HCV5‘NCR和结构蛋白编码序列的克隆及对PA317细胞的转染

目的 进一步研究丙型肝炎病毒５‘ＮＣＲ对结构蛋白编码基因的表达调控。方法 以拼接好的ＨＣＶ５’ＮＣＲ〈Ｃ，Ｅ１和Ｅ２／ＮＳ１区基因共长２５４７个核苷酸片段克隆于逆转录病毒载体ＬＮＳＸ得重组体ｐＬＨＣ２５４７，用聚合酶锭反应（ＰＣＲ）及核苷酸序列分析等方法对重组体进行鉴定。通过磷酸钙－ＤＮＡ共沉淀法把经鉴定的重组体转染入ＰＡ３１７细胞，用Ｇ４１进行克隆筛选，以ＮＩＨ３Ｔ３细胞测其病毒滴度，提取转染细     

丙型肝炎病毒感染的不同人群HCV RNA定量研究

目的了解丙型肝炎病毒感染的不同人群血清HCV RNA水平,比较定性和定量PCR结果,探讨HCV RNA含量与血清ALT的相关性.方法采用荧光定量PCR和逆转录巢式定性PCR同时检测136例丙型肝炎病毒感染者血清HCV RNA,并测定定量PCR(+)者血清ALT.用相关系数分析HCV RNA含量与血清ALT的关系.结果定性PCR阳性率为80.88%,定量PCR阳性率为77.94%.两者相对符合率为94.12%.定量PCR(+)血清(106例)HCVRNA含量在107.04～1010.96拷贝·L-1.无症状献血员HCV RNA含量显著低于慢性丙型肝炎、肝硬变和肝细胞癌患者(P＜0.05),肝细胞癌患者血清ALT水平显著低于慢性丙型肝炎(P＜0.05).HCVRNA滴度与血清ALT呈正相关(r=0.61,P＜0.01).结论定性和定量检测结果有很好的一致性.病毒复制水平上升在肝损伤和肝病进展中可能起重要作用.     

HCV 5′NCR调控抑制活性的反义寡核苷酸的筛选

目的寻找高效特异的新型抗HCV药物,探讨针对HCV基因的硫代修饰反义寡核甘酸（S-ASODNs）最佳作用靶序列。方法参考HCV5′NCR计算机预测的二级结构图,设计合成了15条S-ASODN、3条正义寡核苷酸及1条随机序列,采用HCV5′NCR调控荧光素酶基因的瞬时表达系统,将S-ASODN与pHCV-neo4共转染,通过荧光素酶活性表达高低,反映S-ASODN对HCV调控基因的抑制活性。结果5条S-ASODN即HCV65、HCV279、HCV363、HCV349及HCV352在25～100nmol/L浓度范围内,对HCV调控基因具有特异性的剂量依赖性抑制活性。当浓度为100nmol/L时,这些ASODN的抑制率可达80％以上,IC50值提示HCV363的抑制活性最强。通过阴性对照实验包括正义寡核苷酸、随机序列及不含HCV序列的靶载体pGL3提示ASODN仅存在轻度非特异性作用。此外,实验结果还提示不同作用靶位的ASODN之间具有一定的协同作用。结论HCV5′NCR第二茎环结构Ⅱa、第三茎环结构Ⅲd和翻译起始区可能是ASODN作用的重要靶序列。     

HCV 5′NCR和结构蛋白编码序列的克隆及对PA317细胞的转染

目的进一步研究丙型肝炎病毒５′ＮＣＲ对结构蛋白编码基因的表达调控。方法以拼接好的ＨＣＶ５′ＮＣＲ，Ｃ，Ｅ１和Ｅ２／ＮＳ１区基因共长２５４７个核苷酸片段克隆于逆转录病毒载体ＬＮＳＸ得重组体ｐＬＨＣ２５４７，用聚合酶链反应（ＰＣＲ）及核苷酸序列分析等方法对重组体进行鉴定。通过磷酸钙－ＤＮＡ共沉淀法把经鉴定的重组体转染入ＰＡ３１７细胞，用Ｇ４１８进行克隆筛选，以ＮＩＨ３Ｔ３细胞测其病毒滴度。提取转染细胞ＤＮＡ及培养上清ＲＮＡ分别用ＰＣＲ和逆转录ＰＣＲ进行鉴定。结果培养上清的病毒滴度为２×１０５ＣＦＵ／ｍｌ，ＰＣＲ及逆转录ＰＣＲ（ＲＴ－ＰＣＲ）分别可以从转染的细胞ＤＮＡ及培养上清中扩增出ＨＣＶ的基因片段。结论目的基因已整合到细胞的基因组中并得以表达     

