双位点核酶细胞内抑制HBV基因表达的初步研究

乙型肝炎病毒(hepaitis B Virus,HBV)是最小的DNA病毒之一,不仅它的HBsAg、HBeAg、     

乙型肝炎病毒准种研究的意义

一、准种 (ｑｕａｓｉｓｐｅｃｉｅｓ)的基本概念病原微生物 ,特别是核酸成分为ＲＮＡ的病原微生物 ,在自身复制过程中由于ＲＮＡ聚合酶 /逆转录酶缺乏严密的自我校正功能 ,或同时在感染宿主的免疫压力或抗微生物药物的选择压力下 ,其核酸成分在少数位点上发生突变是一种十分常见的现象。在长期的生存环境中 ,早期来源于同一个祖先的病原微生物 ,因为这种突变现象的不断累积 ,就可能发展为核酸成分差别很大的病原微生物群 ,以至于造成核酸成分的显著差别 ,最终逐渐形成不同的基因型。如果某些核酸序列位点的改变导致其编码产物抗原位点的改…     

DNA疫苗诱导小鼠特异性细胞免疫及抗乙型肝炎病毒皮下移植瘤的研究

目的观察乙型肝炎病毒(HBV)DNA疫苗(pCR3 1-S)诱导Balb/c小鼠(H-2d)的特异性细胞免疫应答及其对稳定表达HBsAg的小鼠肥大细胞瘤P815细胞(P815 HBV-S)(H-2d)成瘤性的影响.方法肌肉注射DNA疫苗,背部皮下接种P815-HBV S细胞,观察成瘤情况,4h 51Cr释放法检测小鼠脾细胞细胞毒T细胞(CTL)杀伤活性.结果DNA疫苗可以降低成瘤率,抑制肿瘤生长,延长小鼠平均存活期,提高小鼠存活率.CTL细胞杀伤活性明显增加(P＜0.001).结论DNA疫苗可以诱导细胞免疫应答,对体内HBV感染可能具有预防及治疗作用.     

丁型肝炎病毒载体携带核酶在小鼠体内抑制乙型肝炎病毒复制

目的:研究用丁型肝炎病毒(HDV)作为载体携带乙型肝炎病毒(HBV)特异性的锤头状核酶所构建的重组体,在细胞体系及转染动物模型中对HBV基因表达和复制的影响.方法:将HDV-核酶重组体和HBV的共表达质粒转染Huh-7细胞以分析HDV-核酶重组体对HBV基因表达的影响;用小鼠尾静脉快速注射法将共表达质粒转染到小鼠体内,检测重组体在动物体内对HBV基因表达和复制的抑制作用.结果:转染细胞中,重组体对HBsAg的抑制与HDV重组位点和核酶靶位都有关;水压法注射的质粒在小鼠肝内得到表达,与对照相比重组HDV-核酶可有效抑制在肝和血清中HBV的基因表达以及复制,与细胞中的结果一致.结论:此项体内实验为进一步构建治疗性重组HDV病毒,发现靶向性抗病毒基因治疗手段奠定基础.     

乙型肝炎病毒基因分型与疾病病情的关系

目的探讨乙型肝炎病毒基因分型与疾病病情的关系。方法采用PCR微板核酸杂交ELISA技术检测299例慢性乙型肝炎患者HBV基冈型,并分析其与HBVDNA定量和HBeAg阳性率的关系。结果本组患者HBV基因分型结果以c型143例最多,B型119例次之,其他类型少见;肝衰竭患者HBVC基因型（53．3％）显著高于B基因型（34．7％,P〈O．05）;C型患者HBVDNA定量（7．0±1．1lgcopies／m1）和HBeAg阳性率（68．5％）均明显高于B型（6．1±1．01gcopies／ml和45．4％,P〈0．05）。结论本组乙型肝炎病毒基冈分型以c型和B型居多,在肝衰竭患者HBVC型感染者多于B型。     

