无关于ESR1SNP与CHB患者抗病毒疗效的相关性研究，因此，本研究在扩大样本量、延长随访时间以及增加相关SNPs的基础上进一步分析基因单核苷酸多态性、基因mRNA表达水平以及干扰素抗病毒疗效之间的相关性，IFN-a和恩替卡韦(ETV)均因疗效好耐药率低而被视为CHB初治患者的一线用药，我们对ESR1SNP与恩替卡韦抗病毒治疗的可能相关性也进行了分析，以期为CHB患者干扰素和恩替卡韦抗病毒治疗提供新的预测因素。目的以IFN-a和ETV初始抗病毒治疗的CHB患者为研究对象，选取了3条基因0识7、MxA和e/F-2a)和7个SNP(rs2077647、rs9340799、rs2234693、rs322354、rs2071430、rs3759755以及rs3759756)，，对CHB患者抗病毒治疗(IFN-a和ETV)疗效与宿主基线特征、基因单核苷酸多态性以及基因mRNA表达水平的相关性进行了探讨。方法本课题共收集了248例初始抗病毒治疗的CHB患者,包括172例使用IFN-α抗病毒治疗的HBeAg阳性患者和76例使用ETV抗病毒治疗的HBeAg阳性或阴性的患者。我们对患者抗病毒治疗第4周、12周、24周、48周、72周以及96周的病毒学应答进行评估,采用QIAGEN DNA抽提试剂盒对所有病例进行了基因组DNA的提取,综合使用限制性片段长度多态性(PCR-RFLP)及质谱分析的方法对ESR1rs2077647、rs9340799、rs2234693和rs322354,eIF-2a rs3759755和rs3759756以及MxA rs2071430进行了基因型分型,采用荧光染料实时定量(SYBR Green RT-PCR)法检测了103例IFN-α抗病毒治疗患者治疗前、中、后各时间点外周血单个核细胞(PBMCs)中的3条基因(ESR1、eIF-2α和MxA)mRNA的表达水平以及采用凝胶移动抑制分析(EMSA)实验检测了eIF-2α基因rs3759755不同等位的核蛋白结合能力。采用SPSS13.0软件对数据资料进行统计分析。结果 1.通过多因素分析,我们发现基线HBVDNA水平与IFN-α快速病毒学应答(OR=1.718,95%CI:1.184-2.492, P=0.004)和早期病毒学应答(OR=1.457,95%CI:1.008-2.107, P=0.045)均有显著相关性,且发生快速病毒学应答(RVR)的患者更易发生早期病毒学应答(OR=9.123,95%CI：3.507-23.735,P0.001)；接受IFN-α治疗的患者中基线HBsAg水平(OR：1.000,95%CI：1.000-1.000,P=0.018)、基线HBeAg水平≤200S/CO (OR:0.009,95%CI:0.001-0.077,P0.001)、12周HBeAg≤10S/CO (OR:0.196,95%CI:0.069-0.560,P=0.002)以及早期病毒学应答(OR：0.101,95%CI：0.022-0.470,P=0.003)与72周病毒学应答均有显著相关性。基线HBVDNA水平对恩替卡韦24周、48周病毒学应答有重要的预测意义(P0.05)。 2.我们在172例IFN-a初治HBeAg阳性CHB患者中对3条基因上的7个SNPs进行了分型(ESR1基因的rs2077647、rs9340799、rs2234693和rs322354; MxA基因的rs2071430;eIF-2α基因的rs3759755和rs3759756),结果发现早期病毒学应答率在rs2077647TT、TC和CC基因型组间存在显著差异(χ2=6.240,P)=0.044),且携带至少一个rs2077647T等位基因的患者早期病毒学无应答率显著高于CC基因型(TT+TC vs.CC,χ2=6.189, P=0.013),但在校正基线ALT、基线HBVDNA以及RVR之后差异均无统计学意义(分别为OR：0.727,95%CI：0.279-1.889,P=0.512和OR：1.585,95%CI：0.277-9.060,P=0.604)；我们的单因素分析结果还显示72周病毒学应答率在rs2234693TT、TC和CC基因型组中存在显著差异(χ2=6.478,P=0.039),且携带至少一个rs2234693C等位基因的患者72周病毒学应答率显著高于CC基因型(66.4%vs.46.4%,χ2=6.258,P=0.012); rs2071430GG、GT和TT基因型组间的72周病毒学应答率也存在显著差异(χ2=8.079,P=0.018),携带TT基因型患者72周病毒学无应答率显著高于至少携带至少一个C等位基因的患者(66.6%vs.37.0%,χ2=5.899,P=0.015)；rs3759756AG基因型组中的72周病毒学无应答率明显高于AA基因型(77.3%vs.34.7%,2=14.498,P0.001),但在校正基线HBsAg水平、基线HBeAg水平、12周HBeAg水平、RVR及EVR之后,只有.(?)IF-2α基因上的rs3759756与IFN-α72周病毒学应答显著相关(OR=37.988,95%CI：1.665-86.6737,P=0.023),提示携带rs3759756AG基因型的患者在72周发生病毒学无应答的几率远远高于AA基因型患者。 3.我们在76例恩替卡韦初治的CHB患者中检测了ESRl基因上的2个SNPs(rs2234693和rs9340799),单因素分析结果显示48周、96病毒学应答率在rs2234693TT、TC和CC基因型组间均存在显著性差异(分别为57.1%vs.87.8%vs.58.3%,P=0.012和64.3%vs.96.7%vs.87.5%,P=0.018),在校正了基线TBIL水平后发现,至少携带一个rs2234693C等位基因的患者48周、96周病毒学应答率均明显高于TT基因型患者(分别为81.1%vs.57.1%,95%CI：1.026-14.785,P=0.046和94.7%vs.64.3%,95%CI：1.456-57.509,P=0.018)。 