人精子携带的HBs和HBc基因在早期胚胎细胞中的蛋白表达

发布时间：2018-01-23 04:11

本文关键词： 人精子胚胎细胞垂直传播 HBsAg和HBcAg　出处：《汕头大学》2008年硕士论文　论文类型：学位论文

【摘要】： 【背景与目的】乙型肝炎是危害人类健康的全球性疾病。由于该病流行面广,危害性大,因此其传播途径的研究一直是各国政府和科学家们高度重视的课题。1985年Hadchouel等提出“乙肝病毒有可能通过生殖细胞垂直传播”的假设。但由于没有合适的动物模型和细胞培养系统,10多年过去了没有人能证实这个假设。理论上,用乙肝患者的精子与正常人卵母细胞受精,研究从精子、受精到胚胎发育各阶段中HBV基因的行为是最理想的途径,但这条途径既存在法律、伦理问题,也存在获取人卵母细胞的困难。2002年,黄建民等引入了在生殖医学领域中广泛用于检测人精子受精能力的异种体外受精实验系统,应用荧光原位杂交技术(FISH)首次提供了HBV DNA在人精子染色体上整合的直接证据,证实了携带HBV基因的人精子在受精时不会受到选择性淘汰,能够与卵母细胞正常地完成受精过程。继后,Ali等应用同样的异种体外受精实验系统,获得2-细胞胚胎,用FISH技术证实了整合在人精子基因组中的HBx基因进入受精卵后,能随胚胎细胞分裂而复制;用RT-PCR技术证实了由人精子携带的HBx、HBs和HBc基因能够在胚胎细胞中转录。但是人精子携带的乙肝病毒基因在胚胎细胞中能否在蛋白水平表达,迄今为止未见文献报道。本研究以此为目的进行了研究。【材料与方法】1)材料①pIRES2-EGFP-HBV重组质粒;②精子,取自健康男性自愿者;③卵母细胞,取自成熟雌性金黄地鼠。2)方法①用pIRES2-EGFP-HBV转染的人精子和地鼠去透明带卵母细胞离体受精并继续培养至2-细胞胚胎;②在荧光显微镜下收集带有绿色荧光的2-细胞胚胎,此类胚胎表示含有由精子带入的HBV基因。③制备精子核、受精卵雄原核和带绿色荧光的2-细胞胚间期核制片,用FISH检测在上述样本中是否有HBs与HBc DNA阳性杂交信号;④收集带绿色荧光的2-细胞期胚胎,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及大致细胞定位。⑤收集带绿色荧光的2-细胞胚,用ELISA方法分别对乙肝表面抗原(hepatitis B surface antigen, HBsAg)和乙肝核心抗原(hepatitis B core antigen, HBcAg)进行半定量和定性分析。【结果】①FISH:在精子核和2-细胞胚两个间期核内均观察到HBs与HBc DNA阳性杂交信号;②免疫荧光检测:在2-细胞胚细胞浆内观察到HBsAg和HBcAg阳性表达。HBsAg位于胞浆内靠近胞膜,HBcAg位于胞浆内。③ELISA: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】①HBs和HBc DNA能整合到人精子基因组内;②通过受精,整合在精子基因组中的HBs和HBc基因被带入受精卵内,并在胚胎细胞中复制;③由人精子带入受精卵内的HBs和HBc基因可在早期胚胎细胞中表达HBsAg和HBcAg。④本研究为HBV经人精子在父婴间垂直传播新途径的研究提供了病毒蛋白表达方面的实验证据。
[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.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R321

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