乙型肝炎病毒表面抗原变异的临床意义

乙型肝炎病毒（ＨＢ）感染是全球性的公共卫生问题，携带者超过３亿５千万人。ＨＢＶ的复制需经复制中间体ＲＮＡ反转录，且病毒的ＤＮＡ聚合酶缺乏校对酶活性，不能修正核苷酸错配，因而具有较高的变异性。乙型肝炎病毒表面抗原（ＨＢｓＡｇ）包括主蛋白（Ｓ蛋白）、中蛋白（Ｍ蛋白）和大蛋白（Ｌ蛋白）。Ｓ蛋白是其最主要成分，能诱发机体产生保护性免疫反应，是目前制备疫苗的基础。Ｓ蛋白氨基酸（ａａ）１２４～１４７，由１２４与１３７、１３９与１４７位点的半胱氨酸以二硫键相连形成两个ｌｏｏｐ构成ａ决定簇，是乙型肝炎疫苗产生保…     

慢性乙型肝炎病毒血症水平与临床关系的研究

乙型肝炎病变活动是宿主免疫对复制病毒及其抗原的应答状态,乙型肝炎病毒（ＨＢＶ）复制启动病变活动。病毒在体内持续复制,是引起肝炎慢性化的主要因素。我们采用定量聚合酶链反应（ＰＣＲ）观察不同类型慢性ＨＢＶ感染患者的病毒血症水平及其与病变活动的关系,并探讨...     

乙型肝炎病毒前c区1896点突变及临床意义

为了解兰州地区ＨＢＶ前ｃ区１８９６位点突变的存在及临床意义,我们对８６例乙肝抗体阳性而抗原阴性慢乙肝患者进行了错配ＰＣＲ限制片长度多态性分析。结果：显示ＨＢＶＤＮＡ阳性２４．４２％,变异者１９．０５％。说明体内ＨＢＶ并未完全清除,兰州地区存在ＨＢＶ前ｃ区１８９６位点突变且与病情似有一定关系。     

核酶在2.2.15细胞内抗乙肝病毒作用

目的:研究针对乙型肝炎病毒前基因组RNA双靶位自剪切核酶在细胞内对HBV复制和表达的抑制作用,进一步探讨如何提高核酶在细胞内的催化效率。方法:人工合成核酶基因片段,利用分子克隆技术,定向克隆到pGEM3ZF(-)质粒的EcoR I、HindⅢ位点中,构建出核酶的自剪切转录载体,再切下核酶基因片段,克隆到pBBS212质粒上构建核酶的真核表达载体。通过脂质体介导的基因转染方法将其导入2.2.15细胞中,观察核酶在细胞内对HBsAg、HBeAg表达的抑制作用。结果:核酶对HBsAg、HBeAg表达的抑制率分别达55％、28％。结论:研究结果表明该双靶位核酶可以在2.2.15细胞内对HBV的复制和表达有明显的抑制作用。     

丁型肝炎病毒载体携带核酶在小鼠体内抑制乙型肝炎病毒复制

目的：研究用丁型肝炎病毒（HDV）作为载体携带乙型肝炎病毒（HBV）特异性的锤头状核酶所构建的重组体，在细胞体系及转染动物模型中对HBV基因表达和复制的影响。方法：将HDV-核酶重组体和HBV的共表达质粒转染Huh-7细胞以分析HDV-核酶重组体对HBV基因表达的影响；用小鼠尾静脉快速注射法将共表达质粒转染到小鼠体内，检测重组体在动物体内对HBV基因表达和复制的抑制作用。结果：转染细胞中，重组体对HBsAg的抑制与HDV重组位点和核酶靶位都有关；水压法注射的质粒在小鼠肝内得到表达，与对照相比重组HDV-核酶可有效抑制在肝和血清中HBV的基因表达以及复制。与细胞中的结果一致。结论：此项体内实验为进一步构建治疗性重组HDV病毒，发现靶向性抗病毒基因治疗手段奠定基础。     

拉米夫定治疗前后乙型肝炎病毒P区基因的动态变化

目的研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒P基因区的变异情况。方法从5例慢性乙型肝炎患者拉米夫定治疗前和治疗12个月的血清标本中，扩增目的片段，阳性结果双酶切后克隆至JM105感受态细胞。每份标本随机挑取20个阳性克隆，以错配聚合酶链反应一限制性片段长度多态性分析法检测YMDD基因序列，出现变异者进行双向测序。结果5例慢性乙型肝炎患者在拉米夫定治疗12个月后，其中2例HBVDNA为阴性，2例HBVDNA转阴后又转阳，测序结果提示出现M5521变异；1例HBVDNA始终阳性，和治疗前一样存在D553G的变异。结论D553G变异可能是慢性乙型肝炎患者拉米夫定治疗无效的原因之一，拉米夫定治疗后出现的乙型肝炎病毒基因组P区YMDD变异是抗病毒药物诱导的结果。     