4.通过检测103例接受FN-α治疗的HBeAg阳性ICHB患者中的ESR1、MxA和.?IF-2α基因在IFN-α治疗前、中及治疗后个时间点1nRNA的表达水平,发现女性患者72周病毒学应答组的基线mRNA水平显著高于无应答组(9.2722±6.448vs.6.041±6.738,Z=-0.207,P=0.038),且基线ESR1RNA表达水平与基线HBVDNA呈负相关(r=-0.750,P0.001)。男性患者随访至72周时,病毒学应答组的ESR1mRNA水平显著高于无应答组(21.838±39.775vs.1.929±1.174,Z=-2.162,P=0.031)。MxA和eIF-2a基因的1nRNA表达水平在IFN-α治疗前后均无显著变化(P0.05)。 5.携带eIF-2α rs3759756AG基因型的CHB患者中IFN-a72周病毒学无应答率显著高于AA基因型患者,凝胶移动抑制分析(EMSA)实验发现rs3759756A、G特异性等位位点存在核蛋白结合差异,且A等位与核蛋白的结合量约为G等位的3倍,AA和AG基因型患者中eIF-2α mRNA表达水平的差异可能与此有关。结论 1.基线HBVDNA水平和ESR1rs2234693对恩替卡韦的病毒学应答均有重要的预测意义。 2.在IFN-a初治的HBeAg阳性CHB患者中,基线HBVDNA水平和RVR对快速和早期病毒学应答均有预测意义；基线HBsAg水平、HBeAg水平≤200S/CO、12周HBeAg水平≤10S/CO、EVR以及.?IF-2a rs3759756对72周病毒学应答有重要的预测意义；女性患者中,治疗前PBMC ESR1mRNA表达水平对72周病毒学应答有预测意义,且与基线HBVDNA水平负相关；干扰素能显著增加72周病毒学应答男性患者中ESR1mRNA的表达水平。 3. EIF-2αmRNA表达水平与IFN-α病毒学应答无显著相关性,但在rs3759756AA和AG基因型组中的分布存在显著差异,可能与rs3759756两种等位对核蛋白的结合能力的差异有关。
[Abstract]:background
Chronic hepatitis B virus (HBV) infection after the classical three steps of chronic hepatitis B (CHB), liver cirrhosis and hepatocellular carcinoma. Antiviral is the main method for the treatment of chronic hepatitis B (IFN-ot), including interferon and nucleoside analogues (acid), to remove the virus, delay the progression of hepatocellular carcinoma and liver cirrhosis. To improve the survival rate of the long-term goals of treatment. For a long time, liver function, five items of hepatitis B and hepatitis B virus load (HBVDNA) and other laboratory indicators has been a classic sign of assessment of hepatitis B patients. With the progress of science and technology, many patients with CHB antiviral efficacy of genetic background markers began to appear. Ourprevious by a prospective study found that estrogen receptor 1 (ESR1) single nucleotide polymorphism (SNP) associated with IFN-a initial treatment of early virological response patients. In addition to this group, there is no effect on the ESR1SNP and CHB patients with antiviral The correlation studies, therefore, this study in a larger sample and longer follow-up times based on further analysis of SNPs related gene polymorphism, the correlation between the expression levels of mRNA gene and interferon antiviral efficacy, IFN-a and entecavir (ETV) with good curative effect and low rate of resistance is regarded as the initial treatment of CHB patients with first-line drugs, we may on the correlation between ESR1SNP and entecavir were also analyzed, in order to provide a new predictor for CHB patients with interferon and entecavir.
