Inhibition of hepatitis B virus via selective apoptosis modulation by Chinese patent medicine Liuweiwuling Tablet

BACKGROUND Liuweiwuling Tablet(LWWL)is a Chinese patent medicine approved for the treatment of chronic inflammation caused by hepatitis B virus(HBV)infection.Previous studies have indicated an anti-HBV effect of LWWL,specifically in terms of antigen inhibition,but the underlying mechanism remains unclear.AIM To investigate the potential mechanism of action of LWWL against HBV.METHODS In vitro experiments utilized three HBV-replicating and three non-HBV-replicating cell lines.The in vivo experiment involved a hydrodynamic injectionmediated mouse model with HBV replication.Transcriptomics and metabolomics were used to investigate the underlying mechanisms of action of LWWL.RESULTS In HepG2.1403F cells,LWWL(0.8 mg/mL)exhibited inhibitory effects on HBV DNA,hepatitis B surface antigen and pregenomic RNA(pgRNA)at rates of 51.36%,24.74%and 50.74%,respectively.The inhibition rates of LWWL(0.8mg/mL)on pgRNA/covalently closed circular DNA in HepG2.1403F,HepG2.2.15 and HepG2.A64 cells were 47.78%,39.51%and 46.74%,respectively.Integration of transcriptomics and metabolomics showed that the anti-HBV effect of LWWL was primarily linked to pathways related to apoptosis(PI3K-AKT,CASP8-CASP3 and P53 pathways).Apoptosis flow analysis revealed that the apoptosis rate in the LWWL-treated group was significantly higher than in the control group(CG)among HBV-replicating cell lines,including HepG2.2.15(2.92%±1.01%vs 6.68%±2.04%,P<0.05),HepG2.A64(4.89%±1.28%vs 8.52%±0.50%,P<0.05)and HepG2.1403F(3.76%±1.40%vs 7.57%±1.35%,P<0.05)(CG vs LWWL-treated group).However,there were no significant differences in apoptosis rates between the non-HBV-replicating HepG2 cells(5.04%±0.74%vs 5.51%±1.57%,P>0.05),L02 cells(5.49%±0.80%vs 5.48%±1.01%,P>0.05)and LX2 cells(6.29%±1.54%vs 6.29%±0.88%,P>0.05).TUNEL staining revealed a significantly higher apoptosis rate in the LWWL-treated group than in the CG in the HBVreplicating mouse model,while no noticeable difference in apoptosis rates between the two groups was observed in the non-HBV-replicating mouse model.CONCLUSION Preliminary results suggest that LWWL exerts a potent inhibitory effect on wild-type and drug-resistant HBV,potentially involving selective regulation of apoptosis.These findings offer novel insights into the anti-HBV activities of LWWL and present a novel mechanism for the development of anti-HBV medications.

Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes

AIM: To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells. METHODS: HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by Iipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 μg/mL G418 and named HL-7702/HBV-encoded X protein (HBx) cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively. RESULTS: RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semi-quantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than those in control. CONCLUSION: HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.

Recombinant adeno-associated virus delivered human thioredoxin-PR39 prevents hypoxia-induced apoptosis of ECV304 cells

Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.

Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

Cloning of Chicken Anemia Virus vp3 Gene and Apoptosis Inductive Effect of vp3 Gene In Vitro

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE TM mediated transfection in vitro with pcDNA vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L 02, and RT PCR was performed afterward. The results of RT PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.

Invertebrate Iridovirus Modulation of Apoptosis

Programmed cell death (apoptosis) is a key host response to virus infection. Viruses that can modulate host apoptotic responses are likely to gain important opportunities for transmission. Here we review recent studies that demonstrate that particles of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae, genus Iridovirus), or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off host macromolecular synthesis. Induction of apoptosis depends on endocytosis of one or more heat-sensitive virion component(s). Studies with a JNK inhibitor (SP600125) indicated that the JNK signaling pathway is significantly involved in apoptosis in IIV-6 infections of Choristoneura fumiferana cells. The genome of IIV-6 codes for an inhibitor of apoptosis iap gene (193R) that encodes a protein of 208 aa with 15% identity and 28% similarity in its amino acid sequence to IAP-3 from Cydia pomonella ganulovirus (CpGV). Transcription of IIV-6 iap did not require prior DNA or protein synthesis, indicating that it is an immediate-early class gene. Transient expression and gene knockdown studies have confirmed the functional nature of the IIV-6 iap gene. We present a tentative model for IIV-6 induction and inhibition of apoptosis in insect cells and discuss the potential applications of these findings in insect pest control.

