本文關(guān)鍵詞： HIV 抗逆轉(zhuǎn)錄病毒治療 耐藥 低病毒載量 非核苷類抑制劑 補(bǔ)償性位點(diǎn) 病毒進(jìn)化　出處：《中國(guó)疾病預(yù)防控制中心》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：艾滋病抗逆轉(zhuǎn)錄病毒治療(Antiretroviral Therapy, ART)自20世紀(jì)90年代起在全世界范圍內(nèi)已廣泛開展,已由最初的齊多夫定(Zidovudine, AZT)發(fā)展至七大類33種藥物。ART的開展給全世界的艾滋病患者帶來了福音,幫助其延緩了病情的發(fā)展,并提高了生活質(zhì)量。我國(guó)自2003年開始逐步推廣免費(fèi)的抗逆轉(zhuǎn)錄病毒治療,艾滋病的病死率已由2003年的9.5死亡/100人年降低至了2009年的5.2死亡/人年。雖然我國(guó)的ART已初步取得了一定的成績(jī),但是由于抗病毒治療早期藥物資源有限,相當(dāng)數(shù)量患者在接受ART治療后不得不長(zhǎng)期使用同一種治療方案。而眾多研究表明長(zhǎng)期治療下HIV耐藥株的產(chǎn)生則是影響抗病毒治療效果的主要因素。HIV病毒的生物學(xué)特點(diǎn)之一是較高的遺傳突變率,它使HIV的基因序列產(chǎn)生較多的突變,這些突變病毒以準(zhǔn)種形式在患者體內(nèi)存在。在HIV患者接受抗病毒治療的過程中,HIV的高突變率使病毒能夠產(chǎn)生有效的突變位點(diǎn),攜帶耐藥位點(diǎn)的病毒株可以從藥物作用下逃逸。中國(guó)疾病預(yù)防控制中心性病艾滋病預(yù)防控制中心與河南省疾病預(yù)防控制中心于2003年底即在河南省某確山縣對(duì)抗病毒患者進(jìn)行了長(zhǎng)期的隨訪觀察,本文利用該隊(duì)列嘗試探尋HIV耐藥突變對(duì)長(zhǎng)期抗病毒治療的影響。 本文應(yīng)用科室自建的基因型耐藥檢測(cè)方法(in-house)和單模板擴(kuò)增方法(SingleGenomeAmplification,SGA)獲得HIV-1病毒poL區(qū)的蛋白酶和逆轉(zhuǎn)錄酶基因區(qū)片段(2253bp-3553bp,1.3kb).應(yīng)用Sequencher (4.10.1)和Bioedit(7.0)軟件對(duì)序列進(jìn)行編輯并利用美國(guó)斯坦福大學(xué)HIV耐藥數(shù)據(jù)庫(Stanfrod University HIV Drug Resistance Database, HIVDB)進(jìn)行耐藥程度分析。 世界衛(wèi)生組織(WHO)公布的HIV耐藥檢測(cè)指南中,HIV耐藥檢測(cè)的病毒載量檢測(cè)線為1000拷貝/ml,故國(guó)內(nèi)外對(duì)于病毒載量在50-1000拷貝/ml時(shí)患者的耐藥研究報(bào)道較少,而這一范圍往往是藥物抑制效果開始降低的時(shí)期。對(duì)低載量耐藥的研究可能會(huì)有助于提早預(yù)警病毒抑制失敗和耐藥的發(fā)生,有助于提高患者的治療效果。本研究利用上述耐藥隊(duì)列中曾出現(xiàn)過50-1000拷貝/ml的患者,研究其發(fā)生耐藥后對(duì)后期治療效果的影響及耐藥的發(fā)展。結(jié)果發(fā)現(xiàn)在28例病毒抑制失敗的患者中有18人在低病毒載量時(shí)期發(fā)生了耐藥。在耐藥毒株出現(xiàn)的初期,非核苷類抑制劑(Non-Nucleoside Reverse Transcriptase Inhibitor, NNRTI)是主要耐受藥物,對(duì)奈韋拉平(Nevirapine NVP)的耐藥是導(dǎo)致患者耐藥的主要因素,且耐藥水平已達(dá)到高度。在病毒載量大于150拷貝/mL低于1000拷貝/mL的患者中,隨著治療時(shí)間延長(zhǎng)其耐藥位點(diǎn)增加,耐藥譜擴(kuò)大(耐受藥物種類與大于1000拷貝/mL的治療失敗患者相似)并且原本耐藥程度較低的核苷類抑制齊(Nucleoside Reverse Transcriptase Inhibitor, NRTI)類藥物的耐藥水平有所增加。 NVP是我國(guó)早期一線抗病毒治療方案中NNRTI類藥物的重要組成部分,在早期藥物缺乏的情況下部分患者在產(chǎn)生NVP耐藥后仍在服用。許多研究已證實(shí)NVP相關(guān)的耐藥位點(diǎn)可以叫持久的存在于患者體內(nèi),但另外一些較新的論文則發(fā)現(xiàn)了NVP相關(guān)位點(diǎn)K103N隨治療時(shí)間而產(chǎn)生的減少。