精子携带的乙肝病毒基因在金黄地鼠胚胎中的复制与表达

背景与目的:乙型肝炎是危害人类健康的全球性疾病。本研究旨在探索乙肝病毒(HepatitisBvirus,HBV)通过精子垂直传播的可能性。方法:将金黄地鼠精子与pBR322-HBVDNA重组质粒进行共培养后,与金黄地鼠正常卵母细胞受精,取2-细胞胚分别制片和提取RNA,用荧光原位杂交(Fluorescenceinsituhybridization,FISH)、RT-PCR和免疫荧光检测技术研究精子携带的HBV基因在胚胎细胞中的复制与表达。结果:①FISH:用全长HBVDNA探针与128个2-细胞胚进行FISH,其中3个胚胎FISH结果阳性,每个胚胎的2个间期核上均有HBVDNA杂交信号;②RT-PCR:在检测样本中可见特异的HBxDNA阳性条带,阴性对照1(模板为灭菌水)和阴性对照2(未加反转录酶)均为阴性;③免疫荧光检测:2-细胞胚经血清封闭后,分成两组。一组作检测样本,另一组用PBS代替一抗(小鼠抗人HBsAg)作为阴性对照,检测样本中可见阳性免疫荧光信号,阴性对照未见信号。结论:以精子为载体,携带到卵内的HBV基因能够在胚胎细胞中复制和表达,提示HBV能够通过男性生殖细胞由父亲垂直传递给子代。     

HIV-1 gag基因经卵母细胞垂直传递模型的建立

目的：建立HIV-1gag基因经卵母细胞垂直传递的模型,为HIV通过雌性生殖细胞垂直传递的研究奠定实验基础。方法：将含有HIV-1gag基因的重组质粒pIRES2-EGFP-gag注射到小鼠双侧卵巢,转染生殖细胞。应用超排获得实验用卵母细胞,收集显示绿色荧光的卵母细胞,将一部分呈现绿色荧光的卵母细胞裂解,提取基因组DNA,采用PCR检测gagDNA是否存在卵母细胞中;再将另一部分带有绿色荧光的卵母细胞常规制备中期染色体片,用荧光原位杂交（FISH）技术检测HIV-1gag基因在成熟卵母细胞染色体上的整合。结果：转染pIRES2-EGFP-gag后,在荧光显微镜下能够很清楚地看到带有绿色荧光的卵母细胞;PCR结果显示在带有绿色荧光的卵母细胞中可检测到HIV-1gag基因特异的阳性条带;FISH检测结果显示在卵母细胞的中期染色体上检测到HIV-1gagDNA的阳性信号。结论：HIV-1gag基因能够通过卵透明带和细胞膜并整合到卵母细胞基因组内,本研究为HIV-1gag基因可经过雌性生殖细胞垂直传递给子代奠定了实验基础。     

HBV DNA重组质粒转染小鼠卵母细胞的研究

背景与目的:乙型肝炎是危害人类健康的全球性疾病.为了探索乙肝病毒通过卵母细胞垂直传递的可能性,对HBV DNA重组质粒能否转染小鼠卵母细胞进行了研究.材料与方法:将小鼠卵母细胞与pBR322-HBV DNA重组质粒进行共培养后,分别提取卵母细胞DNA及制备小鼠卵母细胞中期染色体.用PCR、Southern、斑点杂交及FISH技术证实HBV DNA能否转染小鼠卵母细胞.结果:PCR试验在受检样本中观察到HBV DNA阳性条带.Southern试验在受检PCR产物中观察到明显的阳性杂交信号.用每次实验的最后3次洗液进行斑点杂交未发现HBV DNA阳性信号,排除了PCR和Southern阳性结果来自洗液污染的可能性.荧光原位杂交在1 000个卵母细胞中的36个中期相内发现HBV DNA杂交信号.结论:HBV DNA序列能够通过卵母细胞的透明带和细胞膜,进入卵母细胞内并整合到卵母细胞染色体上.此类卵母细胞与正常精子受精时,有可能作为载体将HBVDNA带进胚胎.     

人精子携带的HBs和HBc基因在早期胚胎细胞中的蛋白表达

背景与目的:探讨由人精子带入受精卵中的HBs和HBc基因能否在早期胚胎细胞中进行蛋白表达.材料与方法:人精子经重组质粒pIRES2-EGFP-HBV转染后,与金黄地鼠去透明带卵母细胞离体受精,选择带绿色荧光的2-细胞胚,用免疫荧光技术检测HBs和HBc基因在胚胎细胞中的蛋白表达,用ELISA方法分别对HBsAg和HBcAg进行半定量和定性分析.结果:在带绿色荧光的2-细胞胚中免疫荧光检测可见清楚的HBsAg和HBcAg阳性信号;ELISA结果表明,单个2-细胞胚内HBsAg的量小于0.064 ng/ml,对HBcAg的检测得到阳性结果.结论:人精子携带到卵内的HBV基因可在早期胚胎细胞中表达表面抗原和核心抗原.     