A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

Hypoxia-inducible factor 1 （HIF-1） attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we construct-ed a recombinant adeno-associated virus （rAAV） vector expressing the human HIF-1αgene （rAAV-HIF-1α）, and tested the assumption that rAAV-HIF-1αrepresses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results conifrmed that rAAV-HIF-1αsigniifcant-ly reduces apoptosis induced by amyloid-beta protein in primary cultured hippocampal neurons. Direct intracerebral rAAV-HIF-1αadministration also induced robust and prolonged HIF-1αproduction in rat hippocampus. Single rAAV-HIF-1αadministration resulted in decreased apoptosis of hippocampal neurons in an Alzheimer＇s disease rat model established by intrace-rebroventricular injection of aggregated amyloid-beta protein （25-35）. Our in vitro and in vivo ifndings demonstrate that HIF-1 has potential for attenuating hippocampal neuronal apoptosis induced by amyloid-beta protein, and provides experimental support for treatment of neurode-generative diseases using gene therapy.

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells. METHODS: The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBV X gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colonyforming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells. RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the Ad0 and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice. CONCLUSION: Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBV X and HCV C genes on hepatocarcinogenesis in athymic nude mice.

Experimental Study on the Antitumor Effect of Chicken Anemia Virus vp3 Gene against Liver Carcinoma In Vivo

In order to testify the antitumor effect, especially its effect against liver carcinoma in vivo, of VP3 protein, one kind of protein coded by chicken anemia virus, recombinants pcDNA-vp3 containing chicken anemia virus vp3 gene, and control vector pcDNA3 were mixed with murine liver carcinoma cell lines H22 respectively. The mixture was injected subcutaneously into Balb/C mice. Some days later, the mice were killed and the solid tumor weighed. The antitumor efficiency was evaluated. The manners of VP3 protein in vivo inducing tumor cell death were identified by using TUNEL assay. All the results suggested that the injection of pcDNA-vp3 and H22 mixture resulted in a significant reduction of tumor growth in mice when compared with the results of control groups. TUNEL assay revealed that VP3 induced apoptosis in vivo. All these indicated that CAV vp3 might be a potential new gene in reducing the growth rate of tumor cells in liver carcinoma or in other kind of solid tumors in vivo.

PLASMID VACCINE ENCODING HN GENE FROM NEWCASTLE DISEASE VIRUS HAS MARKED ANTITUMORAL EFFECT IN VITRO

Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.

大豆异黄酮的降血糖活性研究

本文研究了大豆异黄酮纯级分(SI-Ⅱ)的降血糖作用。结果表明:SI-Ⅱ能显著(p<0.01)降低四氧嘧啶糖尿病小鼠的血糖水平,显著(p<0.01)提高其血清胰岛素浓度,缓解糖尿病症状;抑制胰岛细胞Fas蛋白表达,促进胰岛β细胞的恢复;而且能增加糖尿病小鼠的免疫器官重量。提示大豆异黄酮具有降血糖作用,其作用机制可能是通过抑制胰岛细胞凋亡、提高免疫功能等途径促进胰岛β细胞功能恢复。

