本文關(guān)鍵詞： 人精子 胚胎細(xì)胞 垂直傳播 HBsAg和HBcAg　出處：《汕頭大學(xué)》2008年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 【背景與目的】乙型肝炎是危害人類(lèi)健康的全球性疾病。由于該病流行面廣,危害性大,因此其傳播途徑的研究一直是各國(guó)政府和科學(xué)家們高度重視的課題。1985年Hadchouel等提出“乙肝病毒有可能通過(guò)生殖細(xì)胞垂直傳播”的假設(shè)。但由于沒(méi)有合適的動(dòng)物模型和細(xì)胞培養(yǎng)系統(tǒng),10多年過(guò)去了沒(méi)有人能證實(shí)這個(gè)假設(shè)。理論上,用乙肝患者的精子與正常人卵母細(xì)胞受精,研究從精子、受精到胚胎發(fā)育各階段中HBV基因的行為是最理想的途徑,但這條途徑既存在法律、倫理問(wèn)題,也存在獲取人卵母細(xì)胞的困難。2002年,黃建民等引入了在生殖醫(yī)學(xué)領(lǐng)域中廣泛用于檢測(cè)人精子受精能力的異種體外受精實(shí)驗(yàn)系統(tǒng),應(yīng)用熒光原位雜交技術(shù)(FISH)首次提供了HBV DNA在人精子染色體上整合的直接證據(jù),證實(shí)了攜帶HBV基因的人精子在受精時(shí)不會(huì)受到選擇性淘汰,能夠與卵母細(xì)胞正常地完成受精過(guò)程。繼后,Ali等應(yīng)用同樣的異種體外受精實(shí)驗(yàn)系統(tǒng),獲得2-細(xì)胞胚胎,用FISH技術(shù)證實(shí)了整合在人精子基因組中的HBx基因進(jìn)入受精卵后,能隨胚胎細(xì)胞分裂而復(fù)制;用RT-PCR技術(shù)證實(shí)了由人精子攜帶的HBx、HBs和HBc基因能夠在胚胎細(xì)胞中轉(zhuǎn)錄。但是人精子攜帶的乙肝病毒基因在胚胎細(xì)胞中能否在蛋白水平表達(dá),迄今為止未見(jiàn)文獻(xiàn)報(bào)道。本研究以此為目的進(jìn)行了研究。 【材料與方法】1)材料①pIRES2-EGFP-HBV重組質(zhì)粒;②精子,取自健康男性自愿者;③卵母細(xì)胞,取自成熟雌性金黃地鼠。2)方法①用pIRES2-EGFP-HBV轉(zhuǎn)染的人精子和地鼠去透明帶卵母細(xì)胞離體受精并繼續(xù)培養(yǎng)至2-細(xì)胞胚胎;②在熒光顯微鏡下收集帶有綠色熒光的2-細(xì)胞胚胎,此類(lèi)胚胎表示含有由精子帶入的HBV基因。③制備精子核、受精卵雄原核和帶綠色熒光的2-細(xì)胞胚間期核制片,用FISH檢測(cè)在上述樣本中是否有HBs與HBc DNA陽(yáng)性雜交信號(hào);④收集帶綠色熒光的2-細(xì)胞期胚胎,用免疫熒光技術(shù)檢測(cè)HBs和HBc基因在胚胎細(xì)胞中的蛋白表達(dá)及大致細(xì)胞定位。⑤收集帶綠色熒光的2-細(xì)胞胚,用ELISA方法分別對(duì)乙肝表面抗原(hepatitis B surface antigen, HBsAg)和乙肝核心抗原(hepatitis B core antigen, HBcAg)進(jìn)行半定量和定性分析。 【結(jié)果】①FISH:在精子核和2-細(xì)胞胚兩個(gè)間期核內(nèi)均觀察到HBs與HBc DNA陽(yáng)性雜交信號(hào);②免疫熒光檢測(cè):在2-細(xì)胞胚細(xì)胞漿內(nèi)觀察到HBsAg和HBcAg陽(yáng)性表達(dá)。HBsAg位于胞漿內(nèi)靠近胞膜,HBcAg位于胞漿內(nèi)。③ELISA:單個(gè)2-細(xì)胞胚內(nèi)HBsAg的量小于0.064 ng/ml,對(duì)HBcAg的檢測(cè)得到陽(yáng)性結(jié)果。 【結(jié)論】①HBs和HBc DNA能整合到人精子基因組內(nèi);②通過(guò)受精,整合在精子基因組中的HBs和HBc基因被帶入受精卵內(nèi),并在胚胎細(xì)胞中復(fù)制;③由人精子帶入受精卵內(nèi)的HBs和HBc基因可在早期胚胎細(xì)胞中表達(dá)HBsAg和HBcAg。④本研究為HBV經(jīng)人精子在父嬰間垂直傳播新途徑的研究提供了病毒蛋白表達(dá)方面的實(shí)驗(yàn)證據(jù)。
[Abstract]:[background & objective] Hepatitis B is a global disease harmful to human health. In 1985, Hadchouel et al proposed that "hepatitis B virus may be transmitted vertically through germ cells". But there is no suitable animal model and cell culture system. No one has been able to confirm this hypothesis for more than 10 years. In theory, the sperm of hepatitis B patients was fertilized with normal human oocytes and studied from sperm. The behavior of HBV gene in all stages of fertilization and embryo development is the most ideal pathway, but this approach has not only legal and ethical problems, but also the difficulty of obtaining human oocytes. 2002. Huang Jianmin et al introduced a heterologous in vitro fertilization (IVF) system which is widely used in the field of reproductive medicine to test the fertilization ability of human spermatozoa. Fluorescence in situ hybridization (fish) for the first time provides direct evidence for the integration of HBV DNA on human sperm chromosomes. It was confirmed that the human sperm carrying HBV gene could not be selectively eliminated during fertilization, and could complete the fertilization process normally with oocytes. Later, Ali et al used the same heterologous in vitro fertilization system. 2-cell embryos were obtained. FISH technique confirmed that the HBx gene integrated into the human sperm genome could replicate with the division of the embryo cells when it entered the fertilized eggs. HBx carried by human sperm was confirmed by RT-PCR technique. HBs and HBc genes can be transcribed in embryonic cells, but whether the hepatitis B virus genes carried by human spermatozoa can be expressed at the protein level in embryonic cells. So far, no literature has been reported. The purpose of this study is to study. [materials and methods] 1) material 1pIRES2-EGFP-HBV recombinant plasmid; (2) sperm from healthy male volunteers; 3 oocytes. Methods (1) pIRES2-EGFP-HBV transfected human sperm and hamster without pellucida oocytes were fertilized in vitro and cultured to 2-cell embryos. 22-cell embryos with green fluorescence were collected under fluorescence microscope, which indicated that the embryos contained HBV gene 3. 3 brought in by spermatozoa to prepare sperm nucleus. The male prokaryotes of fertilized eggs and the 2-cell interembryonic nuclear slices with green fluorescence were prepared. FISH was used to detect the positive hybridization signals between HBs and HBc DNA in the above samples. (4) the 2-cell embryos with green fluorescence were collected, the protein expression of HBs and HBc genes in the embryonic cells was detected by immunofluorescence technique, and the 2-cell embryos with green fluorescence were collected by roughly locating .5 cells. Hepatitis B surface antigen was detected by ELISA. HBsAg and hepatitis B core antigen-HBcAg were analyzed by semi-quantitative and qualitative analysis. [results] 1FISH: positive hybridization signals of HBs and HBc DNA were observed in sperm nucleus and 2-cell embryo interphase nucleus. 2Immunofluorescence assay: the positive expression of HBsAg and HBcAg was observed in the cytoplasm of 2-cell embryos. HBcAg was located in cytoplasm. 3 ELISAThe amount of HBsAg in single 2-cell embryos was less than 0.064 ng / ml, and the positive results of HBcAg were obtained. [conclusion] 1HBs and HBc DNA can be integrated into human sperm genome; (2) by fertilization, the HBs and HBc genes integrated into the spermatozoa genome were brought into the fertilized egg and replicated in the embryonic cells. (3) HBs and HBc genes carried by human spermatozoa into fertilized eggs can express HBsAg and HBcAg.4 in early embryonic cells. This study is a new way for the vertical transmission of HBV between father and baby via human spermatozoa. The experimental evidence of viral protein expression is provided.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 熊小芳,黃天華,謝慶東,王曉梅,陳桂蘭;精子攜帶的HBV DNA在小鼠早期胚胎中的復(fù)制與表達(dá)[J];癌變.畸變.突變;2005年03期

