摘要
目前,全世界有大约3.5亿的慢性乙型肝炎病毒(Hepatitis B virus,HBV)感染者,我国约有9300万的HBV感染者。HBV慢性感染可以引起慢性乙型肝炎、肝硬化、肝衰竭进而诱发为肝癌。临床上治疗HBV慢性感染的药物主要有:1.α-干扰素(IFN-α);2.核苷(酸)类似物(NA)。α-干扰素主要是通过诱导细胞产生抗病毒蛋白和免疫调节来达到抗病毒的作用。与核苷(酸)类似物相比,α-干扰素治疗应答持续更久,但是α-干扰素治疗慢性乙型肝炎(Chronic hepatitis B,CHB)有效率仍然较低,仅为30%～50% ,且具有副作用多,价格昂贵等缺点,并且有其他因素决定着α-干扰素抗病毒疗效。
核苷(酸)类似物是近些年来临床治疗慢性乙型肝炎患者的重要药物,在体内核苷(酸)类似物可在磷酸激酶作用下形成三磷酸化合物,拮抗HBV所需的天然底物脱氧鸟苷三磷酸(dGTP)。核苷(酸)类似物作用靶点是HBV逆转录酶,其可以在3个环节上抑制HBV逆转录酶的活性,依次是:碱基引导(base priming)、从mRNA前体反转录成负链DNA、HBV DNA正链合成。目前,在我国临床应用的核苷(酸)类似物药物有拉米夫定(LAM)、阿德福韦(ADV)、恩替卡韦(ETV)、替比夫定(L-dT)和替诺福韦(TDF)。
HBV基因组易产生变异,原因在于HBV的多聚酶/逆转录酶(Reverse transcriptase,RT)缺乏必要的校对功能,其变异速度可达到10-7 bp/d,而远大于其他DNA病毒的平均变异水平。所以,临床长期应用核苷(酸)类似物治疗HBV感染者的过程中,在药物的压力选择下HBV准种中存在的极少量的耐药变异株成为优势株,从而出现病情的反复或加剧。HBV的耐药类型按照发生顺序可依次分为:1.基因型耐药(Genotypic Resistance),表现为HBV基因组产生某些特定位点突变,并且这些突变的位点已证明与耐药相关;2.病毒学突破(Virological Breakthrough),表现为患者血清病毒裁量出现>1 log10水平的反弹;3.生化学突破(Biochemical breakthrough),表现为患者出现丙氨酸氨基转移酶(Alanine Aminotransferase,ALT)上升和(或)病理上出现肝脏组织学损伤加重。要确定某种HBV基因变异是否与耐药相关,需要用表型耐药(Phenotype Resistance)分析证实,基本策略是构建HBV DNA变异株与野生株基因组重组载体,转染肝癌细胞系后定量检测HBV DNA复制中间体,比较在不同药物浓度下病毒复制力的受抑制程度。由于HBV表型耐药分析技术方法比较复杂,用传统Southern Blotting杂交法结果重复性较差,国内仅有少数实验室初步建立了方法。
目的建立实用的表型耐药分析方法,为鉴定定临床可能出现新型与复杂HBV耐药变异提供方法基础。具体包括:
1.构建1.1倍HBV野生型和LAM耐药突变型的重组载体。
2.将1.1倍HBV野生型和LAM耐药突变型的重组载体转染肝癌细胞系HepG2细胞,通过检测在不同药物浓度下的病毒复制力水平检测变异病毒的耐药表型。
3.建立实时荧光PCR定量法检测病毒复制力,并与Southern杂交法比较。
方法从临床上获得的LAM耐药慢性乙型肝炎患者血清中提取HBV DNA,经基因工程技术获得具有HBV基因组耐药突变位点的质粒载体,在体外培养系统分析慢性乙型肝炎患者血清HBV分离株对拉米夫定(LAM)的敏感性。
1.从临床检测确诊1例拉米夫定耐药患者(耐药位点为:rtL180M+rtM204V)血清内提取HBV DNA,运用巢式PCR扩增HBV DNA RT区,获得扩增RT区基因,命名为9077,将9077克隆至pGEM-Teasy载体,获得含有RT区耐药基因的质粒9077-p-m,将9077-p-m进行扩增,采用定点突变方法,把9077-p-m的RT区所含的耐药突变位点回复为野生型,野生型质粒命名为9077-p-w,并把9077-p-w克隆扩增。运用Nco I/Xho I双酶切质粒9077-p-m和9077-p-w回收RT区片段,将两条RT区片段替换至pTriEx-1.1-HBV载体,替换为可以表达野生型的载体,命名为:S9077-1;表达耐药突变型的载体,命名为:S9077-2。分别转染至HepG2细胞,ELISA法检测上清中HBsAg和HBeAg的水平。
2.将构建成功的pTriEx-1.1-HBV野生型载体S9077-1和pTriEx-1.1-HBV拉米夫定耐药突变型载体S9077-2分别转染至HepG2细胞,加用拉米夫定,按0μmol/L、0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L和100μmol/L的浓度梯度,每天换液一次,连续加用拉米夫定5天,提取HBV核心颗粒DNA,分别用荧光定量PCR和Southern杂交法检测HBV复制中间体水平,后者产生的图像用核酸定量软件进行灰度扫描定量。
结果初步建立了体外HBV表型分析方法,该方法对监测HBV耐药性具有可行性,对指导临床医生制订更合理的抗病毒治疗方案及筛选新型抗病毒药物具有重要意义。具体如下:
