精子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：2018-06-09 11:47

本文选题：精子 + 早期胚胎　；参考：《汕头大学》2005年硕士论文

【摘要】：【背景与目的】乙型肝炎是危害人类健康的全球性疾病。我国是该病高发区,乙肝病毒携带者约占全国人口的10%。由于该病危害性大,流行面广,因此其传播途径、发病机理及诊疗手段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 等发现乙肝患者的精子基因组中存在HBV DNA序列整合,提出“乙肝病毒有可能通过生殖细胞垂直传播”的假设。但10多年来,相关研究由于没有适用的实验动物与细胞培养模型而止步不前。2002年黄建民等应用人精子/金黄地鼠去透明带卵异种体外受精和荧光原位杂交技术,证实染色体上整合了HBV DNA序列的人精子能够与卵母细胞正常地完成受精过程。Ali等也在离体实验中发现由精子携带的HBV基因能够在早期胚胎中复制和转录。本研究首次从活体动物的整体效应探讨携带了HBV DNA的精子能否顺利完成受精过程,以及通过受精由精子携带到胚胎的HBV基因能否进行复制、转录和蛋白水平的表达,以期为HBV真正意义上的父婴垂直传播途径提供直接证据。【材料与方法】 1)材料①pBR322-HBV DNA质粒;②精子,取自性成熟金黄地鼠与昆明小鼠附睾;③早期胚胎,取自雌性金黄地鼠和昆明小鼠输卵管。 2)方法①双侧睾丸内注射HBV DNA质粒;②用PCR、Southern杂交、斑点杂交和荧光原位杂交技术(FISH) 证实HBV DNA是否进入精子细胞并与精子细胞基因组整合;③收集受精卵、2-细胞胚胎, 制备间期核片,用FISH检测HBV DNA在间期核上的整合与复制;④收集2-细胞胚胎, 用RT-PCR检测HBx与HBs基因在胚胎细胞中的转录;⑤收集2-细胞期胚胎,用免疫荧光技术检测HBsAg蛋白在胚胎细胞中的表达。【结果】①PCR、Southern在精子样本中可见HBx基因阳性带,洗涤液样本斑点杂交结果阴性,排除了PCR和Southern阳性结果来自洗液污染的可能性;②FISH在精子核、受精卵中雄原核、2-细胞胚中两个间期核观察到HBV DNA阳性杂交信号;③RT-PCR 2- 细胞胚样本可见HBx和HBs基因特异带;④免疫荧光检测在2-细胞胚细胞浆内观察到 HBsAg阳性表达。【结论】①HBV基因能够通过精子膜进入精子细胞内并整合到精子基因组内;②携带有 HBV基因的精子在自然受精时能够将病毒基因带入胚胎中;③由精子带入的HBV基因能够在早期胚胎中复制、转录和表达。④本研究为HBV可经过雄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一个理想的实验模型,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 objective hepatitis B is a global disease that endangers human health. China is a high incidence area of hepatitis B virus.
Drug carriers account for about 10%. of the national population. Because of the high risk and wide spread of the disease, the carriers are infected by the virus.
The research on mechanism and diagnosis and treatment has been a topic highly regarded by governments and scientists all over the world,.1985 Hadchouel
It was found that HBV DNA sequence integration existed in the sperm genome of hepatitis B patients.
Hypothesis of vertical transmission of germ cells ", but for more than 10 years, due to lack of suitable experimental animals and Xi Baopei
In the.2002 years, Huang Jianmin and other people applied the human sperm / hamster to the zona pellucida eggs in vitro fertilization.
Fluorescence in situ hybridization confirmed that human spermatozoa integrated with HBV DNA sequences on chromosomes were normal to oocytes.
.Ali and so on also found in vitro experiments that the HBV gene carried by spermatozoa could be found in early embryos.
Replication and transcription. This study is the first to explore whether the sperm carrying HBV DNA can be successfully completed from the holistic effect of living animals.
The process of fertilization, and the HBV gene carried by spermatozoa to embryos can be replicated, transcribed and protein water.
The expression of Ping is intended to provide direct evidence for the true father child vertical transmission of HBV.
[materials and methods] 1) materials: pBR322-HBV DNA plasmid;
The epididymis of Ming mice; the third embryo was taken from female golden hamster and Kunming mouse oviduct. 2) method: bilateral testes.
Intramuscular injection of HBV DNA plasmid; PCR, Southern hybridization, dot blot hybridization and fluorescence in situ hybridization (FISH).
To confirm whether HBV DNA enters the sperm cell and integrate with the sperm cell genome; (3) to collect fertilized eggs and 2- cell embryos.
Interphase nuclei were prepared, and the integration and replication of HBV DNA on the interphase nucleus were detected by FISH. 4. 2- cell embryos were collected.
RT-PCR was used to detect transcription of HBx and HBs genes in embryonic cells. 5. 2- cell stage embryos were collected and immunofluorescence was used.
The expression of HBsAg protein in embryonic cells was detected by light technology.
[results] (1) HBx gene positive bands were found in sperm samples of PCR and Southern, dot blot hybridization of scour samples.
The negative results excluded the possibility of PCR and Southern positive results from the contamination of lotion, and FISH in the sperm nucleus.
HBV DNA positive hybridization signals were observed in the two nuclei of the zygote prokaryotic cells and 2- cell embryos; and RT-PCR 2-
The specific bands of HBx and HBs genes were observed in cell embryo samples. 4. Immunofluorescence detection was observed in 2- cell cytoplasm.
HBsAg positive expression.
[Conclusion] HBV gene can enter spermatozoa through sperm membrane and integrate into the sperm genome.
The sperm of HBV gene can bring the virus gene into the embryo during natural fertilization, and the HBV gene can be brought into the sperm by sperm.
It can replicate, transcribe and express in early embryos. 4. HBV can be transferred vertically through male germ cells in this study.
Direct scientific evidence is provided. This study provides an ideal alternative for similar studies in other viral infectious diseases.
The experimental model may further develop a new field for 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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