鸡贫血病毒vp3基因体内抑瘤效应的实验研究

目的 观察鸡贫血病毒 ( chicken anem ia virus,CAV) vp3基因的体内抑瘤效应。方法 分别将 pc DNA- vp3(含 vp3基因的真核表达载体 )、pc DNA3 (空载体 )和小鼠腹水型肝癌细胞株 H2 2混合后 ,接种于 BAL B/ c小鼠皮下。几天后 ,处死动物 ,取肿瘤组织称重 ,比较试验组和对照组瘤重差异 ,确定 vp3基因的抑瘤效应。TU NEL 法鉴定 vp3在体内诱导肿瘤细胞死亡的方式。结果 注射 vp3基因的试验组肿瘤生长率明显低于注射空载体的对照组。TUNEL 试验的结果也表明 ,鸡贫血病毒 VP3蛋白在体内以凋亡方式诱导肿瘤细胞死亡。结论 预示

新城疫病毒HN基因诱导人肺癌细胞SPC-A1凋亡的作用机制

为了探索新城疫病毒血凝素神经氨酸酶(HN)基因对人肺癌细胞SPC-A1的作用及机制,将含有HN基因的重组质粒pVHN经脂质体介导转染人肺癌细胞SPC-A1,通过MTT方法检测细胞活力;采用吖啶橙/溴化乙锭(AO/EB)染色分析肿瘤细胞凋亡;罗丹明123和DCFA染色测定线粒体跨膜电位(ΔΨm)和活性氧水平变化;流式细胞仪分析MHC-Ⅰ分子表达;底物染色反应检测caspase-3活性.结果重组质粒pVHN转染人肺癌细胞SPC-A148h后,细胞活力明显降低;AO/EB染色可见明显的细胞凋亡形态学变化;与空质粒对照相比,线粒体ΔΨm下降(P<0·01),活性氧水平升高(P<0·05);细胞表面MHC-Ⅰ分子表达上调(P>0·05);caspase-3活性增强(P<0·01).以上结果提示,新城疫病毒HN基因能够上调SPC-A1细胞表面MHC-Ⅰ分子表达,并通过上调ROS水平,下调线粒体ΔΨm,进而激活caspase-3,最终诱导人肺癌细胞凋亡.

细胞凋亡与小鼠病毒性心肌炎发生发展的关系

通过给ＢＡＬＢ／Ｃ小鼠腹腔接种ＣＶＢ３ｍ后３、６、９、１２、１５、３０天分批随机处死小鼠，取心脏进行检测发现：感染后９～１５天，病毒性心肌炎（ＶＭＣ）检出率可达９２．３０％。感染后３～９天，心肌中可分离出病毒，而病毒核酸（ＣＶＢＲＮＡ）可持续存在于感染后３个月。应用电镜、原位末端标记法（ＴＵＮＥＬ）及免疫组化法检测发现：ＶＭＣ鼠心肌中可见心肌细胞和血管内皮细胞凋亡及ＴＧＦ－β１和Ｃ－ｍｙｃ蛋白的表达。感染后９～１５天，三者阳性率分别为７５．８６％、７２．４１％和８２．７６％，且分布区域基本一致。Ｐ５３蛋白摄影性率为１．９２％，无统计学意义。实验结果表明：ＣＶＢ３ｍ可诱发ＢＡＬＢ／Ｃ小鼠ＶＭＣ，而细胞凋亡可能参与ＶＭＣ的发生与发展。ＣＶＢ３ｍ、ＣＶＢＲＮＡ、ＴＧＦ－β１和Ｃ－ｍｙｃ蛋白可能在ＶＭＣ细胞凋亡调控机制中起一定作用。进而揭示细胞凋亡可能与ＤＣＭ。

呼吸道合胞病毒感染对细胞凋亡及FasL、Fas、Bcl-2、Bax基因表达的研究

目的:研究呼吸道合胞病毒(RSV)感染与细胞凋亡的关系及对凋亡相关基因FasL、Fas、bcl-2和bax表达的影响。方法:采用A549细胞,在RSV感染后不同时点收集细胞,流式细胞仪和透射电镜检测细胞凋亡,免疫组织化学法检测凋亡相关基因FasL、Fas、Bcl-2和Bax表达情况。结果:流式细胞仪检测结果RSV感染后72h(6.61%)、120h(10.94%)的细胞凋亡指数明显高于对照组(4.32%、5.31%);免疫组化法检测结果对照组FasL、Bax基因呈现无表达或局部弱表达;随着RSV感染时间的延长,bax、Fas、FasL基因的表达均高于对照细胞,bcl-2基因呈现弱表达或无表达。结论:RSV在感染后期能诱导宿主细胞发生凋亡,促凋亡基因FasL、Fas、Bax和抗凋亡基因Bcl-2表达水平的差异是RSV诱导凋亡的机制之一。