1.从拉米夫定耐药患者血清中提取HBV DNA RT区经克隆后测序,其耐药位点符合要求。将该HBV DNA RT区定点突变,克隆后测序,确证回复为野生型,重组到pTriEx-1.1-HBV载体上,转染至HepG2细胞检测上清抗原表达量检测符合要求。
2. HBV DNA水平检测,加用不同浓度的LAM后,随药物浓度增加,野生株细胞内HBV复制中间体水平明显下降,IC50为0.04±0.01μmol/L;而rtL180M+M204V突变株转染细胞后,随药物浓度的增加,HBV复制中间体水平并没有明显下降, IC50 > 100μmol/L。与野生型毒株相比,rtL180M+M204V突变株的IC50增加了约2500倍。Southern Blotting检测结果与实时荧光PCR测定一致。
结论1.本研究构建体外表达的1.1倍HBV野生型和LAM耐药突变型重组载体,将载体转染入HepG2细胞后,该载体能够在体外稳定地高表达HBsAg和HBeAg抗原水平,为HBV耐药体外研究提供了载体基础。
2.本研究采用脂质体体外转染的技术手段,进行HBV DNA在体外细胞进行抗HBV药物作用的研究,建立了HBV表型耐药分析方法,对鉴定临床发现的病毒基因变异与HBV耐药性的关系提供了方法学基础。
Currently, there are more than 350 million people with chronic hepatitis B virus(HBV) infection, including 93 million people in China. Chronic HBV infection could cause chronic hepatitis B (CHB), live cirrhosis, liver failure and liver cancer. Two major kinds of anti-HBV agents have been used for the treatment of HBV infection, i.e.,α-interferon (IFN-α) and nucleoside/nucleotide analogues. The role of IFN-αis mainly via inducing host cells to produce antiviral proteins and regulating immunity against HBV. The virologic response of IFN-αis longer than that of nucleos(t)ide analogues, but clinic response of IFN-αis not high which is around 30% to 50%. Furthermore, there are many side effects and high costing. The other important anti-HBV agent is nucleos(t)ide analogues.
Nucleos(t)ide analogues can be catalysised by the phosphokinase to form 3-phosphorylation compounds, which can compete with natural deoxyguanosine triphosphate(dGTP)to bind with HBV. The target of nucleos(t)ide analogues is HBV polymerase/reverse transcriptase (RT). Nucleos(t)ide analogues could inhibit HBV replication in three steps: base priming, negative strand synthesis by hnRNA reverse transcription, and positive strand synthesis. In our country, four nucleos(t)ide analogues are used in clinic. i.e., lamivudine (LAM), adefovir (ADV), entecavir (ETV), and telbivudine (L-dT). HBV has high frequency genetic variation because its reverse transcriptase is lack of proof reading function. The variation rate of HBV is about 10-7 bp/d,significantly higher than the average error rate of DNA viruses. During long-term clinical use of the nucleos(t)ide analogues, HBV stains with drug-resistant variation could be selected from HBV quasispecis pool to become dominant strains and cause disease relapse.