objective
The IFN-a and ETV of initial therapy in CHB patients as the research object, selects 3 genes MxA and e/F-2a 0 general 7, 7) and SNP (rs2077647, rs9340799, rs2234693, rs322354, rs2071430, rs3759755, CHB and rs3759756) on patients with antiviral therapy (IFN-a and ETV) and the effect of host baseline characteristics, correlation gene polymorphism and gene expression level of mRNA was discussed.
Method
This paper collected a total of 248 cases of initial treatment of CHB patients, including patients with positive HBeAg IFN- alpha antiviral therapy in 172 cases and 76 cases of the use of antiviral therapy for ETV HBeAg positive or negative patients. We in patients with antiviral therapy for fourth weeks, 12 weeks, 24 weeks, 48 weeks, 72 weeks and 96 virological response week assessment, QIAGEN was extracted by DNA extraction kit of genomic DNA in all cases, using restriction fragment length polymorphism (PCR-RFLP) method and rs9340799 mass spectrometry analysis of ESR1rs2077647, rs2234693, and rs322354, eIF-2a and rs3759755 rs3759756 and MxA rs2071430 were genotype by real-time fluorescent dye quantitative (SYBR Green RT-PCR) were detected in patients treated with IFN- alpha antiviral therapy in 103 patients, in different time points after peripheral blood mononuclear cells (PBMCs) in 3 genes (ESR1, eIF-2 and Mx A) mRNA expression level and gel mobility inhibition assay (EMSA) were used to detect the binding ability of eIF-2 rs3759755 gene to different alleles. Data were analyzed by SPSS13.0 software.
Result
1. by multivariate analysis, we found that the baseline level of HBVDNA and IFN- a rapid virological response (OR=1.718,95%CI:1.184-2.492, P=0.004) and early virological response (OR=1.457,95%CI:1.008-2.107, P=0.045) had significant correlation, and the occurrence of rapid virological response (RVR) were more prone to early virological response (OR=9.123,95%CI:3.507-23.735, P0.001); the baseline level of HBsAg received IFN- treatment patients (OR:1.000,95%CI:1.000-1.000, P=0.018), the baseline level of HBeAg = 200S/CO (OR:0.009,95%CI:0.001-0.077, P0.001), 12 week HBeAg is less than or equal to 10S/CO (OR:0.196,95%CI:0.069-0.560, P=0.002) and early virological response (OR:0.101,95%CI:0.022-0.470, P=0.003) and 72 week virologic response. There were significant correlation between baseline HBVDNA levels of entecavir for 24 weeks, 48 weeks virologic response is important the predictive significance (P0.05).