Construction of HCV—core gene vector and its expression in cholangiocarcinoma

AIM: To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. METHODS: Recombinant plasmid of HCV-core gene was constructed with molecular cloning technique and transfected into QBC939 cells with lipofection.After it was selected with G418, resistant colonies were obtained. The colonies were analysed by immunocytochemistry and Western blotting.The morphology was observed under transmission electron microscope(TEM) and microscope. RESULTS: The recombinant plasmid was proved to carry the target gene by PCR and restriction enzymed mapping. Moreover, it could express HCV-C protein efficiently in QBC939 cells. The HCV-like particles were found in the cytoplasm by electron microscope, which were spherical with a diameter of 50 nm-80 nm possessing outer membrane.The transfected cells had lower differentiation and higher malignant degree under microscope. CONCLUSION: Because HCV-core gene could express steadily in cholangiocarcinoma cells,the transfected tumor cells(QBC939-HCVC) could be used to study the effect of HCV in the development of cholangiocarcinoma.     

重组体pCD—HCV1诱发小鼠免疫反应的研究

目的探讨丙型肝炎病毒（ＨＣＶ）基因重组体诱发小鼠免疫反应。方法将编码Ⅱ型ＨＣＶ结构蛋白的基因片段Ｃ、Ｅ１和大部分Ｅ２插入到真核细胞表达载体ｐＣＤＳＲα１中,构建成重组体ｐＣＤＨＣＶ１。经肌内注射将此重组体免疫Ｂａｌｂ／ｃ小鼠。结果ｐＣＤＨＣＶ１（１００μｇ／只,ｎ＝１２）３～４次接种小鼠后,血清抗体水平达０．７１～０．７７（Ａ值,下同）,空载体ｐＣＤＳＲα１免疫鼠（ｎ＝８）,血清抗体阴性;对其中８只免疫鼠持续检测１８周,未见抗体水平有下降趋势（０．６８～０．７５）。重组体ｐＣＤＨＣＶ１免疫鼠（ｎ＝１２）的脾细胞对ＨＣＶ合成肽ＣＰ９、基因重组抗原Ｃ、Ｅ１刺激后,均出现增殖反应（ｃｐｍ）,ＳＩ分别为４．１４±０．９,３．９８±１．６,４．０２±１．２,与ｐＣＤＳＲα１免疫鼠（ｎ＝８）相比,差异有显著性（Ｐ＜０．００１）。结论构建的ＨＣＶＤＮＡ重组体可诱发Ｂａｌｂ／ｃ小鼠产生免疫反应。     

Management of hepatitis C virus infection in HIV/HCV co-infected patients： Clinical review

Nearly one fourth of individuals with human immunodeficiency virus （HIV） infection have hepatitis C virus （HCV） infection in the US and Western Europe. With the availability of highly active antiretroviral therapy and the consequent reduction in opportunistic infections, resulting in the prolongation of the life span of HIV-infected patients, HCV co-infection has emerged as a significant factor influencing the survival of HIV patients. Patients with HIV/HCV co-infection have a faster rate of fibrosis progression resulting in more frequent occurrences of cirrhosis, end-stage liver disease, and hepatocellular carcinoma. However, the mechanism of interaction between the two viruses is not completely understood. The treatment for HCV in co-infected patients is similar to that of HCV monoinfection; i.e., a combination of pegylated interferon and ribavirin. The presence of any barriers to anti- HCV therapy should be identified and eliminated in order to recruit all eligible patients. The response to treatment in co-infected patients is inferior compared to the response in patients with HCV mono-infection. The sustained virologic response rate is only 38% for genotype-1 and 75% for genotype-2 and -3 infections. Liver transplantation is no longer considered a contraindication for end-stage liver disease in coinfected patients. However, the 5 year survival rate is lower in co-infected patients compared to patients with HCV mono-infection （33% vs 72%, P = 0.07）. A betterunderstanding of liver disease in co-infected patients is needed to derive new strategies for improving outcome, and survival.     

