本文關(guān)鍵詞： 乙型肝炎病毒 基因變異 耐藥 表型耐藥分析　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景我國(guó)目前有9300萬慢性乙型肝炎病毒(hepatitis B virus, HB V)感染者,每年有30萬人死于乙肝相關(guān)肝病。目前中國(guó)已上市的抗HBV藥核苷(酸)類(nucleos(t)itde analogs, NA)類藥物包括核苷類的拉米夫定(Lamivudine, LAM)、替比夫定(Telbivudine, LdT)、恩替卡韋(Entecavir, ETV)和核苷酸類的阿德福韋(Adefovir, ADV)、替諾福韋(Tenfovir, TDF)。NA藥物能夠有效抑制病毒,副作用小,但長(zhǎng)期治療過程中病毒易產(chǎn)生耐藥變異。HBV耐藥變異可引起病毒學(xué)突破和生化學(xué)突破,是抗HBV治療失敗或療效不佳的重要原因,可以造成肝炎再發(fā)和肝病進(jìn)展,甚至可引起暴發(fā)性肝衰竭導(dǎo)致患者死亡。本研究針對(duì)臨床上HBV耐藥變異中存在的難點(diǎn)和熱點(diǎn),綜合利用臨床大樣本測(cè)序、動(dòng)態(tài)隨訪、克隆測(cè)序、質(zhì)粒瞬時(shí)轉(zhuǎn)染細(xì)胞評(píng)價(jià)復(fù)制力和表型耐藥特性等多種研究手段,重點(diǎn)對(duì)以往研究結(jié)果不夠明確的HBV rtA181T、rtN238T 和 HB V基因型對(duì)耐藥的影響進(jìn)行了分析；同時(shí),對(duì)以往爭(zhēng)議較大的NA未治療患者的預(yù)存耐藥變異進(jìn)行了研究。結(jié)果可以加深對(duì)我國(guó)臨床實(shí)踐中的HBV耐藥變異情況的了解,向臨床優(yōu)化管理耐藥變異提供重要依據(jù)。目的本研究旨在：①研究較大樣本量NA經(jīng)治慢性HBV感染患者rtA181T/sW172的臨床流行情況,明確rtA181T變異的發(fā)生特點(diǎn)與臨床意義。② 分析ADV非經(jīng)典耐藥變異的臨床流行情況,對(duì)其中檢出率較高的HBV變異毒株進(jìn)行動(dòng)態(tài)克隆分析和表型耐藥檢測(cè)。③ 闡明我國(guó)流行的HBV B基因型和C基因型病毒是否與耐藥變異發(fā)生相關(guān)。④ 明確NA未治療患者預(yù)存耐藥變異的臨床檢出率,揭示預(yù)存耐藥變異毒株的表型耐藥特性和臨床演變規(guī)律。方法(1)通過聚合酶鏈?zhǔn)椒磻?yīng)(polymerase chain reaction, PCR)直接測(cè)序法對(duì)18419例慢性HBV感染患者進(jìn)行逆轉(zhuǎn)錄酶(reverse transcriptase, RT)基因測(cè)序,明確rtA181T/sW172各種變異形式所占比例,分析不同rtA181T/sW172變異形式患者HBV DNA和表面抗原(hepatitis B surface antigen, HBsAg)水平的差異。(2)從第一部分病例中選取11143例單獨(dú)應(yīng)用NA藥物治療或未應(yīng)用NA治療的慢性HBV感染者,分析其7種ADV非經(jīng)典耐藥位點(diǎn)的變異情況,即rtV84M, rtS85A, rtV214A, rtQ215S, rt1233V, rtP237H和rtN238T：對(duì)1例rtN238T患者進(jìn)行動(dòng)態(tài)克隆測(cè)序分析;構(gòu)建含有rtN238T+A181V、rtN238T、rtA181V和無耐藥變異野生株的pTriEx-1.1-HBV載體,瞬時(shí)轉(zhuǎn)染HepG2細(xì)胞,檢測(cè)上清中的病毒復(fù)制中單位本以評(píng)價(jià)各病毒株的復(fù)制力和對(duì)核苷(酸)類似物的耐藥性。(3)從第一部分病例中選取13847例經(jīng)歷過NA治療的非肝癌(hepatocacinoma, HCC)患者,應(yīng)用Mega 6軟件構(gòu)建系統(tǒng)進(jìn)化樹確定病毒基因型,分析不同基因型病毒中各種耐藥變異的分布情況。(4)從第一部分病例中選取845例未經(jīng)NA治療的住院患者,確定原發(fā)性耐藥變異的檢出率；構(gòu)建rtL80I+M204I、rtL180M+M204I 和 rtA181V+N236T3種變異株的pTriEx-1.1-HBV表達(dá)質(zhì)粒,并進(jìn)行復(fù)制力和表型耐藥分析；克隆測(cè)序分析1例預(yù)耐藥變異患者體內(nèi)病毒的動(dòng)態(tài)演變情況。結(jié)果(1)共檢出750例(4.1%)rtA181T變異,其發(fā)生率呈逐年升高的趨勢(shì)。發(fā)現(xiàn)rtA181T對(duì)應(yīng)sW172共有9種變異形式,其中166例(22.1%)為sW172*單獨(dú)存在,525例(69.9%)為sW172終止變異與野生株共存；不同sW172變異形式患者的HBV DNA水平和HBsAg水平并無差異。(2)在7種ADV非經(jīng)典耐藥變異中,rtN238T變異檢出率在ADV治療患者中顯著高于應(yīng)用其它NA藥物治療的患者。對(duì)1例患者動(dòng)態(tài)克隆分析發(fā)現(xiàn),發(fā)生rtN238T+A181V變異后,應(yīng)用ETV治療33個(gè)月,患者體內(nèi)病毒全部轉(zhuǎn)變?yōu)橐吧�。病毒株�?fù)制力檢測(cè)結(jié)果顯示rtN238T+A181V顯著高于rtA181V (P0.01)。表型耐藥分析顯示,與野生株比較,rtN238T+A181V 和 rtA181V變異株對(duì)ADV敏感性顯著下降,rtN238T變異株對(duì)ADV敏感,但1tN238T+A181V變異株較rtA181V株的復(fù)制力顯著提高。(3)LAM相關(guān)耐藥變異發(fā)生率為HBV/C型顯著高于HBV/B型(31.67% vs.25.26%)。整體來看ADV耐藥變異在HBV/C和HBV/B型之間并無差異(8.96%vs.9.87%)；具體來看,rtA181V的發(fā)生率為HBV/C型高于HBV/B型(5.29% vs.1.36%),但rtN236T變異則相反(2.70% vs. HBV/B 6.54%)。