Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma

Hepatitis B virus (HBV) may be one of the agents involved in the aetiology of human primary liver cancer. This hypothesis is supported by (1) the similarity between the geographical distribution of chronic carriers of the viral surface antigen (HBsAg) and that of hepatocellular carcinoma (HCC); (2) the increase in the prevalence of HBV markers in serum of patients with primary liver cancer when compared with the general population; (3) the observation that HBV infection precedes the development of the tumour. Moreover, these epidemiological indications of an association between HBV infecton and hepatocellular carcinoma are supported by the detection of HBV markers such as HBsAg or viral DNA sequences, although in a non-integrated form in tumour tissue. To study the relationship between HBV and primary liver cancer further, we looked for the presence of free or integrated viral DNA in tumour tissue of human hepatocellular carcinomas and in a HBsAg-producing human hepatoma cell line. Using the blot-transfer hybridization technique and cloned HBV DNA as a probe, we have now demonstrated that the viral DNA is integrated in the cellular genome both in tumour tissue and in a hepatoma cell line.     

NVP-BEZ235诱导自噬在肝癌细胞凋亡中的作用研究

目的 以Hep3B和PLC/PRF/5两种肝癌细胞为研究对象,探讨自噬在NVP-BEZ235中引起细胞凋亡的作用.方法 人类肝癌细胞Hep3B和PLC/PRF/5在含有10%FBS的DMEM培养基中进行培养,然后再进行细胞活性检测,用于Western blotting检测及线粒体膜电位(MMP)分析.结果 NVP-BEZ235能够有效增加LC3-Ⅱ的表达,并同时降低p62的表达,由此推断,NVP-BEZ235也能够诱导产生自噬,与Atg5的siRNA或自噬抑制剂3-甲基腺嘌呤(3-MA)联合应用时,肝癌细胞的生长被抑制.结论 NVP-BEZ235与Atg5的siRNA或者3-MA的联合应用能够促进细胞凋亡,NVP-BEZ235和自噬抑制剂的联合应用可为肝癌和其他恶性肿瘤临床治疗提供新的方法.     

治疗性乙型肝炎疫苗研究反思

 “治疗性乙肝疫苗”是以乙肝病毒(HBV)的表面抗原(s抗原)为基础的生物制剂,目的是激发抗s抗原免疫反应,终止HBV慢性感染.HBV的e抗原与s抗原无抗原性关联,对e抗原的反应也与病毒及感染细胞的清除无关,因此以II期临床试验者血清有关病毒e抗原的数据结果,尚不能判断“治疗性乙肝疫苗”是否有效.疫苗的应用是抵御病毒入侵,而治疗性乙肝疫苗是在病毒已经进入人体后应用,在患者肝细胞可能广泛受累的情况下,疫苗一旦打破耐受激发抗s抗原免疫反应,除能清除血清中游离的病毒和s抗原颗粒外,将直接攻击被感染的肝细胞.由于无法估计慢性HBV感染者肝细胞感染程度,所以无法推测免疫反应发生后,免疫病理所致的肝损程度以及相应的风险.在应用上隐含如此风险,是“治疗性乙肝疫苗”走不出实验室的重要因素之一.     

武汉地区肝癌患者中HBV基因型分析

目的:分析武汉地区原发性肝癌患者HBV感染情况及基因型分析。方法:利用HBV检测引物及分型引物对肝癌患者血清进行PCR,通过电泳检测PCR特异性产物。结果:50例患者血清中44例HBV DNA呈阳性,占总数的88%;其中30例为C型占68%,14例为B型占32%。结论:武汉地区原发性肝癌患者多为HBV感染患者,而且以HBV C型感染为主。     

Biosynthesis of hepatitis B virus surface antigen in Escherichia coli

Hepatitis B is a widespread viral disease. In the absence of cell cultures capable of propagating the virus (HBV) an efficient vaccine has been prepared from viral envelopes isolated from the plasma of chronic carriers. The major polypeptide of the envelope is one of molecular weight 25,000 which carries the surface antigen (HBsAg). Therefore, the biosynthesis of this polypeptide in Escherichia coli may offer an alternative procedure to produce HbsAg free from human proteins. Recently, the HBV genome has been cloned in E.coli. Determination of its primary structure allowed the localization of the gene (called gene S) coding for HBsAg and the synthesis of the core antigen in E.coli has been reported. We have constructed a derivative of bacteriophage lambda carrying a fusion between the beta-galactosidase gene (lacZ) and the HBsAg coding sequence (lambdalacHBs-1). Infection of E.coli with lambdalacHBs-1 leads to the biosynthesis of a polypeptide of molecular weitht 138,000 carrying antigenic determinants of HBV surface antigen.     