對(duì)于這一現(xiàn)象,本研究針對(duì)長(zhǎng)期治療的患者,研究NVP相關(guān)耐藥位點(diǎn)的變化和發(fā)展情況,并希望借此幫助患者在更換新NNRTI類藥物時(shí)了解自身的優(yōu)勢(shì)株NNRTI類耐藥位點(diǎn),避免或延遲再次耐藥的發(fā)生。此外,本研究希望能夠借助對(duì)NNRTI類主要耐藥位點(diǎn)變化發(fā)展的研究,幫助現(xiàn)階段以PCR探針技術(shù)為基礎(chǔ)的快速檢測(cè)手段,更好更準(zhǔn)確的針對(duì)不同治療時(shí)間的患者使用與其優(yōu)勢(shì)耐藥株符合的探針。 本文以上述建立的耐藥隊(duì)列為基礎(chǔ),應(yīng)用in-house基因型檢測(cè)技術(shù)對(duì)全部研究對(duì)象進(jìn)行耐藥分析,并應(yīng)用單模板擴(kuò)增技術(shù)(Single Genome Amplification, SGA),對(duì)其中三例患者的連續(xù)血漿樣本進(jìn)行準(zhǔn)種毒株的耐藥進(jìn)化分析。結(jié)果顯示存活的患者群體中K103N1位點(diǎn)的頻率保持相對(duì)穩(wěn)定,而Y181C和G190A位點(diǎn)的頻率則從5%左右升高至了15%左右。3例隨訪患者中的情形則略有不同,K103N位點(diǎn)在準(zhǔn)種毒株中的頻率隨治療時(shí)間顯著降低,在兩例患者中甚至從100%降到了0%。Y181C和G190A位點(diǎn)則同人群中相似,其頻率有較明顯的升高。 耐藥位點(diǎn)在降低毒株對(duì)藥物敏感性的同時(shí),會(huì)降低毒株的復(fù)制適應(yīng)性,但是補(bǔ)償性位點(diǎn)可以部分恢復(fù)這種被降低的復(fù)制能力。這種補(bǔ)償性位點(diǎn)雖然自身不能產(chǎn)生耐藥性,但有助于耐藥毒株在藥物壓力下存活和發(fā)展,因此其會(huì)對(duì)HIV抗病毒治療,特別是長(zhǎng)期治療帶來不利因素和風(fēng)險(xiǎn)。本文對(duì)3例患者(HENDRC604, HENDRC622, HENDRC593)連續(xù)6年的系列血漿樣本進(jìn)行單模板擴(kuò)增分析,并獲得HIV-1準(zhǔn)種毒株pol區(qū)序列,每個(gè)時(shí)間點(diǎn)至少分析20條以上序列,每個(gè)患者累計(jì)100條以上的序列,使用貝葉斯軟件(Beast, v1.7.0)結(jié)合分子鐘方法構(gòu)建Bayesian MCMC系統(tǒng)進(jìn)化樹,按樣本采樣時(shí)間完成分子鐘的計(jì)算,并利用核苷酸替代數(shù)學(xué)模型對(duì)病毒序列數(shù)據(jù)進(jìn)行計(jì)算和模擬,結(jié)合3D結(jié)構(gòu)模擬、Ka/Ks率等方面的計(jì)算描述患者體內(nèi)HIV準(zhǔn)種的進(jìn)化和耐藥株的發(fā)展。本研究發(fā)現(xiàn)了兩個(gè)與主要耐藥位點(diǎn)相關(guān)的補(bǔ)償性突變位點(diǎn),K43E和L228R。推測(cè)K43E是一個(gè)能夠與鄰近的M41L耐藥位點(diǎn)協(xié)同產(chǎn)生作用的補(bǔ)償性突變位點(diǎn)。它可能通過提高HIV-1毒株適應(yīng)性從而使毒株在抗病毒藥物壓力下生存并進(jìn)化。此外,還發(fā)現(xiàn)L228R位點(diǎn)是一個(gè)與常見非核苷類耐藥位點(diǎn)H221Y功能相近的補(bǔ)償性位點(diǎn)。它可以通過協(xié)同作用,協(xié)同其他非核苷類耐藥位點(diǎn)產(chǎn)生耐藥作用。 本文針對(duì)長(zhǎng)期抗病毒治療下HIV耐藥株變異的特點(diǎn)及其對(duì)抗病毒治療影響的研究,將為進(jìn)一步提高我國(guó)的HIV抗病毒治療效果提供參考數(shù)據(jù)�？傊�,為實(shí)現(xiàn)“十二五”降低艾滋病新發(fā)感染和病死率的目標(biāo),治療作為預(yù)防正在我國(guó)逐步推廣,接受終生治療的患者會(huì)越來越多,尋找措施如何更好的預(yù)防HIV耐藥的發(fā)生和傳播的工作將面臨著新的挑戰(zhàn)。