ETV藥變異也有類似的現(xiàn)象,整體來看ETV耐藥變異在HBV/C和HBV/B型之間并無差異(4.32%vs.3.62%);具體來看,rtM204V/I+rt184/202的發(fā)生率為HBV/C型高于H1BV/B型(3.66%vs.2.16%),但rtM204V/I+rt250變異則相反(0.67% vs.1.46%)。多重耐藥變異發(fā)生率也為HBV/C型顯著高于HBV/B型(0.83%vs.0.35%),主要變異形式是rtM204V/I+rtA181V(0.72% vs.0.30%)。(4)共有17例(2.01%)NA未用藥患者檢出原發(fā)耐藥變異,直序法和克隆測(cè)序法結(jié)果顯示所有患者均為耐藥變異株與野生株共存。通過克隆測(cè)序共發(fā)現(xiàn)了13種耐藥變異株,包括rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T, and rtN236T。表型耐藥分析顯示2種LAM預(yù)存耐藥變異株rtL80I+M204I 和 rtL180M+M204V與野生株相比,對(duì)LAM的耐藥倍數(shù)都大于1000;1種ADV預(yù)存耐藥變異株 rtA181V+N236T 對(duì) ADV的耐藥倍數(shù)為15.4。動(dòng)態(tài)隨訪發(fā)現(xiàn),預(yù)存耐藥變異rtM204I在ETV治療后在病毒池中的比例上升(20%→85%)。結(jié)論rtA181T變異株在臨床患者中常與rt181非變異株共存,單獨(dú)出現(xiàn)該變異未對(duì)HBsAg和HBV DN A水平有明顯影響;rtN238T變異可以增加rtA181V經(jīng)典耐藥變異株的復(fù)制力,是1種ADV耐藥相關(guān)的復(fù)制力補(bǔ)償變異;HBV/C基因型患者更易發(fā)生LAM和多重耐藥變異;在NA未治療患者中,預(yù)存耐藥變異的檢出率并不高,該類患者開始NA治療時(shí)耐藥檢測(cè)并非必要。
[Abstract]:The background of our country at present has 93 million chronic hepatitis B virus (hepatitis B virus, HB V) infection, 300 thousand people died of HBV related liver disease each year. At present, China has listed the anti HBV drug class of nucleoside (acid) (nucleos (T) itDe analogs, NA) drugs include nucleoside Rami J Ding (Lamivudine, LAM) telbivudine, (Telbivudine, LdT), entecavir (Entecavir, ETV) and nucleotides (Adefovir, ADV), A Duff Vee Nuo Fuwei (Tenfovir, TDF).NA drugs can effectively inhibit the virus, the side effect is small, but the treatment process of long period virus resistance mutation can cause.HBV mutations and virological breakthrough biochemical breakthrough, is the failure of anti HBV treatment or poor efficacy of the important reasons, can cause hepatitis recurrence and progress of liver disease, even can cause fulminant hepatic failure leads to death of the patient. This study for HBV mutations in clinically difficult And hot spots, the comprehensive utilization of a large clinical sample sequencing, dynamic follow-up, cloning and sequencing of plasmid transfected cell evaluation replication and phenotypic resistance characteristics of a variety of research methods, research results are not clear HBV rtA181T focus on the past, the effects of rtN238T and HB genotype of V gene on resistance are analyzed; at the same time, the resistance variation of deposit the controversial NA pre untreated patients were studied. The results can enhance the resistance to HBV variation in clinical practice in China to provide an important basis to clinical optimization management of drug resistance. The purpose of this study aims at: 1. Study of larger sample size NA by clinical epidemiological treatment of chronic HBV infection in patients with rtA181T/sW172. Clear the characteristics and clinical significance of rtA181T mutation. The clinical analysis of the prevalence of drug-resistant mutations in non classical ADV, of which HBV variation strain high