人肿瘤细胞株中细小病毒H—1的DNA扩增与非结构蛋白NS—1基因的表达

研究三株人癌细胞和两株对照细胞对细小病毒Ｈ－１杀伤作用敏感性的分子机制．表明了在感染复数ｍｏｉ（ｍｕｌｔｉｐｉｃｉｔｙｏｆｉｎｆｅｃｔｉｏｎ）为５ｐｆｕ（ｐｌａｑｕｅｆｏｒｍｉｎｇｕｎｉｔ）／细胞的情况下，作为Ｈ—１病毒复制受纳细胞的人肝癌细胞株ＯＧＹ－７７０３和人胃癌细胞株ＳＧＣ—７９０１，能够支持病毒ＤＮＡ扩增和非结构蛋白ＮＳ—１基因的表达，这和作为阳性对照的由ＳＶ４０转化的新生儿肾细胞株ＮＢ—Ｋ一样，但对Ｈ—１病毒感染有抗性的人肾癌细胞株ＯＵＲ—１０和它的对照人胎肾细胞株ＨｕＫ—１，并不支持病毒ＤＮＡ扩增和ＮＳ—１蛋白的表达．本文结果指出，细小病毒Ｈ—１的杀伤作用与细胞中的病毒ＤＮＡ扩增及ＮＳ—１基因表达的程度相关．     

赛若金对HepG2.2.15细胞株HBV复制的抑制作用

以转染有ＨＢＶ的ＨｅｐＧ２．２．１５细胞株为实验模型,用ＭＴＴ法测定赛若金对２．２．１５细胞的生长抑制作用,流式细胞仪测定药物对２．２．１５细胞周期时相的改变,通过ＥＬＩＳＡ方法观察赛若金对２．２．１５细胞分泌ＨＢｅＡｇ及ＨＢｓＡｇ的影响,ＰＣＲ－Ｈｙｂ杂交法检测赛若金对２．２．１５细胞ＨＢＶ ＤＮＡ复制的抑制作用。结果显示,赛若金对转染乙肝病毒的２．２．１５细胞有生长抑制作用,抑制率随药物浓度呈     

935例HBsAg阳性血清检测PHSAR的临床意义

采用ＥＬＩＳＡ法检测９３５例乙肝病毒（ＨＢＶ）患者血清ＰＨＳＡＲ，并对其与有关ＨＢＶ标志物的关系进行了探讨．结果表明，ＰＨＳＡＲ检出率分别与ＨＢｓＡｇ、ＨＢｅＡｇ及抗－ＨＢｃ有密切关系，尤以ＨＢｅＡｇ的存在更为相关，是乙肝病毒复制，具有高度传染性的指标之一．     

RNA interference-mediated inhibition of Hepatitis B Virus replication

Persistent and recurrent infection of hepatitis B virus (HBV) represents one of the most common and severe viral infections of humans, and has caused a formidable health problem in the affected countries. Currently used antiviral drugs have a very limited success on controlling HBV replication and infection. RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of target mRNA in mammalian and plant cells, has recently been used to knockdown gene expression in various species. In this study, we sought to determine whether RNAi-mediated silencing of HBV viral gene expression could lead to the effective inhibition of HBV replication. We first developed RNAi vectors that expressed small interfering RNA (siRNA) and targeted the HBV core or surface gene sequence. Our results demonstrated that these specific siRNAs efficiently reduced the levels of corresponding viral RNAs and proteins, and thus suppressed viral replication. Treatment with siRNA gave the greatest reduction in the levels of HBsAg (92%) and in HBeAg (85%) respectively in the cultured cell medium. Our findings further demonstrated that the RNAi-mediated antiviral effect was sequence-specific and dose-dependent. Therefore, our findings strongly suggest that RNAi-mediated silencing of HBV viral genes could effectively inhibit the replication of HBV, hence RNAi-based strategy should be further explored as a more efficacious antiviral therapy of HBV infection.     