HBV基因经生殖细胞垂直传播与DNA甲基化研究进展

乙肝病毒(HBV)基因可能通过生殖细胞垂直传播并在早期胚胎中复制与表达.DNA甲基化模式的改变是胚胎发育的关键,HBV感染可导致宿主细胞表观基因组修饰异常,诱发染色体不稳定,影响胚胎的正常发育并增加肿瘤发生的风险.本文中对近年HBV基因的垂直传播及其与DNA甲基化的关系的研究进行综述.     

pIRES2-EGFP-DAZ3质粒构建及金黄地鼠2-细胞胚中人DAZ基因的复制与表达

背景与目的：人Yq11.2上AZF区 (内含DAZ基因家族) 的缺失是多数男性不育的病因。本研究旨在探讨DAZ3基因导入精子的可能性以及导入基因在2-细胞胚中的功能。材料与方法：构建pIRES2-EGFP-DAZ3质粒,经鉴定无误后将其导入金黄地鼠精子。将携带DAZ3基因的精子与金黄地鼠卵母细胞进行体外受精。用FISH、PCR、RT-PCR技术分别检测DAZ3基因在精子头部、雄原核上的整合以及在2-细胞胚中的复制与表达。结果：构建的pIRES2-EGFP-DAZ3质粒经酶切、PCR和绿色荧光蛋白表达鉴定构建成功。采用FISH在精子头部、单细胞胚雄原核和2-细胞胚两个间期核上观察到阳性杂交信号。采用RT-PCR在2-细胞胚样本中观察到DAZ3 cDNA的特异条带。结论：外源性DAZ3基因能被导入精子基因组。携带DAZ3基因的精子可与卵母细胞完成正常的受精过程。导入的DAZ3基因在早期胚胎细胞中能够随细胞分裂自我复制并表达其功能。本研究结果为DAZ基因缺失所致男性不育的治疗提供了新的线索。     

转基因小鼠及其在体内突变研究中的应用

早在十五年前,就有人想到将外源性DNA引入哺乳动物体细胞和生殖细胞中,例如将胚胎从体内移出后在体外培养,再植入假孕的母体内使其完成正常的胚胎发育,这一技术使一个动物的胚胎细胞与其它动物的结合以形成嵌合动物成为可能。他们还发明了将生殖细胞或早期胚胎来源的畸胎瘤培养细胞引入到发育胚胎中的方法。与此同时,遗传学家和病毒学家正在探索将选择的基因引入组织培养细胞的方法。于是便可将基因转入畸胎瘤细胞,并将这些细胞导入发育胚胎的囊胚以产生嵌合动物。到了七十年代中期,有人用小鼠白血病病毒(MuLV)     

TCRγ独特型DNA疫苗诱导特异性免疫反应的实验研究

目的：观察TCRγ独特型DNA疫苗诱导BALB／c小鼠的抗人类Jurkat淋巴肿瘤免疫反应的情况。方法：碱裂解法大量提取质粒，制备DNA疫苗。选用16只BALB／c小鼠随机分为pcDNA3组、pcDNA3／TCRγV1组，双侧股四头肌内注射疫苗免疫，于第0、2、4周各免疫1次，共3次。每次免疫前及免疫开始后至第8周取鼠血，用间接免疫荧光法检测小鼠血清中抗体生成情况；RT-PCR法检测重组质粒在小鼠肌肉中的mRNA表达。结果：pcDNA3／TCRγV1组小鼠血清中全部产生了特异性抗独特型抗体，抗体滴度在第4周开始增高，第6周时达到高峰(1：160)。在pcDNA3／TCRγV1组小鼠中用RT—PCR法检测到了重组质粒在肌肉中的mRNA表达。结论：TCRγV1独特型DNA疫苗可以诱导小鼠产生特异性抗淋巴瘤细胞独特型抗体。     

小鼠后肾集合管水通道蛋白表达及其上皮细胞超微结构研究

目的观察小鼠后肾集合管水通道蛋白(AQP)-4的表达及其上皮细胞超微结构变化。方法应用透射电镜、免疫组织化学及图像分析技术观察并检测小鼠后肾不同发育阶段集合管的超微结构及AQP-4表达。结果小鼠胚龄18 d见发育早期集合管主细胞,生后7 d～21 d,其形态结构发育基本完善。A型闰细胞于胚胎18 d出现,B型闰细胞生后21天出现。AQP-4于胚龄14 d始见表达,分布于集合管管壁上皮细胞侧基底细胞膜,随胚龄增加表达逐渐增强,生后1 d达高峰。结论集合管管壁上皮细胞于胚胎时出现,但其形态结构在生后才逐渐完善。AQP-4对小鼠胚胎时期肾水平衡调节可能起重要的作用。     

