本文關(guān)鍵詞：慢性乙型肝炎患者抗病毒治療療效相關(guān)的遺傳因素研究　出處：《安徽醫(yī)科大學(xué)》2013年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 慢性乙型肝炎 單核苷酸多態(tài)性 外周血單個(gè)核細(xì)胞 抗病毒治療 療效

【摘要】：背景 慢性乙型肝炎病毒(HBV)感染后的經(jīng)典三步曲即慢性乙型肝炎(CHB)、肝硬化和原發(fā)性肝癌。抗病毒是慢性乙型肝炎的主要治療方法，包括干擾素(IFN-ot)和核苷(酸)類似物，以清除病毒、延遲肝硬化、肝癌的進(jìn)展以及提高生存率為長(zhǎng)期治療目標(biāo)。長(zhǎng)期以來(lái)，肝功能、乙肝五項(xiàng)和乙型肝炎病毒載量(HBVDNA)等實(shí)驗(yàn)室指標(biāo)一直是評(píng)估乙肝患者病情的經(jīng)典標(biāo)志。隨著科技的進(jìn)步，許多與CHB患者抗病毒療效相關(guān)的遺傳背景標(biāo)志物開始出現(xiàn)。本課題組前期采用前瞻性研究發(fā)現(xiàn)雌激素受體1(ESR1)單核苷酸多態(tài)性(SNP)與IFN-a初治患者早期病毒學(xué)應(yīng)答相關(guān)。除了本課題組之外，尚無(wú)關(guān)于ESR1SNP與CHB患者抗病毒療效的相關(guān)性研究，因此，本研究在擴(kuò)大樣本量、延長(zhǎng)隨訪時(shí)間以及增加相關(guān)SNPs的基礎(chǔ)上進(jìn)一步分析基因單核苷酸多態(tài)性、基因mRNA表達(dá)水平以及干擾素抗病毒療效之間的相關(guān)性，IFN-a和恩替卡韋(ETV)均因療效好耐藥率低而被視為CHB初治患者的一線用藥，我們對(duì)ESR1SNP與恩替卡韋抗病毒治療的可能相關(guān)性也進(jìn)行了分析，以期為CHB患者干擾素和恩替卡韋抗病毒治療提供新的預(yù)測(cè)因素。 目的 以IFN-a和ETV初始抗病毒治療的CHB患者為研究對(duì)象，選取了3條基因0識(shí)7、MxA和e/F-2a)和7個(gè)SNP(rs2077647、rs9340799、rs2234693、rs322354、rs2071430、rs3759755以及rs3759756)，，對(duì)CHB患者抗病毒治療(IFN-a和ETV)療效與宿主基線特征、基因單核苷酸多態(tài)性以及基因mRNA表達(dá)水平的相關(guān)性進(jìn)行了探討。 方法 本課題共收集了248例初始抗病毒治療的CHB患者,包括172例使用IFN-α抗病毒治療的HBeAg陽(yáng)性患者和76例使用ETV抗病毒治療的HBeAg陽(yáng)性或陰性的患者。我們對(duì)患者抗病毒治療第4周、12周、24周、48周、72周以及96周的病毒學(xué)應(yīng)答進(jìn)行評(píng)估,采用QIAGEN DNA抽提試劑盒對(duì)所有病例進(jìn)行了基因組DNA的提取,綜合使用限制性片段長(zhǎng)度多態(tài)性(PCR-RFLP)及質(zhì)譜分析的方法對(duì)ESR1rs2077647、rs9340799、rs2234693和rs322354,eIF-2a rs3759755和rs3759756以及MxA rs2071430進(jìn)行了基因型分型,采用熒光染料實(shí)時(shí)定量(SYBR Green RT-PCR)法檢測(cè)了103例IFN-α抗病毒治療患者治療前、中、后各時(shí)間點(diǎn)外周血單個(gè)核細(xì)胞(PBMCs)中的3條基因(ESR1、eIF-2α和MxA)mRNA的表達(dá)水平以及采用凝膠移動(dòng)抑制分析(EMSA)實(shí)驗(yàn)檢測(cè)了eIF-2α基因rs3759755不同等位的核蛋白結(jié)合能力。采用SPSS13.0軟件對(duì)數(shù)據(jù)資料進(jìn)行統(tǒng)計(jì)分析。 結(jié)果 1.通過(guò)多因素分析,我們發(fā)現(xiàn)基線HBVDNA水平與IFN-α快速病毒學(xué)應(yīng)答(OR=1.718,95%CI:1.184-2.492, P=0.004)和早期病毒學(xué)應(yīng)答(OR=1.457,95%CI:1.008-2.107, P=0.045)均有顯著相關(guān)性,且發(fā)生快速病毒學(xué)應(yīng)答(RVR)的患者更易發(fā)生早期病毒學(xué)應(yīng)答(OR=9.123,95%CI：3.507-23.735,P0.001)；接受IFN-α治療的患者中基線HBsAg水平(OR：1.000,95%CI：1.000-1.000,P=0.018)、基線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)以及早期病毒學(xué)應(yīng)答(OR：0.101,95%CI：0.022-0.470,P=0.003)與72周病毒學(xué)應(yīng)答均有顯著相關(guān)性。基線HBVDNA水平對(duì)恩替卡韋24周、48周病毒學(xué)應(yīng)答有重要的預(yù)測(cè)意義(P0.05)。 2.