免疫接种乙肝疫苗防治效果探讨

目的：了解免疫接种乙肝疫苗后产生乙肝病毒表面抗体（抗-HBs）的阳性率和人群乙肝病毒（HBV）的感染率,来评价1992年我国乙肝疫苗纳入儿童计划免疫管理和2002年乙肝疫苗纳入儿童计划免疫以及卫生部/全球疫苗免疫联盟乙肝疫苗合作项目实施的效果。方法：对所有调查对象均采集静脉血4ml,由中国疾病预防控制中心病毒病所统一使用酶联免疫吸附试验（ELISA）方法对标本进行检测,统一检测表面抗原（HBsAg）,抗-HBs,核心抗体（抗-HBc）,对HBsAg阳性者再做e抗原（HBeAg）和e抗体（抗-HBe）检测。根据乙肝疫苗推广的时间分为1～4岁组69人,5～14岁组89人,15～59岁157人,对检测结果进行相关分析。结论：乙肝疫苗的推广和乙肝疫苗纳入儿童计划免疫对全人群的免疫水平都有很大的提高,尤其对婴儿预防乙肝病毒感染有显著的效果。     

树对人乙型肝炎病毒易感性的验证

为验证树对人乙型肝炎病毒 (HBV)的易感性 ,用含 HBV的人血清接种给 10只成年树 (雌雄各半 )。然后每周抽血 1次 ,每只动物共抽血 11次。用不同公司生产的 EL ISA试剂检测接种后的动物血清感染指标。实验观察至 13周 ,结果分别有 9只和 7只动物出现 HBs Ag阳性 ,持续最短 1周 ,最长 7周。用 PCR检测 ,结果有 1只动物连续 4周在血清中检测出 HBV DNA。表明树能感染人 HBV,但实验有待改进以延长感染持续时间     

树鼩对人乙型肝炎病毒易感性的验证

为验证树Ｑｕ对人乙型肝炎病毒（ＨＢＶ）的易感性,用含ＨＢＶ的人血清接种给１０只成年树Ｑｕ（雌雄各半）。然后每周抽血１次,每只动物共抽血１１次。用不同公司生产的ＥＬＩＳＡ试剂检测接种后的动物血清感染指标。实验观察至１３周,结果分别有９只和８只动物出现ＨＢｓＡｇ阳性,持续最短１周,最长７周。用ＰＣＲ检测,结果有１只动物连续４周在血清中检测出ＨＢＶ ＤＮＡ。表明树Ｑｕ能感染人ＨＢＶ,但实验有待改进以延长感染持续时间。     

大理吸毒人群HBV感染的调查及基因型研究

目的：调查大理吸毒人群乙型肝炎病毒（HBV）的基因型和亚型。方法：采集大理市某戒毒所吸毒人群血清共192份，采用酶联吸附免疫法（ELISA）测定HBV血清标志物（HBVM），再采用基因型特异性引物PCR和聚合酶链反应-限制性片段长度多态性（PCR—RFLP）方法测定HBV感染血清基因型和亚型。结果：192份血清中，HBsAg阳性率8．85％（17／192）。17份HBsAg阳性标本中基因型分别为：B型3例，均为Ba型；C型1例，为Cs型；B＋C混合型3例（Ba＋Cs）；未扩增10例。结论：大理吸毒人群HBV感染率较高；HBV感染的基因型为B型、C型和B＋C型，基因亚型为Ba和Cs。     

Aberrant activity of Wnt/Frizzled signaling pathway in renal cancer cell lines

The expression ofWnt, Wntreceptor-Frizzled, and several other key components in Wnt pathway in renal cancer cell lines was studied. The result of semi-quantitative RT-PCR has shown that the expression level ofWnt5A andhFz5 mRNA were higher in renal cancer cell line (GRC-1) than in normal renal cell line (HK-2). This result has been confirmed byin situ hybridization. The expression of β-catenin protein was obviously higher in GRC-1 than in HK-2 (P< 0.01), but there were no different expressions of its mRNA between 3 lines. The reasons of the overexpression of β-catenin has been investigated by means of immunocytochemistry, SSCP and so on, no mutation ofβ-catenin gene and APC were found. That means that the overexpression ofWnt5A/hFz5 might be the reason of overexpression of β-catenin. It was concluded that the aberrant activity of Wnt pathway might play an important role in renal cell carcinoma.     