A transactivating function encoded in the hepatitis B virus X gene is conserved in the integrated state

Recently we have shown that the hepatitis B virus (HBV) X gene encodes a transactivating factor. Here we report on the construction of a series of HBx expression plasmids which were tested for a transactivating function by cotransfections with a plasmid expressing the CAT gene under control of the SV40 early promoter. One of the plasmids, expressing a HBx specific protein, is shown to transiently transactivate CAT gene expression after cotransfection into the human cell line CC113. Furthermore, a cloned integrated HBV DNA sequence is also shown to transactivate several viral promoters and LTRs. By sequence analyses we can demonstrate that only the HBx ORF is intact and that it is fused to an ORF of at least 228 bp in the flanking cellular DNA. The HBx gene is cotranscribed with the flanking cellular DNA, resulting in a RNA of approximately 10 kb. By subcloning of the integrate we can demonstrate that the presence of the HBx gene and its expression by the HBV enhancer and/or the HBx promoter is required for the transactivating function of the integrated HBV DNA.     

Hepatitis B x antigen (HBx) is an important therapeutic target in the pathogenesis of hepatocellular carcinoma

Hepatitis B virus (HBV) is a human pathogen that has infected an estimated two billion people worldwide. Despite the availability of highly efficacious vaccines, universal screening of the blood supply for virus, and potent direct acting anti-viral drugs, there are more than 250 million carriers of HBV who are at risk for the sequential development of hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma (HCC). More than 800,000 deaths per year are attributed to chronic hepatitis B. Many different therapeutic approaches have been developed to block virus replication, and although effective, none are curative. These treatments have little or no impact upon the portions of integrated HBV DNA, which often encode the virus regulatory protein, HBx. Although given little attention, HBx is an important therapeutic target because it contributes importantly to (a) HBV replication, (b) in protecting infected cells from immune mediated destruction during chronic infection, and (c) in the development of HCC. Thus, the development of therapies targeting HBx, combined with other established therapies, will provide a functional cure that will target virus replication and further reduce or eliminate both the morbidity and mortality associated with chronic liver disease and HCC. Simultaneous targeting of all these characteristics underscores the importance of developing therapies against HBx.     

乙型肝炎病毒感染在肝细胞癌发生机制中的研究进展

肝细胞癌（HCC）是世界范围内常见的恶性肿瘤之一,乙型肝炎病毒（HBV）感染是引起HCC的最主要原因。HBV 通过DNA整合影响插入点附近宿主基因的功能,促使宿主基因组的不稳定,产生具有致癌作用的融合蛋白;HBV的x基因产物（HBx蛋白）具有广泛的反式激活功能,如调控细胞的凋亡,抑制细胞DNA的修复,干扰细胞内信号通路及改变表观遗传学等。本文结合近年相关报道对HBV相关HCC流行和发生、HBV DNA整合、HBx蛋白等相关研究作一综述。     

Downregulation of DNA excision repair by the hepatitis B virus-x protein occurs in p53-proficient and p53-deficient cells

Synergism between exposure to chemical carcinogens and infection with the hepatitis B virus (HBV) has been implicated in the high incidence of hepatocellular carcinoma. In this study we report that the HBV protein HBx, inhibits cellular DNA repair capacity in a p53-independent manner. Two alternative assays were used: the host cell reactivation assay, which measures the cell's capacity to repair DNA damage in a reporter plasmid, and unscheduled DNA synthesis, which measures the overall DNA repair capacity in damaged cells. Two p53-proficient cell lines, the hepatocellular carcinoma cell line HepG2 and liver epithelial cell line CCL13, were co-transfected with the pCMV-HBx reporter plasmid and the pCMV-CAT plasmid damaged with UVC radiation. Compared with cells transfected with control plasmid, the presence of HBx resulted in approximately 50% inhibition of the cell's capacity to reactivate CAT activity of UVC-damaged plasmid, and approximately 25% inhibition of unscheduled DNA synthesis in cells treated with either aflatoxin B1 epoxide or UVC radiation. Using the p53-deficient cell line Saos-2, we demonstrated that expression of HBx also resulted in diminished overall cellular DNA repair of damage induced by both aflatoxin B1 epoxide and UVC radiation, using both the host cell reactivation and unscheduled DNA synthesis assays. In summary, this study provides evidence for p53-independent regulation of DNA repair by HBx.