乙型肝炎病毒DNA多聚酶RNase H对细胞凋亡易感基因表达的上调作用

目的探讨乙型肝炎病毒DNA多聚酶(HBV DNA P)结构域蛋白RNase H对细胞凋亡易感基因(CAS)的转录激活作用。方法以HBV DNA P结构域蛋白RNase H的反式调节因子的基因表达谱芯片结果为基础,利用生物信息学技术确定CAS的启动子区域(CASp),扩增CASp并克隆至真核报告载体pCAT3-Basic中,构建pCAT3-CASp报告载体。分别以该质粒单独或与pcDNA3.1(-)-RH共转染肝癌细胞系HepG2细胞,ELISA法检测氯霉素乙酰转移酶(CAT)的表达活性,并以pCAT3-Basic空载体、pCAT3-TXNRD1p分别转染HepG2细胞作为阴性和阳性对照。结果pCAT3-CASp和pcDNA3.1(-)-RH瞬时共转染的HepG2细胞的CAT表达活性是pCAT3-CASp的1.5倍,是pCAT3-Basic空载体的2.7倍。结论本实验进一步验证了我室利用基因表达谱技术研究RNase H蛋白反式激活作用的结果。我室克隆的CAS启动子具有顺式激活下游基因的活性;HBV的RNase H蛋白具有对CAS的反式激活作用。

乙型肝炎病毒X基因对正常肝细胞HL7702凋亡影响的初步探讨

目的研究乙型肝炎病毒X基因(HBX基因)对HL7702肝细胞凋亡及凋亡相关因子表达的影响。方法用脂质体转染法将HBx真核表达载体pcDNA3.1(+)-X瞬时转入HL7702细胞,以转染空质粒pcDNA3.1(+)的细胞及未转染的HL7702细胞为对照。转染后72 h收集细胞标本,作以下处理:RT-PCR法检测HBXmRNA的表达,免疫荧光鉴定HBx蛋白的表达;流式细胞术检测细胞凋亡率;以β-actin为内参,半定量RT-PCR法检测凋亡相关基因Bax、Bcl-2 mRNA的表达量变化。结果转染72 h后,转染pcDNA3.1(+)-X真核表达载体的HL7702细胞经RT-PCR扩增出HBX片段,免疫荧光结果显示细胞内出现HBx蛋白的较强荧光;而转染空质粒组及未转染对照组经RT-PCR法及免疫荧光检测均显示无HBx表达。通过流式细胞术和半定量RT-PCR法检测细胞凋亡情况,结果显示:转染HBX的细胞凋亡率(3.38%±0.67%)相对于转染空质粒组(1.25%±0.30%)及未转染质粒组(1.40%±0.37%)凋亡率增高,差异均有显著性意义(P<0.05);转染HBx的细胞Bax mRNA相对表达量较转染空质粒组和未转染质粒组明显增高,差异均有显著性意义(P<0.05),Bcl-2 mRNA相对表达量较转染空质粒组和未转染质粒组明显降低,差异均有显著性意义(P<0.05)。转染空质粒组和未转染质粒组之间,对细胞凋亡率、Bax mRNA、Bcl-2 mRNA进行比较,差异无统计学意义(P>0.05)。结论成功将HBX转染入HL7702细胞,并在细胞中表达;转染后72 h HBx可上调Bax表达,而下调Bcl-2表达,表明HBx能促进HL7702细胞凋亡。