2. of our patients with initial treatment in 172 cases of IFN-a HBeAg positive CHB in the 7 SNPs 3 gene were divided into type (ESR1 gene rs2077647, rs9340799, rs2234693 and rs322354; MxA rs2071430 gene; eIF-2 gene rs3759755 and rs3759756), the early virological response rate in rs2077647TT, there was significant the difference between TC and CC genotype groups (2=6.240, P) =0.044), and patients with at least one early virologic carrying rs2077647T allele non response rate was significantly higher than CC genotype (TT+TC, vs.CC, X 2=6.189, P=0.013), but the school based line ALT, HBVDNA and RVR after the baseline differences were not statistically significant (respectively OR:0.727,95%CI:0.279-1.889, P=0.512 and OR:1.585,95%CI:0.277-9.060, P=0.604); single factor analysis also shows the results of our 72 week virologic response rate in rs2234693TT, there was significant difference between TC and CC genotype group (x2 2=6 .478, P=0.039), and with at least one rs2234693C allele in patients with 72 week virologic response rate was significantly higher than that of the CC genotype (66.4%vs.46.4%, X 2=6.258, P=0.012); rs2071430GG, GT and TT 72 week virologic response rates between genotype groups have significant difference (2= 8.079, P=0.018), TT gene patients carrying 72 week virologic response rate was significantly higher than that without carrying at least one C allele in the patients (66.6%vs.37.0%, X 2=5.899, P=0.015); rs3759756AG genotype group in the 72 week virologic nonresponse rate was significantly higher than that of the AA genotype (77.3%vs.34.7%, 2=14.498, P0.001), but after adjustment for baseline HBsAg levels, baseline the level of HBeAg, 12 weeks after RVR, HBeAg level, and EVR only. (?) rs3759756 was significantly correlated with IFN- alpha 72 week virologic response on IF-2 alpha gene (OR=37.988,95%CI:1.665-86.6737, P=0.023), suggesting that carrying rs3759756AG genotype in 72 patients Zhou Fasheng's virology is far more likely to be unresponsive than the AA genotype.
3. among the 76 cases of entecavir initiallytreating CHB patients detected 2 SNPs of ESRl gene (rs2234693 and rs9340799), the single factor analysis showed that the 48 week virologic response rate in rs2234693TT 96, there were significant differences in TC and CC genotype groups (respectively 57.1%vs.87.8% and vs.58.3%, P=0.012 64.3%vs.96.7%vs.87.5%, P=0.018), found in the baseline level of TBIL after correction, carry at least one rs2234693C allele in 48 weeks, 96 weeks virologic response rate were significantly higher in patients with TT genotype (81.1%vs.57.1%, 95%CI:, 1.026-14.785, P=0.046 and 94.7%vs.64.3%, 95%CI:1.456-57.509, P=0.018).
4. through the detection of 103 cases accepted FN- treatment of HBeAg positive patients with ICHB in ESR1, MxA and IF-2.? alpha gene in IFN- alpha before treatment, the expression level of 1nRNA in time and after treatment, found that women with 72 week virologic response group baseline mRNA level was significantly higher than that of the non response group (9.2722 + 6.448vs.6.041 + 6.738, Z=-0.207, P=0.038), and the baseline ESR1RNA expression was negatively correlated with baseline HBVDNA (r=-0.750, P0.001). Male patients with follow-up at 72 weeks, virological response group ESR1mRNA were significantly higher than that of the non response group (21.838 + 39.775vs.1.929 + 1.174, Z=-2.162, P=0.031) expression levels of.MxA and eIF-2a gene 1nRNA no significant changes before and after IFN- treatment (P0.05).
No IFN-a72 weeks virologic response rate was significantly higher than that of AA genotype 5. patients carrying eIF-2 alpha rs3759756AG gene type CHB patients, analysis of mobile gel (EMSA) inhibition test showed that rs3759756A, G specific alleles and nuclear protein binding differences, and combined with the A allele and nuclear protein was about 3 times higher than that of G allele the differences may be related to this eIF-2 alpha mRNA expression of AA and AG genotype patients.
conclusion
1. the baseline HBVDNA level and ESR1rs2234693 have important predictive value for the virological response of entecavir.
2. IFN-a in the early treatment of HBeAg positive CHB patients, baseline HBVDNA levels and RVR on rapid and early virological response had prognostic significance; baseline HBsAg levels, HBeAg levels less than or equal to 200S/CO, 12 weeks HBeAg level less than or equal to 10S/CO, EVR and IF-2a.? rs3759756 has important prognostic significance for the 72 week virologic response; female patients. Before treatment, PBMC expression level of ESR1mRNA has significance for prediction of 72 week virologic response, and baseline HBVDNA levels negatively correlated; interferon could significantly increase the expression level of ESR1mRNA 72 week virologic response to male patients.
3., there was no significant correlation between the expression level of EIF-2 mRNA and the IFN- virological response, but there was significant difference in the distribution of rs3759756AA and AG genotypes. It may be related to the difference of the two kinds of alleles of rs3759756 on the binding capacity of nucleoprotein.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

【共引文献】