2 張秋菊;黃天華;謝慶東;譚小方;劉戈飛;陳德宇;周小玲;;人精子攜帶的HBs和HBc基因在早期胚胎細(xì)胞中的蛋白表達(dá)[J];癌變.畸變.突變;2008年03期

3 許廷貴 ,劉鳳華;慢性HBV感染者HBsAg基因區(qū)變異的臨床意義[J];國(guó)外醫(yī)學(xué).病毒學(xué)分冊(cè);2002年05期

4 王厚照;田豐豐;馬莉;周友泉;;HBcAg ELISA法測(cè)定的臨床分析[J];臨床軍醫(yī)雜志;2006年03期

5 何啟強(qiáng),楊進(jìn)波,肖成峰,鄔堂春;雙抗夾心ELISA法檢測(cè)血漿HSP70的建立及初步應(yīng)用[J];環(huán)境與職業(yè)醫(yī)學(xué);2004年01期

6 韓俊峰,吳玉章;HBcAg病毒樣顆粒與疫苗設(shè)計(jì)[J];免疫學(xué)雜志;2005年S1期

7 何進(jìn)球,袁松華,冼革堅(jiān),陳亦明,梁業(yè)麗;乙型肝炎病毒陽(yáng)性父-嬰垂直傳播的臨床研究[J];實(shí)用婦產(chǎn)科雜志;2005年05期

8 李文玲,彭萍,張艷玲;乙型肝炎病毒父嬰垂直傳播的臨床研究[J];實(shí)用醫(yī)學(xué)雜志;2005年03期

9 陳國(guó)武;不育癥病人精子功能試驗(yàn)的應(yīng)用與評(píng)估[J];生殖與避孕;1995年05期

10 徐志強(qiáng),成軍,張鴻飛;HBcAg生物學(xué)特性研究進(jìn)展[J];世界華人消化雜志;2004年12期

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