HBx-GFP DN突变子长期稳定表达抑制2.2.15细胞乙型肝炎病毒基因的表达

目的　探索和寻找更有效的乙型肝炎治疗新的靶点和新的治疗方法 ,研究其抗病毒基因表达作用。方法　运用基因工程技术 ,建立了HBx GFP及其作对照的表达野生X蛋白、绿色荧光蛋白 (GFP)的长期稳定表达细胞克隆 ,Northernblot检测细胞内的乙型肝炎病毒相关基因RNA转录 ,应用RIA检测细胞上清液中HBsAg和HBeAg表达 ,观察对乙型肝炎病毒基因表达的影响。 结果所构建的X GFP突变子 ,Xwt,GFP质粒均能在 2 .2 .15细胞株中稳定高效表达并使细胞上清液中HBsAgHBeAg表达水平分别较 2 .2 .15组的 ( 10 1± 5.5)ng/ml、 ( 12 1± 8.6)ng/ml显著降低为平均( 7.6± 11.5)ng/ml、 ( 3 5± 3 .5)ng/ml (P <0 .0 1) ,细胞内的病毒 3 .5kb ,2 .1kb及 2 .4kb的RNA与各对照组比较均有显著降低 ,以 2 .1kb及 2 .4kb的mRNA下降最为显著。结论　乙型肝炎X基因DN突变子X GFP能显著抑制乙型肝炎病毒基因转录和S ,C基因的表达。提示 ,X基因亦是乙型肝炎治疗的一个靶基因     

Engineering immune therapy against hepatitis B virus

Approximately 350–400 million people worldwide are chronically infected with the hepatitis B virus (HBV). These individuals harbor the virus for their whole life and they transmit the virus to uninfected individuals. In addition, considerable numbers of chronic HBV carriers develop progressive liver diseases like chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma. At present, antiviral agents like type-1 interferons, lamivudine, adefovir and entacavir are used to treat a selected population of chronic HBV carriers. These antiviral treatments are not satisfactory in that they are unable to eradicate HBV, only partially efficient in less than 30% subjects, expensive, can have debilitating side-effects and require constant monitoring. In addition, once treatment is stopped, the virus and clinical conditions return in many patients. Recent advancements in cellular and molecular biology indicate that the host's immune responses to HBV play cardinal roles during acquisition, pathogenesis, progression, and complications of chronic HBV infection. Immune responses are also important in the context of antiviraltherapy and clinical recovery. This explains why the efficacy of antiviral drugs is limited even in some selected patients with chronic HBV infection. Various published work now state that HBV-specific immunity may be beneficial for patients with chronic HBV infection and non-HBV-specific immunity may be related to flare up of liver diseases. Accordingly, a new few field of immunological research and clinical application of prophylactic vaccines (vaccine therapy) has been started in chronic HBV carriers. Vaccine therapy has inspired optimism as a new therapeutic approach, but it is unlikely that the present regimen of vaccine therapy will stand the test of time. Based on present understandings about vaccine/host interactions, we provide herein an outline for engineering more potent regimen of HBV-specific immune therapy against HBV.     

Induced immunity against hepatitis B virus

Prevention of hepatitis B virus (HBV) infection with its consequent development of HBV chronic liver disease and hepatocellular carcinoma is a global mandatory goal. Fortunately, safe and effective HBV vaccines are currently available. Universal hepatitis B surface antigen HBV vaccination coverage is almost done. Growing knowledge based upon monitoring and surveillance of HBV vaccination programs has accumulated and the policy of booster vaccination has been evaluated. This review article provides an overview of the natural history of HBV infection, immune responses and the future of HBV infection. It also summarizes the updated sources, types and uses of HBV vaccines, whether in the preclinical phase or in the post-field vaccination.     

