本文關(guān)鍵詞： 卵母細胞 早期胚胎 HBV 整合 復制和表達 垂直傳播　出處：《汕頭大學》2005年碩士論文　論文類型：學位論文

【摘要】：【研究背景與意義】乙型肝炎病毒(hepatitis B virus,HBV)對人類健康危害嚴重,是 引起急、慢性肝炎的病因,與肝硬化和肝癌的發(fā)生密切相關(guān),因此研究HBV傳播途徑 無論是對人類健康還是對病毒學本身的發(fā)展都具有十分重要的意義。掘調(diào)查,我國嬰 幼兒HBV感染約1/3系由乙肝表面抗原陽性的母親將HBV傳給嬰兒引起感染所至,因此 母嬰垂直傳播途徑引起人們特別關(guān)注。目前已經(jīng)清楚的母嬰垂直傳播途徑有;①妊娠 時經(jīng)胎盤傳播;②分娩期外傷或吸入羊水;③分娩后母乳喂養(yǎng)等。而真正意義的母 嬰垂直傳播(true vertical transmission),即“由HBV感染的卵母細胞,作為病毒 基因的載體,與正常精子受精,在胚胎發(fā)育過程中,HBV基因隨人基因組復制與表達, 從而使子代患病”,目前仍是一種尚待證實的假說。本研究旨在證實這一假說。 【材料與方法】研究用金黃地鼠和小鼠建立實驗動物模型:將卵巢內(nèi)注射HBV DNA 的實驗動物分成兩組,一組注射后進行超排卵,收集卵巢和輸卵管的卵母細胞,用PCR、 Southern雜交,斑點雜交和熒光原位雜交(fluorescence in situ hybridization、FISH)檢測 HBV在卵母細胞內(nèi)的存在和染色體上的整合;另一組超排雌鼠與正常雄鼠合籠,收集 受精卵(金黃地鼠)和2-細胞胚(小鼠),用FISH、RT-PCR和免疫熒光檢測技術(shù)分別 研究HBV基因在受精卵雌原核上的整合以及在2-細胞胚中的復制與表達。 【結(jié)果】①PCR實驗在金黃地鼠和小鼠的卵母細胞受檢樣本中均發(fā)現(xiàn)HBV DNA陽性 條帶;Southern試驗在待檢PCR產(chǎn)物中觀察到明顯的雜交信號。用最后幾次洗液進行斑 點雜交未發(fā)現(xiàn)HBV DNA陽性信號,排除了PCR和Southern陽性結(jié)果來自洗液污染的可 能性。②FISH在一些卵母細胞的染色體、受精卵的雌原核和一些2-細胞胚的兩個間期 核上檢測到HBV DNA陽性雜交信號;③RT-PCR在受檢樣本中發(fā)現(xiàn)HBx和HBs DNA陽 性條帶;④免疫熒光檢測在受檢2-細胞胚的胞漿中發(fā)現(xiàn)HBsAg陽性信號。 【小結(jié)】①HBV DNA能夠進入卵母細胞內(nèi)并整合在染色體上;②正常受精時,卵母細 胞能夠作為載體將HBV基因帶入受精卵內(nèi);③卵母細胞攜帶的HBV基因能夠在小鼠 早期胚胎中復制和表達;④本研究為HBV可經(jīng)過雌性生殖細胞垂直傳遞提供了直接的 科學證據(jù)。⑤本研究為在其他病毒性傳染病中進行類似研究提供了理想的實驗?zāi)Ｐ? 進而有可能為臨床醫(yī)學基礎(chǔ)研究展開一個新的領(lǐng)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.
【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R363

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