Mutations in carboxy-terminal part of E2 including PKR/eIF2α phosphorylation homology domain and interferon sensitivity determining region of nonstructural 5A of hepatitis C virus 1b： Their correlation with response to interferon monotherapy and viral load

AIM: To study the amino acid substitutions in the carboxy (C)-terminal part of E2 protein and in the interferon (IFN) sensitivity determining region (ISDR) and their correlation with response to IFN and viral load in 85 hepatitis C virus (HCV)-1b-infected patients treated with IFN. METHODS: The C-terminal part of E2 (codons 617-711) including PKR/eIF2alpha phosphorylation homology domain (PePHD) and ISDR was sequenced in 85 HCV-1b-infected patients treated by IFN monotherapy. RESULTS: The amino acid substitutions in PePHD detected only in 4 of 85 patients were not correlated either with response to IFN or with viral load. The presence of substitutions in a N-terminal variable region (codons 617-641) in the C-terminal part of E2 was significantly correlated with both small viral load (33.9% vs 13.8%, P = 0.0394) and sustained response to IFN (25.0% vs 6.9%, P = 0.0429). Four or more substitutions in ISDR were significantly correlated with both small viral load (78.6% vs 16.2%, P < 0.0001) and sustained response to IFN (85.7% vs 2.9%, P < 0.0001). In multivariate analysis, ISDR in nonstructural (NS) 5A (OR = 0.39, P < 0.0001) and N-terminal variable region (OR = 0.51, P = 0.039) was selected as the independent predictors for small viral load, and ISDR (OR = 39.0, P < 0.0001) was selected as the only independent predictor for sustained response. CONCLUSION: The N-terminal variable region in the C-terminal part of E2 correlates with both response to IFN monotherapy and viral load and is one of the factors independently associated with a small viral load.     

HBV cccDNA in patients＇ sera as an indicator for HBV reactivation and an early signal of liver damage

AIM: To evaluate the covalently closed circle DNA (cccDNA)level of hepatitis B virus (HBV) in patients‘ liver and sera. METHODS: HBV DNA was isolated from patients‘ liver biopsies and sera. A sensitive real-time PCR method, which is capable of differentiation of HBV viral genomic DNA and cccDNA, was used to quantify the total HBV cccDNA. The total HBV viral DNA was quantitated by real-time PCR using a HBV diagnostic kit (PG Biotech, LTD, Shenzhen, China)described previously. RESULTS: For the first time, we measured the level of HBV DNA and cccDNA isolated from ten HBV patients‘ liver biopsies and sera. In the liver biopsies, cccDNA was detected from all the biopsy samples. The copy number of cccDNA ranged from from 0.03 to 173.1 per ceil, the copy number of total HBV DNA ranged from 0.08 to 3 717 per cell. The ratio of total HBV DNA to cccDNA ranged from 1 to 3 406. In the sera,cccDNA was only detected from six samples whereas HBV viral DNA was detected from all ten samples. The ratio of cccDNA to total HBV DNA ranged from 0 to 1.77%. To further investigate the reason why cccDNA could only be detected in some patients‘ sera, we performed longitudinal studies. The cccDNA was detected from the patients‘ sera with HBV reactivation but not from the patients‘ sera without HBV reactivation. The level of cccDNA in the sera was correlated with ALT and viral load in the HBV reactivation patients. CONCLUSION: HBV cccDNA is actively transcribed and replicated in some patients‘ hepatocytes, which is reflected by a high ratio of HBV total DNA vs cccDNA. Detection of cccDNA in the liver biopsy will provide an end-point for the anti-HBV therapy. The occurrence of cccDNA in the sera is an early signal of liver damage, which may be another important clinical parameter.     

Positional effect of mutations in 5＇UTR of hepatitis C virus 4a on patients＇ response to therapy

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.
METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.
RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.
CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.     

In vitro assay for HCV serine proteinase expressed in insect cells

AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells. METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cell sand packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS)and purifed by metalchelated-affinity chromatogralphy, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected. RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5&#215;10^7 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients‘‘ sera in indirect ELISA format. In vitro cleavage assay corroborated itse nzymatic activity. CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.     

HCV5‘NCR调控抑制活性的反义寡核苷酸的筛选

目的 寻找高效特异的新型抗ＨＣＶ药物,探讨针对ＨＣＶ基因的硫代修饰反义寡核苷酸（Ｓ－ＡＳＯＤＮｓ）最佳作用靶序列。方法 参考ＨＣＶ５’ＮＣＲ计算机预测的二级结构图,设计合成了１５条Ｓ－ＡＳＯＤＮ、３条正义寡核苷酸及１条随机序列,采用ＨＣＶ５’ＮＣＲ调控荧光素酶基因的瞬时表达系统,将Ｓ－ＡＳＯＤＮ与ｐＨＣＶ－ｎｅｏ４共转染,通过荧光素激活性表达高低,反映Ｓ－ＡＳＯＤＮ对ＨＣＶ调控基因的抑制活性。结果     

Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes

Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection.     