我們?cè)?72例IFN-a初治HBeAg陽(yáng)性CHB患者中對(duì)3條基因上的7個(gè)SNPs進(jìn)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),結(jié)果發(fā)現(xiàn)早期病毒學(xué)應(yīng)答率在rs2077647TT、TC和CC基因型組間存在顯著差異(χ2=6.240,P)=0.044),且攜帶至少一個(gè)rs2077647T等位基因的患者早期病毒學(xué)無(wú)應(yīng)答率顯著高于CC基因型(TT+TC vs.CC,χ2=6.189, P=0.013),但在校正基線ALT、基線HBVDNA以及RVR之后差異均無(wú)統(tǒng)計(jì)學(xué)意義(分別為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)；我們的單因素分析結(jié)果還顯示72周病毒學(xué)應(yīng)答率在rs2234693TT、TC和CC基因型組中存在顯著差異(χ2=6.478,P=0.039),且攜帶至少一個(gè)rs2234693C等位基因的患者72周病毒學(xué)應(yīng)答率顯著高于CC基因型(66.4%vs.46.4%,χ2=6.258,P=0.012); rs2071430GG、GT和TT基因型組間的72周病毒學(xué)應(yīng)答率也存在顯著差異(χ2=8.079,P=0.018),攜帶TT基因型患者72周病毒學(xué)無(wú)應(yīng)答率顯著高于至少攜帶至少一個(gè)C等位基因的患者(66.6%vs.37.0%,χ2=5.899,P=0.015)；rs3759756AG基因型組中的72周病毒學(xué)無(wú)應(yīng)答率明顯高于AA基因型(77.3%vs.34.7%,2=14.498,P0.001),但在校正基線HBsAg水平、基線HBeAg水平、12周HBeAg水平、RVR及EVR之后,只有.(?)IF-2α基因上的rs3759756與IFN-α72周病毒學(xué)應(yīng)答顯著相關(guān)(OR=37.988,95%CI：1.665-86.6737,P=0.023),提示攜帶rs3759756AG基因型的患者在72周發(fā)生病毒學(xué)無(wú)應(yīng)答的幾率遠(yuǎn)遠(yuǎn)高于AA基因型患者。 3.我們?cè)?6例恩替卡韋初治的CHB患者中檢測(cè)了ESRl基因上的2個(gè)SNPs(rs2234693和rs9340799),單因素分析結(jié)果顯示48周、96病毒學(xué)應(yīng)答率在rs2234693TT、TC和CC基因型組間均存在顯著性差異(分別為57.1%vs.87.8%vs.58.3%,P=0.012和64.3%vs.96.7%vs.87.5%,P=0.018),在校正了基線TBIL水平后發(fā)現(xiàn),至少攜帶一個(gè)rs2234693C等位基因的患者48周、96周病毒學(xué)應(yīng)答率均明顯高于TT基因型患者(分別為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.通過(guò)檢測(cè)103例接受FN-α治療的HBeAg陽(yáng)性ICHB患者中的ESR1、MxA和.?IF-2α基因在IFN-α治療前、中及治療后個(gè)時(shí)間點(diǎn)1nRNA的表達(dá)水平,發(fā)現(xiàn)女性患者72周病毒學(xué)應(yīng)答組的基線mRNA水平顯著高于無(wú)應(yīng)答組(9.2722±6.448vs.6.041±6.738,Z=-0.207,P=0.038),且基線ESR1RNA表達(dá)水平與基線HBVDNA呈負(fù)相關(guān)(r=-0.750,P0.001)。男性患者隨訪至72周時(shí),病毒學(xué)應(yīng)答組的ESR1mRNA水平顯著高于無(wú)應(yīng)答組(21.838±39.775vs.1.929±1.174,Z=-2.162,P=0.031)。MxA和eIF-2a基因的1nRNA表達(dá)水平在IFN-α治療前后均無(wú)顯著變化(P0.05)。 5.攜帶eIF-2α rs3759756AG基因型的CHB患者中IFN-a72周病毒學(xué)無(wú)應(yīng)答率顯著高于AA基因型患者,凝膠移動(dòng)抑制分析(EMSA)實(shí)驗(yàn)發(fā)現(xiàn)rs3759756A、G特異性等位位點(diǎn)存在核蛋白結(jié)合差異,且A等位與核蛋白的結(jié)合量約為G等位的3倍,AA和AG基因型患者中eIF-2α mRNA表達(dá)水平的差異可能與此有關(guān)。 結(jié)論 1.基線HBVDNA水平和ESR1rs2234693對(duì)恩替卡韋的病毒學(xué)應(yīng)答均有重要的預(yù)測(cè)意義。 2.在IFN-a初治的HBeAg陽(yáng)性CHB患者中,基線HBVDNA水平和RVR對(duì)快速和早期病毒學(xué)應(yīng)答均有預(yù)測(cè)意義；基線HBsAg水平、HBeAg水平≤200S/CO、12周HBeAg水平≤10S/CO、EVR以及.?IF-2a rs3759756對(duì)72周病毒學(xué)應(yīng)答有重要的預(yù)測(cè)意義；女性患者中,治療前PBMC ESR1mRNA表達(dá)水平對(duì)72周病毒學(xué)應(yīng)答有預(yù)測(cè)意義,且與基線HBVDNA水平負(fù)相關(guān)；干擾素能顯著增加72周病毒學(xué)應(yīng)答男性患者中ESR1mRNA的表達(dá)水平。 3. EIF-2αmRNA表達(dá)水平與IFN-α病毒學(xué)應(yīng)答無(wú)顯著相關(guān)性,但在rs3759756AA和AG基因型組中的分布存在顯著差異,可能與rs3759756兩種等位對(duì)核蛋白的結(jié)合能力的差異有關(guān)。
[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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.62

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào)：1428994