丙型肝炎病毒体外培养细胞模型研究进展

由于丙型肝炎病毒(HCV)的高度变异性以及缺乏高效率的细胞培养体系,人们对HCV生活史的认识、对相关疫苗和特异性抗病毒药物的研究进展缓慢.寻找高效率的HCV体外细胞培养体系一直是HCV研究的重点,现就近年来在相关领域的研究进展作一综述.     

丙型肝炎病毒体外感染正常成人肝细胞的初步研究

一、资料与方法 １．正常成人肝组织标本：取意外死亡的３例成年男性,１例成年女性肝脏,均无肝病史,临床生物化学和病理学检查无异常发现,各种肝炎病毒标志物检测均阴性。 ２．采用体外两步灌流法分离正常成人肝细胞。最后用含１０％小牛血清１６４０培养液按细胞密度 １．０× １０６／ｍｌ稀释后接种至 ２５ ｍｌ培养瓶和加爬片的 ２４孔培养板,置于３７℃、 ５％ ＣＯ２培养箱孵育,２－３ｄ更换完全培养液１次,维持培养１月以上。 ３．加适量感染血清至培养瓶（０．２５ｍｌ／瓶）和培养板（０．１ｍｌ／孔）中,混匀,置于 ３７…     

体外感染实验探讨丙型肝炎病毒的ADE分子机制

目的研究丙型肝炎病毒(HCV)在感染人滋养层细胞的过程中是否存在抗体依赖性感染增强(ADE)作用,以探讨HCV母婴传播的分子机制。方法将HCV阳性血清以4种不同方式感染人滋养层细胞,以免疫电镜观察HCV病毒颗粒在滋养层细胞中的表达,应用RT-PCR法、免疫组化法检测细胞内外的HCVRNA正链、负链,HCVNS5、NS3及C区抗原。结果在滋养层细胞胞浆内发现了HCV病毒颗粒,HCVRNA正链、负链,HCVNS5、NS3、C区抗原仅在全血清感染细胞内或上清中间断测得。结论HCV可以在滋养层细胞中复制。HCV感染滋养层细胞的过程中存在ADE机制,抗体和补体均参与了HCV的跨膜转运过程。     

丙型肝炎病毒体外感染MT-2细胞的研究

目的建立体外丙型肝炎病毒感染的ＭＴ－２细胞模型。方法用ＨＣＶＲＮＡ阳性的丙型肝炎患者血清感染人淋巴细胞株ＭＴ－２以ＨＣＶＲＮＡ阴性血清作对照，用ＲＴ－ＰＣＲ分析ＨＣＶ在体外细胞培养中的复制状态。结果用ＨＣＶ阳性血清感染的ＭＴ－２细胞在培养第７天开始检出ＨＣＶＲＮＡ，并一直持续阳性至第２８天；培养至第１０天可测出ＨＣＶ复制中间体，于第１８天消失。用ＨＣＶ阴性血清感染的ＭＴ－２细胞未检出ＨＣＶＲＮＡ。扩增产物经Ｓｏｕｔｈｅｒｎ转移后用ＨＣＶ探针杂交证实ＨＣＶ特异性序列。结论ＨＣＶ可在ＭＴ—２中短期复制。     

丙型肝炎病毒体外可感染树鼩肝细胞

目的 探讨丙型肝炎病毒（HCV）体外感染树目句原代肝细胞。方法 用HCV RNA阳性血清感染原代树鼩肝细胞，并用感染后细胞培养上清液进行传代感染，通过检测受染肝细胞正、负链HCV RNA、培养上清液中包装后HCV RNA，并对比分析感染前后病毒准种变化等指标，评价感染是否成功。结果 受染树鼩肝细胞自第5～10天可检出负链HCV RNA，而正链RNA至感染后第14天仍可检出；感染后3～14d培养上清液中可检出HCV RNA，且呈RNA酶抗性；培养上清液中病毒可传代感染新的树鼩肝细胞；感染前后HCV准种分析显示树鼩肝细胞可被特定的准种选择性感染，而传代感染后肝细胞可检出新的准种。结论 原代树鼩肝细胞体外可被HCV感染且支持病毒复制。     

丙型肝炎病毒受体研究进展

丙型肝炎病毒(HCV)是丙型肝炎的主要致病因子,目前全球至少有1.7亿人感染HCV.HCV感染具有高度慢性化特点,高达50％～80％的感染者由于不能有效清除体内病毒而发展为慢性感染,以及肝硬化和肝细胞癌,对人类的生活质量构成严重威胁.HCV属黄病毒科单股正链RNA病毒,基因组全长9.6 Kb,共编码包括包膜糖蛋白E1、E2在内的十种蛋白.     