detection rate Dynamic analysis and cloning of resistant phenotype detection. HBV genotype B and genotype C virus illuminate epidemic in China is related to drug resistance mutations. The clinical definite NA patients with pre-existing resistance mutation detection rate, phenotypic resistance characteristics reveal pre-existing resistance variation of strains and clinical evolution method (1. Polymerase chain reaction (polymerase) by chain reaction, PCR) direct sequencing method in 18419 cases of patients with chronic HBV infection by reverse transcriptase (reverse transcriptase RT) gene sequencing, clear rtA181T/sW172 variation form of the proportion of cases, analysis of different rtA181T/sW172 variants in patients with HBV DNA (hepatitis B surface and surface antigen antigen, HBsAg) level the difference. (2) infection selected by NA alone in the treatment of 11143 cases of drug cases from the first part or application of NA for the treatment of chronic HBV, analysis of the 7 kinds of non classical ADV resistance Mutation sites, namely rtV84M, rtS85A, rtV214A, rtQ215S, rt1233V, rtP237H and rtN238T: dynamic analysis of cloning and sequencing of 1 rtN238T patients; to construct rtN238T+A181V rtN238T, rtA181V, and pTriEx-1.1-HBV carrier no resistance of wild strains, transient transfection of HepG2 cells detected in the supernatant of virus replication in this unit evaluation of the virus replication and drug resistance to nucleoside (acid) analogues. (3) a total of 13847 patients with non HCC experienced NA treatment from the first part of the cases in (hepatocacinoma, HCC) patients, viral genotypes should be determined by the Mega 6 software to construct phylogenetic tree analysis, distribution of various resistance variation genotype of the virus. (4) from the first part were selected in 845 cases without NA patients, to determine the detection rate of primary drug resistance; construction of rtL80I+ M204I, rtL180M+M204I and rtA181V+N The 236T3 mutant pTriEx-1.1-HBV expression plasmid, and analyzed replication and phenotypic resistance; dynamic cloning and sequencing analysis of 1 cases of drug resistance in patients with pre virus evolution. Results (1) were detected in 750 cases (4.1%) rtA181T mutation, its incidence was the trend of increased year by year. RtA181T sW172 has found the corresponding 9 kinds of variation form, of which 166 cases (22.1%) are separately for sW172*, 525 cases (69.9%) for the sW172 mutation and wild strains of HBV DNA coexist; and HBsAg level had no difference in different sW172 variant patients. (2) in the 7 kinds of non classical ADV mutation, rtN238T mutation detection rate in ADV treatment group was significantly higher than that of other NA drugs in the treatment of patients. In 1 patients with dynamic clonal analysis showed that the rtN238T+A181V mutation occurred after the