[Abstract]:AIDS antiretroviral treatment (Antiretroviral, Therapy, ART) since 1990s in the world has been widely carried out, from the initial set of AZT (Zidovudine, AZT) to carry out the development of seven kinds of 33 kinds of drug.ART for World AIDS patients brought the gospel, to help the development of delayed disease. And improve the quality of life. China began to gradually promote free antiretroviral treatment of AIDS since 2003, the mortality rate from 9.5 in 2003 to reduce deaths /100 person years in 2009 5.2 deaths per person year. Although China's ART has made some preliminary results, but due to antiviral treatment of early drug resources Co., a considerable number of patients receiving ART after treatment to long-term use of the same kind of treatment. Many studies show that long-term treatment of HIV resistant strains produced is the impact of antiviral treatment One of the main factors of the biological characteristics of fruit.HIV virus is a high genetic mutation rate, which makes the sequence of HIV gene to produce more mutations, these mutant viruses exists as quasispecies in patients. The process of receiving antiretroviral treatment in patients with HIV in HIV, the high mutation rate of the virus can produce mutations effectively. Carrying the virus mutation can escape from drugs. Chinese Center for Disease Control and prevention center of STD and HIV / AIDS prevention and control in Henan Province Center for disease prevention and control at the end of 2003 in a Henan Province on antiviral patients were observed the long-term follow-up in this paper attempts to explore the influence on the resistance of HIV cohort of long-term antiviral therapy mutation.
Genotypic resistance testing method used in this paper were built (in-house) and single template amplification method (SingleGenomeAmplification, SGA) HIV-1 virus poL protease and reverse transcriptase gene fragment (2253bp-3553bp, 1.3kb). The application of Sequencher (4.10.1) and Bioedit (7) software to edit the sequence and the use of the United States HIV resistance database of Stanford University (Stanfrod University HIV Drug Resistance Database, HIVDB) of degree of drug resistance analysis.