丙型肝炎病毒所致胰岛素抵抗相关分子的研究进展

慢性丙型肝炎（CHC）患者易发生胰岛素抵抗（IR）。实验表明CHC相关IR不但与肝脂肪变性、肝纤维化、食管静脉曲张、肝细胞肝癌、肝癌扩散及一些肝外临床表现有关,还降低了以干扰素（IFN）联合利巴韦林为基础的抗病毒治疗早期及持续应答率。已有较多实验利用各种模型分析了丙型肝炎病毒（CHV）导致IR的发病机制,现对当前研究已知的几个分子及其机制做一综述。     

丙型肝炎病毒的检测策略及其应用

丙型肝炎病毒(HCV)引起丙型肝炎,因临床症状不明显,易造成漏诊或误诊,延误治疗;因此,临床医生将HCV实验室检测结果作为主要的诊断依据,以便及时采取相应措施,有利于疾病诊断、治疗和阻断HCV传播;血站通过HCV检测筛查献血员可以有效阻断血液传播这一途径,保障血液及血液制品安全; HCV检测也为公共卫生人员进行人群监测,掌握人群HCV感染情况提供依据[1].各国HCV感染情况有差别,且HCV检测的目的不同,因此,制定的HCV检测策略既需能达到预期目标,又需符合成本-效益原则.     

Cell culture systems for the hepatitis C virus

Since the discovery of HCV in 1989, the lack of a cell culture system has hampered research progress on this important human pathogen. No robust system has been obtained by empiric approaches, and HCV cell culture remained hypothetical until 2005. The construction of functional molecular clones has served as a starting point to reconstitute a consensus infectious cDNA that was able to transcribe infectious HCV RNAs as shown by intrahepatic inoculation in a chimpanzee. Other consen- sus clones have been selected and established in a hu- man hepatoma cell line as replicons, i.e. self-replicating subgenomic or genomic viral RNAs. However, these repli- cons did not support production of infectious virus. Inter- estingly, some full-length replicons could be established without adaptive mutations and one of them was able to replicate at very high levels and to release virus particles that are infectious in cell culture and in vivo. This new cell culture system represents a major breakthrough in the HCV field and should enable a broad range of basic and applied studies to be achieved.     

Progress in the development of vaccines for hepatitis C virus infection

The hepatitis C virus(HCV), first described in 1989, is now a leading cause of liver cirrhosis and hepatocellular carcinoma. With more than 170 million people infected globally, this virus is a major public health issue. The current standard therapy is based on interferon in combination with ribavirin. This costly therapy often fails to completely clear the infection and is associated with adverse side effects. Recent anti-HCV therapies are interferon-free direct-acting antiviral(DAA) regimens for HCV, including simeprevir, sofosbuvir, and ledipasvir, which have effects on non-structural proteins. DAA regimens have several advantages, such as specifically targeting HCV viral replication, accompanied by very high sustained virological response rates and lower side effects like flu-like syndrome. These facts plus the fact that most HCV cases progress to chronic infection suggest the potential need for an efficient HCV vaccine. Different innovative methods, including methods based on peptide, recombinant protein, DNA, vector-based, and virus-like particles, have been introduced for the development of HCV vaccines. An extensive number of studies have been published on these vaccines, and some vaccines were even tested in clinical trials. In the current review, progress in the development of preventive and therapeutic vaccines against the HCV is reviewed in the context of peptide vaccines, recombinant protein vaccines, HCV-like particle, DNA vaccines and viral vectors expressing HCV genes.     