乙型肝炎基因治疗的研究进展

基因治疗是指通过基因水平的操纵达到治疗或预防疾病的疗法,分体内疗法和体外疗法.前者是将外源基因直接导入人体发挥作用;     

乙型肝炎病毒基因变异对慢性乙型肝炎患者病情发展及抗病毒治疗的影响

目的 探讨乙型肝炎病毒(HBV)基因变异对慢性乙型肝炎(慢乙肝)患者病情发展、严重程度以及对抗病毒治疗疗效的影响。方法 采用基因芯片技术，对选取的部分乙型肝炎患者进行HBV基因变异位点的检测。结果 α-干扰素治疗24周以上，HBeAg不发生血清转化，或出现HBeAg(-)／抗-HBe( )，但HBV—DNA定量检测仍持续阳性患者与nt1896、nt1814、nt1762、nt1764位点突变有关；拉米夫定治疗后，HBV—DNA先下降或转阴，后又再度反弹患者与aa528、aa552变异(即YMDD变异)造成拉米夫定耐药有关；拉米夫定治疗52周以上的部分患者易发生YMDD变异(26．4％)；在慢性乙肝患者中nt1896位点变异较普遍(68．5％)；慢性重症肝炎、肝硬化失代偿、原发性肝癌与nt1896、nt1762、nt1764位点突变亦有关。结论 HBV基因变异可加重患者病情，影响抗病毒治疗效果。临床上通过对乙肝患者进行HBV常见变异位点进行检测，对判断疾病预后，调整抗病毒治疗方案具有一定的参考价值。     

Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

AIM To detect hyper-conserved regions in the hepatitis B virus(HBV) X gene(HBX) 5' region that could be candidates for gene therapy.METHODS The study included 27 chronic hepatitis B treatmentnaive patients in various clinical stages(from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeA g-negative and HBeA g-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia 3.5 log IU/m L, the HBX 5' end region [nucleotide(nt) 1255-1611] was PCRamplified and submitted to next-generation sequencing(NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences(haplotypes) obtained by NGS. Conservation at the HBx protein amino acid(aa) level was also analyzed.RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample(2487-9279, IQR 2817). In 14/27 patients(51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region(nt 1255-1286) and the other in the 5' end coding region(nt 1519-1603). This last region coded for a conserved amino acid region(aa 63-76) that partially overlaps a Kunitz-like domain.CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.     

针对HBV—P基因的RNA干扰抑制乙型肝炎病毒的研究

目的构建针对乙型肝炎病毒P基因编码区、能在体内转录产生发夹状小干扰RNA（siRNA）的表达载体psiHBV／p,观察RNA干扰对HBsAg、HBeAg及乙型肝炎病毒DNA复制的抑制作用。方法针对HBV—P基因区特异序列,构建siRNA的表达载体psiHBV／p。采用脂质体介导方法将其与1．3倍HBV真核表达质粒pHBV1．3共转染HepG2细胞。分别于转染后24小时、48小时、72小时用ELISA法对HepG2细胞培养上清液进行HBsAg、HBeAg的检测;于转染后72小时通过FQ-PCR法分析RNA干扰作用对HBVDNA的抑制效果。结果成功构建了针对HBV—P基因区的siRNA的真核表达重组体psiHBV／p,并发现它能明显抑制HBsAg及HBeAg的分泌,转染后第二天抑制率达高峰,分别为84％、65％。FQ—PCR结果也证实了转染72小时后,随psiHBV／p比例的升高,其对HBV DNA的抑制作用也随之增加。结论成功构建的psiHBV／p,它能在体内持续转录产生针对P基因转录体的发夹状siRNA;在细胞水平上,体内转录产生的、针对乙型肝炎病毒P基因区特异序列的siRNA对共转染的重组载体pHBV1．3有显著和特异的抑制作用。     

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Hepatitis delta virus(HDV) seems to strongly suppress hepatitis B virus(HBV)replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein(HBx) is essential for HBV replication, determination of HBV X gene(HBX)quasispecies complexity in HBV/HDV infection compared to HBV monoinfection may provide information on the interactions between these two viruses.AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta(CHD) and chronic HBV mono-infected patients.METHODS Twenty-four untreated patients were included: 7/24(29.2%) with HBeAgnegative chronic HBV infection(CI, previously termed inactive carriers), 8/24(33.3%) with HBeAg-negative chronic hepatitis B(CHB) and 9/24(37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels.The HBX 5' region [nucleotides(nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing(MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidencebased indices(number of haplotypes and number of mutations), abundancebased indices(Hill numbers of order 1 and 2), and functional indices(mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity.RESULTS CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL,interquartile range(IQR) 3.5-7.9] than CHD(3.4 logIU/mL, IQR 3-7.6)(P = n.s.)or CI(3.2 logIU/mL, IQR 2.3-3.5)(P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences(CHB2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3 G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.