application of ETV for 33 months, the virus in patients with completely converted to wild strains. Disease strain replication detection The results showed that rtN238T+A181V was significantly higher than that of rtA181V (P0.01). Phenotypic resistance analysis showed that compared with wild-type, rtN238T+A181V mutant and rtA181V sensitivity to ADV decreased significantly, rtN238T mutants were sensitive to ADV, but the 1tN238T+A181V mutant than rtA181V replication strains increased significantly. (3) LAM mutation incidence of HBV/C significantly higher than HBV/B (31.67% vs.25.26%). The whole ADV mutations between HBV/C and HBV/B had no difference (8.96%vs.9.87%); specifically, the incidence of rtA181V was higher than that of the HBV/B type HBV/C (5.29% vs.1.36%), but the rtN236T variation is opposite (2.70% vs. 6.54% HBV/B).ETV drug variant is also a similar phenomenon, the overall the ETV mutation between HBV/C and HBV/B had no difference (4.32%vs.3.62%); specifically, the incidence of rtM204V/I+rt184/202 was higher than that of the H1BV/B type HBV/C (3.66%vs.2.16%), but rtM204V The /I+rt250 variation is opposite (0.67% vs.1.46%). Multidrug resistant mutation rate for type HBV/C was significantly higher than that of HBV/B (0.83%vs.0.35%), the main form of variation is rtM204V/I+rtA181V (0.72% vs.0.30%). (4) a total of 17 cases (2.01%) of NA were detected in untreated primary resistance variation, direct sequence and clone sequencing method the results showed that all patients were mutant and wild strains coexist. By cloning and sequencing found a total of 13 resistant strains, including rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+, L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T, and rtN236T. analysis showed that the phenotypic resistance of 2 kinds of LAM pre existing resistance rtL80I+M204I and rtL180M+M204V strains and wild strains compared to multiple drug resistance of LAM are greater than 1000; 1 ADV pre existing resistance mutant rtA181V+N236T of A DV multiple drug resistance for the discovery of 15.4. dynamic follow-up, pre-existing resistance variation of rtM204I after ETV treatment in the virus pool increased (20% - 85%). Conclusion rtA181T mutation in patients with rt181 and non mutant often coexist, it appears alone did not have obvious influence on the variation of HBsAg and HBV DN A levels; rtN238T mutation can increase the replication capacity of classic rtA181V resistant mutant, replication is 1 ADV resistance compensation variation; HBV/C genotype were more susceptible to LAM and multidrug resistance variation; in untreated NA patients, the detection rate of pre-existing resistance variation is not high, the patients began treatment resistant NA the test is not necessary.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R512.62

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