HBV resistance sequentially emerges as follows. 1. genotypic resistance, presenting as the emergence of viral populations bearing amino acid substitutions in RT region of the HBV genome that have been shown to confer resistance to antiviral drugs in a phenotypic assay during antiviral therapy. 2. virologic breakthrough, presenting as serum virus load rebound >1 log10. 3. biochemical breakthrough, presenting as the elevation of alanine aminotransferase and/or increase of liver histologic impairment. Phenotypic analysis is an essential tool to identify whether a variation in HBV RT region is associated with drug resistance. It bases on the comparison of HBV replication capacities of variants and wild-type counterparts by cell-transfer of individual viral genomes followed by quantitation of HBV replication intermediates in the presence of antivirals in different concentrations. However, due to complex methodology and the classical Southern blotting assay has low reproducibility, only few of laboratories in China primarily set up the for phenotyping HBV drug resistance.
Objective To establish a practical assay of phenotypic analysis for HBV drug resistance to help identify novel and complex drug-resistance-associated variation from patients. In concrete:
1. To construct the recombinant vectors containing 1.1-fold genomes of HBV wild-type and LAM-resistant mutant strains.
2. To determine resistance phenotype of mutant virus by measuring viral replication levels of wild-type and mutant viral genomes after transfecting them into HepG2 cells and culturing the cells in different concentration of the drug.
3. To develop real-time polymerase chain reaction (PCR) assay for determination of HBV replicative intermediate level and compare with the classical Souther blotting assay.
Method 1. HBV DNA was extracted from serum of a CHB patient resistant to LAM. The resistance mutational pattern was rtL180M+rtM204V. HBV DNA RT gene was obtained by nested PCR. The mutant viral gene was numbered as 9077 and cloned into pGEM-Teasy vector. The plasmid pGEM-Teasy-RT vector was termed as 9077-p-m. Corresponding wild-type viral gene was produced by site-specific mutagenesis and the recombinant pGEM-Teasy vecter was termed as 9077-p-w.Plasmid vectors 9077-p-m, 9077-p-w and pTriEx-HBV(C) were digested by Xho I/Nco I to construct the recombinant vectors of wild-type HBV pTriEx-wRT(S9077-1) and LAM-resistant mutant strain:pTriEx-mRT(S9077-2). pTriEx-wRT(S9077-1) and pTriEx-mRT(S9077-2) were transfected into HepG2 cells. HBsAg and HBeAg levels in the supernatant were measured by ELISA.
2. pTriEx-wRT(S9077-1) and pTriEx-mRT(S9077-2) were transfected into HepG2 Cells. Various concentrations of LAM (0μmol/L, 0.01μmol/L, 0.1μmol/L, 1μmol/L, 10μmol/L, and 100μmol/L) were added 60 hours after transfection. Intracellular HBV DNA was extracted 5 days after continuous LAM treatment. Quantitative real-time PCR and Southern blotting analysis were performed to detect the HBV DNA level.
Result 1. HBV RT gene cloned from the LAM-resistant patients was confirmed containing correct mutational pattern by cloning sequencing. After site-specific mutagenesis, the HBV RT gene successfully became the wild- type. After transfection of recombinant plasmids containing either LAM-resistant or wild-type HBV 1.1-fold genomes into HepG2, the expression levels of HBsAg and HBeAg in the supernatant reached anticipated levels.
2. After adding various concentrations of LAM, intracellular HBV replicative intermediate level decreased significantly along with the increase of drug concentration. The IC50 was 0.04±0.01μmol/L for the wild-type strain. By contrast, no such obvious decrease of HBV replicative intermediate level was observed in the cells transfected with HBV genome containing LAM-resistant rtL180M+M204V mutations. The IC50 was >100μmol/L, which was about 2500-fold higher than that of the wild-type HBV strain.
Conclusion 1. The study constructed recombinant vectors containing 1.1-fold HBV wild-type or LAM-resistant HBV RT genes. After transfecting the recombinant vectors into HepG2, the recombinant vectors could perform high levels of HBsAg and HBeAg in the supernatant.
2. The study take the transfection with liposomes in vitro to research HBV DNA drug resistance. Successfully established the method for accessing drug susceptibility of HBV in vitro. This phenotyping assay will help to identify drug-resistance-associated variants detected from patients.
引文
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