本文選題：精子 + 早期胚胎　； 參考：《汕頭大學(xué)》2005年碩士論文

【摘要】：【背景與目的】 乙型肝炎是危害人類健康的全球性疾病。我國是該病高發(fā)區(qū),乙肝病 毒攜帶者約占全國人口的10%。由于該病危害性大,流行面廣,因此其傳播途徑、發(fā)病 機(jī)理及診療手段的研究一直是各國政府和科學(xué)家們高度重視的課題。1985年Hadchouel 等發(fā)現(xiàn)乙肝患者的精子基因組中存在HBV DNA序列整合,提出“乙肝病毒有可能通過生 殖細(xì)胞垂直傳播”的假設(shè)。但10多年來,相關(guān)研究由于沒有適用的實(shí)驗(yàn)動(dòng)物與細(xì)胞培 養(yǎng)模型而止步不前。2002年黃建民等應(yīng)用人精子/金黃地鼠去透明帶卵異種體外受精和 熒光原位雜交技術(shù),證實(shí)染色體上整合了HBV DNA序列的人精子能夠與卵母細(xì)胞正常 地完成受精過程。Ali等也在離體實(shí)驗(yàn)中發(fā)現(xiàn)由精子攜帶的HBV基因能夠在早期胚胎中 復(fù)制和轉(zhuǎn)錄。本研究首次從活體動(dòng)物的整體效應(yīng)探討攜帶了HBV DNA的精子能否順利完 成受精過程,以及通過受精由精子攜帶到胚胎的HBV基因能否進(jìn)行復(fù)制、轉(zhuǎn)錄和蛋白水 平的表達(dá),以期為HBV真正意義上的父嬰垂直傳播途徑提供直接證據(jù)。 【材料與方法】 1)材料①pBR322-HBV DNA質(zhì)粒;②精子,取自性成熟金黃地鼠與昆 明小鼠附睪;③早期胚胎,取自雌性金黃地鼠和昆明小鼠輸卵管。 2)方法①雙側(cè)睪 丸內(nèi)注射HBV DNA質(zhì)粒;②用PCR、Southern雜交、斑點(diǎn)雜交和熒光原位雜交技術(shù)(FISH) 證實(shí)HBV DNA是否進(jìn)入精子細(xì)胞并與精子細(xì)胞基因組整合;③收集受精卵、2-細(xì)胞胚胎, 制備間期核片,用FISH檢測(cè)HBV DNA在間期核上的整合與復(fù)制;④收集2-細(xì)胞胚胎, 用RT-PCR檢測(cè)HBx與HBs基因在胚胎細(xì)胞中的轉(zhuǎn)錄;⑤收集2-細(xì)胞期胚胎,用免疫熒 光技術(shù)檢測(cè)HBsAg蛋白在胚胎細(xì)胞中的表達(dá)。 【結(jié)果】①PCR、Southern在精子樣本中可見HBx基因陽性帶,洗滌液樣本斑點(diǎn)雜交 結(jié)果陰性,排除了PCR和Southern陽性結(jié)果來自洗液污染的可能性;②FISH在精子核、 受精卵中雄原核、2-細(xì)胞胚中兩個(gè)間期核觀察到HBV DNA陽性雜交信號(hào);③RT-PCR 2- 細(xì)胞胚樣本可見HBx和HBs基因特異帶;④免疫熒光檢測(cè) 在2-細(xì)胞胚細(xì)胞漿內(nèi)觀察到 HBsAg陽性表達(dá)。 【結(jié)論】①HBV基因能夠通過精子膜進(jìn)入精子細(xì)胞內(nèi)并整合到精子基因組內(nèi);②攜帶有 HBV基因的精子在自然受精時(shí)能夠?qū)⒉《净驇肱咛ブ?③由精子帶入的HBV基因能 夠在早期胚胎中復(fù)制、轉(zhuǎn)錄和表達(dá)。④本研究為HBV可經(jīng)過雄性生殖細(xì)胞垂直傳遞提 供了直接的科學(xué)證據(jù)。⑤本研究為在其他病毒性傳染病中進(jìn)行類似研究提供一個(gè)理想的 實(shí)驗(yàn)?zāi)Ｐ?進(jìn)而有可能為臨床醫(yī)學(xué)基礎(chǔ)研究展開一個(gè)新的領(lǐng)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.
【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】：2005
【分類號(hào)】：R363

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本文編號(hào)：1999622