WHO (WHO) HIV drug resistance testing guidelines published in the HIV resistance detection of viral load testing line for 1000 copies of /ml, both inside and outside the country for viral load in patients with 50-1000 copies /ml drug resistance study reported less, and this range is often begins to decrease the inhibitory effect of drugs on the amount of low load period. Drug resistance may contribute to the early warning of viral suppression failure and the occurrence of drug resistance, help to improve patient outcomes. This study uses the queue had resistant 50-1000 copies of /ml patients, the occurrence of drug resistance development research on the treatment effect and drug resistance later. Results the occurrence of drug resistance 18 people in the low load period failed in 28 cases of virus inhibition of virus patients. Early in resistant strains, non nucleoside inhibitors (Non-Nucleoside Reverse, Transcriptase Inhibitor, NNRTI) is The main drug resistance to nevirapine (Nevirapine, NVP) resistance is a major factor in the patients with drug resistance, and the resistance level has reached a high degree. More than 150 copies of /mL less than 1000 copies of /mL in patients with viral load, with prolonged treatment resistant sites increased drug resistance (tolerance of drugs and expand the spectrum of more than 1000 copy of the /mL treatment failure patients were similar and had low level resistance) nucleoside (Nucleoside Reverse Transcriptase Inhibitor inhibitor, NRTI) levels of resistance to drugs increased.
NVP is an important part of China's first NNRTI drugs early antiviral therapy in the absence of drugs in the early part of patients with drug-resistant NVP still in use. Many studies have demonstrated that NVP mutation related call lasting in patients, but also in some of the newer found reduce NVP related sites of K103N due to the time of treatment. For this phenomenon, this study for patients with long-term treatment, the change and development of NVP related drug resistance loci, and hopes to help patients understand their dominant strains of NNRTI resistant loci in replacement of NNRTI drugs, to avoid or delay the resistance again. In addition, this study hopes to help the research and development of NNRTI main types of resistance loci changes, help at the stage of rapid detection method of PCR probe technology as the foundation, better and more It is accurate to use a probe that conforms to its dominant resistant strain for patients with different time of treatment.
This paper is based on the resistance of the queue, to investigate the drug resistance of all subjects using in-house genotyping techniques, and the application of single template amplification technology (Single Genome Amplification, SGA), of which the continuous plasma samples of three patients were analyzed for quasi strains resistance evolution. The results indicated that the survival of patients in the group the frequency of K103N1 locus remained relatively stable, while the Y181C and G190A loci frequency is from about 5% to about 15%..3 patients in the situation is slightly different, the frequency of K103N locus in quasi strains with treatment time significantly decreased in two patients and even dropped from 100% to 0%.Y181C and similar G190A sites are the same people, its frequency is obviously increased.
The mutation in reducing the strain sensitivity to drugs at the same time, will reduce the fitness of replication of viruses, but could partially restore the ability to replicate compensatory sites this is reduced. Although this compensatory site does not produce drug resistance, but help in drug resistant strains under the pressure of survival and development, so the treatment of HIV virus, especially long term treatment of adverse factors and risks. On the 3 patients (HENDRC604, HENDRC622, HENDRC593) for 6 consecutive years, a series of plasma samples were single template amplification analysis, and obtain the HIV-1 quasispecies sequence of strain pol, each time point of at least more than 20 sequences, each with more than 100 cumulative sequences, using Bayesian the software (Beast, v1.7.0) to construct Bayesian MCMC phylogenetic tree with molecular clock method, calculated according to the sampling time to complete the molecular clock, and the use of nucleotide substitution Calculation and Simulation of algebra model of virus sequence data, combined with 3D structure simulation, calculation of Ka/Ks rate for the description of the evolution and development of resistant strains of quasi HIV patients. This study found two main resistance loci associated with compensatory mutations, K43E and L228R. suggest that K43E is a compensatory M41L mutation can cooperative effect and adjacent mutation. It can increase the adaptability of HIV-1 strains so that the strains survival and evolution in antiviral drugs under pressure. In addition, also found that the L228R locus is a common function and non nuclear glycosides resistance loci H221Y similar sites. It can be through compensatory synergy, together with other non nucleoside the mutation of drug resistance.
This paper researches the long-term antiviral treatment characteristics and antiviral treatment effect of HIV resistant strain variation, will provide the reference data for the further improvement of HIV antiviral treatment in our country. In short, reduce new HIV infections and the mortality rate for the realization of "12th Five-Year" treatment as prevention is gradually spread in China, receiving a lifetime patients will be more and more, looking for the occurrence and spread of the measures on how to better prevent the resistance of HIV work will face new challenges.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.91

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相關(guān)期刊論文 前2條

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本文編號(hào)：1464294