丙型肝炎病毒5＇端非编码区和NS3蛋白酶共调控外分泌性碱性磷酸酶表达细胞模型的建立及意义

目的 构建丙型肝炎病毒5'端非编码区(HCV 5'NCR)和NS3丝氨酸蛋白酶共调控外分泌性碱性磷酸酶(SEAP)表达细胞模型,并分析其用于抗HCV药物筛选和评价的可行性。 方法 用聚合酶链反应技术扩增HCV 5'NCR和NS3/4A片段,定向克隆至表达质粒pSEAP2-Control的SEAP基因上游,构建含HCV 5'NCR-NS3/4A-SEAP嵌合基因的重组表达质粒pNCR-NS3/4A-SEAP。将重组质粒转染至肝细胞株QSG7701,用化学发光法检测SEAP的表达,并观察HCV 5'NCR区对应的反义寡聚核苷酸(ASODN)和丝氨酸蛋白酶抑制剂TPCK对SEAP表达的影响。 结果 重组质粒pNCR-NS3/4A-SEAP有高强度SEAP表达,5μmol/L、10μmol/L ASODN和100μmol/L TPCK对SEAP表达有显著抑制作用(t值分别为4.315、6.985、6.949,P值均<0.01)。 结论 重组质粒pNCR-NS3/4A-SEAP的SEAP表达受HCV 5'NCR和NS 3蛋白酶共调控,建立的细胞模型可用于以HCV 5'NCR和NS3蛋白酶为靶位点的药物筛选和评价。     

HepG2细胞中HCV NS5A蛋白对HCV IRES启动蛋白翻译的影响

目的探讨HepG2细胞中HCV非结构蛋白5A（NS5A）对HCV IRES启动蛋白翻译的影响，以了解HCV的复制调控机制。方法将构建的表达双荧光素酶的双顺反子载体pCMV-Rluc-IRES-Fluc和含HCV NS5A基因的表达质粒pcDNA-NS5A共转染HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，细胞免疫荧光技术检测HCV-NS5A蛋白的表达，RT-PCR检测虫荧光素酶基因mRNA水平，并与相应对照做比较，以观察HCV NS5A对HCV IRES介导虫荧光素酶翻译水平的影响。结果转染pcDNA-NS5A的HepG2细胞中虫荧光素酶活性明显高于转染pCDNA3．I-3flag的对照组，并存在剂量依赖关系；而RT-PCR虫荧光素酶基因mRNA水平在两组间差异无统计学意义。转染pcDNA-NS5A的HepG2细胞质中可见HCV NS5A蛋白的表达。结论HCV NS5A蛋白对HCV IRES介导虫荧光素酶的翻译有正调节作用，并存在剂量依赖关系．     

Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. Successful vaccine development is crucial in controlling global HCV infection. We have previously described the generation of HCV-like particles (HCV-LPs) in insect cells using a recombinant baculovirus containing the complementary DNA of the HCV structural proteins. These HCV-LPs had similar morphological and biophysical properties as the putative virions. In this study, we analyzed the structural features, antigenic composition, seroreactivity, and immunogenicity of purified HCV-LPs. METHODS: HCV-LPs were analyzed by electron microscopy and antibody immunolabeling and precipitation. An enzyme-linked immunosorbent assay (ELISA) using HCV-LPs was developed. The humoral response to HCV-LPs in mice was studies by core and envelope ELISAs, Western immunoblotting, and immunofluorescence. RESULTS: Structural and antigenic compositions of HCV-LPs were shown to be similar to those of putative HCV virions. Using the HCV-LP ELISA, high-titer anti-HCV antibodies were detected in individuals infected with various HCV genotypes. In vivo, HCV-LPs elicited a humoral response broadly directed against HCV structural proteins. CONCLUSIONS: HCV-LPs resemble HCV virions and are capable of inducing a humoral response targeted against various regions of HCV structural proteins, suggesting that HCV-LPs may be promising as a potential vaccine candidate.     

HCV NS5B在人肝癌细胞系Huh7细胞内的表达及活性分析

目的为靶向抗HCV药物筛选奠定基础。方法使用脂质体将包含HCV非结构蛋白5B（NS5B）基因的重组载体pcDNA-5B转染至人肝癌细胞系Huh7细胞,分别采用RT-PCR和Western Blot鉴定NS5B mRNA和蛋白表达,采用荧光素酶试验检测NS5B细胞内RNA依赖的RNA聚合酶（RdRp）活性。结果转染的Huh7细胞出现1．8kb的特异性NSSB mRNA目的带及一条相对分子质量约66kD的蛋白目的带,且后者具有细胞内RdRp活性。结论人肝癌细胞系Huh7细胞可成功表达具有细胞内RdRp活性的NS5B,以其为靶标的抗HCV药物可为临床治疗提供新思路。