rL-RVG体外抑制肺癌细胞增殖并促进其凋亡

目的将表达狂犬病毒糖蛋白的重组LaSota株新城疫病毒疫苗(rL-RVG)感染至A549肺腺癌细胞,观察其对肺癌细胞增殖和细胞凋亡的影响。方法采用直接感染的方法将rL-RVG感染至A549细胞,采用Western blot法检测rL-RVG中RVG和NDV蛋白在A549细胞内的表达;MTT法检测rL-RVG对细胞增殖的影响,TUNEL法检测rL-RVG对A549细胞凋亡的影响;AnnexinⅤ-FITC/PI染色结合流式细胞检测术检测A549细胞凋亡情况;Western blot法检测caspase-3的表达,并与对照组LaSota株进行比较,PBS组为空白对照组。结果 A549细胞感染rL-RVG后RVG蛋白和NDV蛋白都稳定表达,MTT法结果显示细胞增殖明显被抑制,rL-RVG组抑制率高于LaSota组。A549细胞凋亡增多,其中流式细胞检测术中显示rL-RVG组早期凋亡细胞较其他2组增多,差异有统计学意义(P<0.05),TUNEL检测凋亡指数增加,rL-RVG组较其他2组增多,差异有统计学意义(P<0.05)。Western blot法显示促凋亡蛋白caspase-3表达增加,加入caspase抑制剂Z-VAD-FMK后,caspase-3蛋白表达明显降低。结论 rL-RVG感染A549细胞后能在细胞内稳定表达,rL-RVG可抑制肺癌细胞生长、促进肺癌细胞凋亡,效果优于野生LaSota株。

凋亡相关基因bcl-2、bax、Fas及FasL在乙型肝炎病毒相关性肝癌组织中的表达

目的 探讨原发性肝癌中乙型肝炎病毒感染与凋亡基因bcl-2、bax、Fas及FasL表达的关系。方法 采用免疫组化技术分析40例原发肝细胞癌组织中乙肝病毒表面抗原(HBsAg) 阳性组和HBsAg阴性组bcl-2、bax、Fas及FasL的表达。结果 癌组织中HBsAg阳性率为30%(12/40)，癌旁组织中HBsAg阳性率为62.5%(25/40)。12例HBsAg阳性的癌组织中bcl-2和bax的阳性率比值为1：1.5(2/12: 3/12)，28例HBsAg阴性的癌组织中bcl-2和bax阳性率比值为1:1.25(4/28: 5/28)；25例HBsAg阳性和15例HBsAg阴性的癌旁肝细胞中bcl-2和bax的阳性率比值分别为1:3.2 (5/25：16/25) 和1:3(3/15: 9/15)。12例HBsAg阳性和28例HBsAg阴性的肝癌中Fas 和FasL阳性例数分别为6、5和1、1；25例HBsAg阳性和15例HBsAg阴性的癌旁肝细胞Fas和FasL阳性例数分别为17、5和4、3。两组间比较均有明显差异(P<0.05)。结论 肝癌组织中HBV感染与bcl-2和bax阳性表达比率无明显关系，而与Fas和FasL的表达有关。

鸡贫血病毒vp3基因的克隆及其体外凋亡诱导效应的研究

用 PCR方法扩增了鸡贫血病毒标准株的 vp3基因 ,并将其克隆于真核表达载体 pc DNA3上 ,构建了重组体pc DNA- vp3。经酶切鉴定及测序分析表明 ,该片段和预期相符。在体外利用 L ipofect AMINETM介导的基因转染 ,将 pc D-NA- vp3、 pc DNA3分别转入肝癌细胞系 Hep G2和二倍体肝细胞系 L- 0 2中 ,转染后的 RT- PCR结果证实 vp3基因在细胞中得到了表达。同时利用筛选稳定表达细胞株的技术和原位细胞凋亡检测法 ,证明了鸡贫血病毒是以凋亡的方式诱导细胞死亡 ,并且只诱导癌细胞的凋亡 ,而不诱导正常或二倍体细胞死亡。表明鸡贫血病毒