丙型肝炎病毒体外感染胎肝细胞的初步研究

目的：探索原代培养胎肝细胞对丙型肝炎病毒（HCV）的易感性,旨在建立较为稳定实用的细胞感染模型。方法：研究血清与培养肝细胞共同孵育6－8h后,收集不同时相点标本,用逆转录聚合酶链反应（RT－PCR）分别检测细胞内或上清液中正负链RNA,免疫组化检测细胞内HCV NS3,NS5特异性抗原的表达情况,以及原位杂交检测细胞内HCV负链RNA。结果：接种感染血清3d后,即可在细胞内或培养上清液中检出HCV正负链RNA,间断检出至感染后第17天,HCV NS3,NS5特异性抗原可在感染细胞内表达,阳性物质位于乐中,原位杂交法证实细胞内存在负链RNA,也位于胞浆中,结论：原代培养的胎肝细胞对HCV不但易感,而且稳定地支持HCV复制。     

Myelopoiesis in vitro is suppressed by hepatitis A virus

Perturbations of hematopoietic regulation ranging from transient granulocytopenia to rare cases of bone marrow failure are associated with infections due to hepatitis A virus (HAV). In an attempt to elucidate the pathogenetic mechanisms we had previously established that HAV has a direct suppressive effect on human bone marrow progenitors (CFU-GM, -GEMM, BFU-E). These studies were extended to long-term bone marrow cultures (LTBMC): Inoculation of bone marrow mononuclear cells with HAV did not interfere with the establishment of an adherent stromal layer, nor did the inoculation of already established layers cause any morphologically recognizable changes to the stroma. In contrast, a significant and progressive decline of the CFU-GM content in the culture supernatants was demonstrated. HAV antigen was detected by APAAP stain in a subpopulation of stromal cells, and sequential estimations of virus titers in the supernatants provided evidence for viral replication in primary bone marrow cultures. Interferon-gamma and tumor necrosis factor-alpha levels of infected cultures did not differ from those of uninfected controls. These findings argue for a direct suppression of (pre-) CFU-GM by HAV in a model system (LTBMC) lacking an immune defense which would limit viral replication.This work was supported byDeutsche Forschungsgemeinschaft SFB 120/C2 and B5c.     

肝特异性自身抗体与丙型肝炎病毒感染的关系

本研究探讨了HCV感染时体内产生免疫应答并出现多种自身抗体的特点,试图通过检测分析HCV感染与自身抗体的相关性,对丙型肝炎的诊断及治疗提供一些实验数据.     

合并乙型肝炎病毒感染对丙型肝炎病毒核心抗原检测的影响

目的 探讨合并HBV感染对慢性HCV感染者血清丙型肝炎病毒核心抗原(HCVcAg)检出情况的影响. 方法 收集2005年12月-2009年10月慢性丙型肝炎患者和HBV/HCV合并感染者资料,检测血清HCVcAg和HCV RNA,对后者血清进行HBV DNA、HBeAg检测,分析HCVcAg检出率与HBeAg、HBV DNA定量检测的关系.用独立两组多分类的X2检验方法进行统计学分析. 结果 共收集88例慢性丙型肝炎患者和62例HBV/HCV合并感染者资料,血清HCVcAg的检出率分别为72.7％(64/88)和38.7％ (24/62),两者比较,x2= 17.358,P＜0.01,差异有统计学意义.HCV RNA检出率分别为81.8％ (72/88)和53.2％ (33/62),两者比较,x2=20.110,P＜0.01,差异有统计学意义.62例HBV/HCV合并感染者血清中,HBeAg阳性和HBeAg阴性感染者HCVcAg检出率分别为28.6％ (12/42)和60.0％ (12/20),两者比较,x2=5.641,P=0.011,差异有统计学意义.HCV RNA阳性率分别为42.9％ (18/42)和80.0％ (16/20),两者比较,X2=7.547,P＜ 0.01,差异有统计学意义.HBV DNA阳性和阴性时HCVcAg检出率分别为39.1％ (18/46)和37.5％ (6/16),两者比较,P＞0.05,差异无统计学意义.与单纯HCV感染者血清HCVcAg检出率72.7％ (64/88)比较,HBeAg阴性合并感染者为60.0％ (12/20),x2=1.266,P=0.261,差异无统计学意义;HBV DNA阴性合并感染者为37.5％ (6/16),x2=7.635,P＜0.01,差异有统计学意义.结论 HBV/HCV合并感染时HCVcAg检出率较低,可能是由于HBeAg抑制HCV的复制,从而减少HCVcAg的表达所致.