Protection of cottontop tamarins against Epstein-Barr virus-induced malignant lymphoma by a prototype subunit vaccine

Epstein-Barr (EB) virus is one of the five herpesviruses of man. Strong links between this agent and the chain of events causing two human cancers, endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, have long been evident (reviewed in ref. 1). Because of this, and because of the very high incidence of nasopharyngeal carcinoma in certain large populations, it was suggested in 1976 that a vaccine should be developed against EB virus to prevent infection and thereby reduce tumour incidence amongst those at risk. The virus-determined membrane antigen (MA) was proposed as immunogen because it was known to elicit naturally occurring virus-neutralizing antibodies in man and because analogous antigens had been shown to act as effective experimental vaccines for preventing the herpesvirus-induced lymphomas of Marek's disease in chickens. Progress has been achieved in defining, quantifying and preparing MA molecules, and in enhancing their immunogenicity; a sensitive assay for antibodies to MA has been elaborated. Here we report that isolated cell membranes expressing MA, or purified MA glycoprotein of relative molecular mass (Mr) 340,000 (gp340), have been used to vaccinate cottontop tamarins (Saguinus oedipus oedipus), and that animals receiving either preparation were protected against the effects of a 100% tumour-inducing challenge dose of EB virus.     

静脉药瘾者血源传播病毒感染情况的调查分析

目的:调查静脉药瘾者主要血源传播病毒HBV、HCV、HGV、CMV和HSV的感染率。方法:采用酶联免疫吸附法、放射免疫法R、T-PCR和荧光定量PCR法,检测了406例静脉药瘾者血清,其中共用注射器者49.01%,同时设立102例正常健康体检者作为对照。结果:静脉药瘾者HBV、HCV、HGV、CMV和HSV的感染率分别是36.45%、69.7%、1.97%、2.71%和3.45%,其中总感染人数321人(79.06%)和重叠感染人数128人(31.53%),尤其有共用注射器者感染率及重叠感染率均高于未共用者,两者差异具有显著性意义(P<0.05)。对照组检测仅发现存在HBV感染(17.6%),其余病毒感染指标均为阴性。结论:静脉药瘾者血源传播病毒感染率明显增高,其中以HCV感染最为突出,并且共用注射器是导致感染扩大的危险要素。     

Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma

Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation.     

发育调控基因Pax5 mRNA在膀胱癌中的表达及意义

了解发育调控基因Pax5在膀胱肿瘤中的表达,探讨其在膀胱肿瘤发生发展中的作用,采用逆转录PCR（RT－PCR）和Southern杂交方法检测13例膀胱移行细胞癌（TCC）组织、4例癌旁组织、3例正常组织和3种癌细胞系（膀胱移行细胞癌、结肠腺癌、肺癌）中Pax5mRNA的表达。结果显示,3种癌细胞系中均有Pax5mRNA表达;13例膀胱肿瘤组织和4例癌旁组织中Pax5mRNA阳性表达分别为10例和3例,阳性率为77％和75％。3例正常组织中仅有1例检出Pax5mRNA表达。Pax5基因在膀胱肿瘤组织中的表达提示Pax5调控通路可能在膀胱肿瘤发生发展中具有重要作用。     

佛甲草中细胞毒活性成分的研究

采用中压柱色谱和制备高效液相色谱法对中国传统民族药佛甲草(Sedum lineare Thunb)的甲醇提取物化学成分进行分离,从中分离得到6个化合物.首次对化合物山奈酚7-O-6″-O-丙二酸单酰-D-吡喃葡萄糖苷(1)的波谱数据进行了归属,用MTT法测试6个单体成分对人肝癌细胞株HepG2和Hep3B的抑制作用.结果显示6个化合物均对HepG2和Hep3B细胞表现出抗肿瘤活性,并且IC50值在22.6～91.5 μg/mL之间,在浓度大于100 μg/mL时也没有表现出对人正常肝细胞L－02的抑制作用.相比于黄酮类化合物1、2,苯丙酸类化合物3～6(IC50＝22.6～44.3 μg/mL)对HepG2和Hep3B细胞表现出更强的抑制作用.其中,顺式苯丙酸类化合物5对HepG2和Hep3B细胞的抑制作用最明显.其IC50值范围在22.6～28.2 μg/mL,能明显抑制人肝癌细胞的增殖,诱导其凋亡.