卵母细胞介导的乙肝病毒基因在早期鼠胚中的复制与表达

发布时间：2018-01-21 10:58

本文关键词： 卵母细胞早期胚胎 HBV 整合复制和表达垂直传播　出处：《汕头大学》2005年硕士论文　论文类型：学位论文

【摘要】：【研究背景与意义】乙型肝炎病毒(hepatitis B virus,HBV)对人类健康危害严重,是引起急、慢性肝炎的病因,与肝硬化和肝癌的发生密切相关,因此研究HBV传播途径无论是对人类健康还是对病毒学本身的发展都具有十分重要的意义。掘调查,我国婴幼儿HBV感染约1/3系由乙肝表面抗原阳性的母亲将HBV传给婴儿引起感染所至,因此母婴垂直传播途径引起人们特别关注。目前已经清楚的母婴垂直传播途径有;①妊娠时经胎盘传播;②分娩期外伤或吸入羊水;③分娩后母乳喂养等。而真正意义的母婴垂直传播(true vertical transmission),即“由HBV感染的卵母细胞,作为病毒基因的载体,与正常精子受精,在胚胎发育过程中,HBV基因随人基因组复制与表达, 从而使子代患病”,目前仍是一种尚待证实的假说。本研究旨在证实这一假说。【材料与方法】研究用金黄地鼠和小鼠建立实验动物模型:将卵巢内注射HBV DNA 的实验动物分成两组,一组注射后进行超排卵,收集卵巢和输卵管的卵母细胞,用PCR、 Southern杂交,斑点杂交和荧光原位杂交(fluorescence in situ hybridization、FISH)检测 HBV在卵母细胞内的存在和染色体上的整合;另一组超排雌鼠与正常雄鼠合笼,收集受精卵(金黄地鼠)和2-细胞胚(小鼠),用FISH、RT-PCR和免疫荧光检测技术分别研究HBV基因在受精卵雌原核上的整合以及在2-细胞胚中的复制与表达。【结果】①PCR实验在金黄地鼠和小鼠的卵母细胞受检样本中均发现HBV DNA阳性条带;Southern试验在待检PCR产物中观察到明显的杂交信号。用最后几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。②FISH在一些卵母细胞的染色体、受精卵的雌原核和一些2-细胞胚的两个间期核上检测到HBV DNA阳性杂交信号;③RT-PCR在受检样本中发现HBx和HBs DNA阳性条带;④免疫荧光检测在受检2-细胞胚的胞浆中发现HBsAg阳性信号。【小结】①HBV DNA能够进入卵母细胞内并整合在染色体上;②正常受精时,卵母细胞能够作为载体将HBV基因带入受精卵内;③卵母细胞携带的HBV基因能够在小鼠早期胚胎中复制和表达;④本研究为HBV可经过雌性生殖细胞垂直传递提供了直接的科学证据。⑤本研究为在其他病毒性传染病中进行类似研究提供了理想的实验模型, 进而有可能为临床医学基础研究展开一个新的领域。
[Abstract]:[background and significance] Hepatitis B virus (HBV) is a serious hazard to human health. The etiology of acute and chronic hepatitis is closely related to the occurrence of liver cirrhosis and liver cancer. Therefore, the transmission of HBV is studied. It is of great significance both to human health and to the development of virology itself. About 1/3 of the HBV infection in young children is caused by the transmission of HBV to their infants by mothers who are HBsAg positive. People pay special attention to the way of vertical transmission from mother to child. It is clear that there are 1 pregnancy in the way of vertical transmission from mother to child When transmitted through the placenta during labor injury or inhaled amniotic fluid after breast-feeding etc. And the true meaning of the mother. Infantile vertically-transmitted true vertical transmission, or "oocyte infected by HBV, as a virus" Gene vector, fertilized with normal spermatozoa, replication and expression of HBV gene with human genome during embryonic development. It is still an unconfirmed hypothesis. This study aims to confirm this hypothesis. [materials and methods] to establish experimental animal model with golden hamster and mouse: intraovarian injection of HBV DNA Two groups of experimental animals were divided into two groups, one group was injected with superovulation, and the oocytes of ovary and fallopian tube were collected, and PCR was used. Detection of fluorescence in situ hybridization by Southern hybridization, dot hybridization and fluorescence in situ hybridization The presence of HBV in oocytes and the integration of chromosomes. Fertilized eggs (golden hamsters) and 2-cell embryos (mouse) were detected by fish RT-PCR and immunofluorescence, respectively. To study the integration of HBV gene in the female pronucleus of fertilized eggs and its replication and expression in 2-cell embryos. [results] 1 HBV DNA was positive in oocytes of golden hamster and mouse by polymerase chain reaction (PCR). Southern blot analysis showed obvious hybridization signals in the PCR products to be tested. No positive signals of HBV DNA were found by dot hybridization, and the positive results of PCR and Southern were excluded from the contamination of washing fluid. Activity. 2FISH in the chromosomes of some oocytes, in the female prokaryotes of fertilized eggs and in the two phases of some 2-cell embryos Detection of HBx and HBs DNA positive signals by RT-PCR with HBV DNA positive in Nucleus HBsAg positive signals were detected in the cytoplasm of 2-cell embryos by immunofluorescence assay. [nodules] 1HBV DNA can enter the oocytes and integrate into the chromosomes in normal fertilization, the oocytes are fine. The HBV gene carried by oocytes can be used as a vector to carry HBV gene into fertilized eggs in mice. Replication and expression of HBV in early embryos this study provides a direct way for vertical transmission of HBV through female germ cells. Scientific evidence .5 this study provides an ideal experimental model for similar studies in other viral infectious diseases, It is possible to develop a new field for the basic research of clinical medicine.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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