【摘要】： 目的乙型肝炎病毒(hepatitis B virus, HBV)垂直傳播是指HBV以生殖細胞作為載體,把HBV DNA帶入胚胎,使子代發(fā)病或成為HBV攜帶者的一種新的、尚待證明的傳播方式。HBV父嬰垂直傳播研究的關(guān)鍵點在于闡明HBV通過血睪屏障轉(zhuǎn)染精子的途徑。本課題通過活體轉(zhuǎn)染不同組的大鼠睪丸,旨在探討機體大量攝入乙醇后,血睪屏障(blood-testis barrier,BTB)的結(jié)構(gòu)完整性和生理機能的改變,以及能否有效阻止含HBV DNA的質(zhì)粒轉(zhuǎn)染生精小管生精上皮細胞,并進一步探討影響B(tài)TB完整性的因素和作用機制。 方法取20只Wistar成年雄性大鼠隨機分為兩組,每組10只。A組為實驗乙醇組,每只按1ml·100g-1·d-1灌胃50%乙醇,連續(xù)40天；B組為空白組,每只按1ml·100g-1·d-1灌胃生理鹽水,連續(xù)40天。向睪丸轉(zhuǎn)染含HBV DNA的質(zhì)粒,應(yīng)用聚合酶鏈反應(yīng)(polymerase chain reaction, PCR)、實時熒光定量PCR(Real-time quantitative PCR, Real-time PCR)、原位雜交(in situ hybridization, ISH)和透射電子顯微鏡(transmission electron microscopy, TEM)檢測睪丸組織HBV DNA的轉(zhuǎn)染情況。 結(jié)果①PCR:A組樣本可見特異的HBV DNA陽性條帶；②通過Real-time PCR的檢測發(fā)現(xiàn)A組HBV DNA平均含量高于B組,差異有統(tǒng)計學意義；③原位雜交：A組發(fā)現(xiàn)陽性雜交信號彌散,可被廣泛發(fā)現(xiàn)于生精上皮基底室和近腔室的生精細胞上；④TEM：A組大鼠睪丸的生精小管基膜厚薄不均,基膜組織疏松增厚,成波浪式皺褶,可見基膜斷裂,精原細胞與支持細胞及生精小管的基膜之間出現(xiàn)較多空泡,生精小管、生精上皮、生精細胞及支持細胞與相鄰細胞之間的間隙擴大。 結(jié)論①乙醇的攝入會破壞大鼠睪丸血睪屏障的完整性,引起生精小管的基膜、支持細胞等超微結(jié)構(gòu)異常；②血睪屏障的損傷及生精小管的結(jié)構(gòu)異常可以導致大鼠睪丸的整個生精上皮易受到HBV DNA的轉(zhuǎn)染,造成轉(zhuǎn)染率的升高。 血睪屏障是支持細胞的主要功能之一,它保護著處于分化狀態(tài)的生精細胞,防止自身免疫反應(yīng),并具有調(diào)控生精細胞分裂和分化,維持曲精小管內(nèi)有利于精子發(fā)生和成熟排放的特殊微環(huán)境等重要作用。血睪屏障的完整性是其起保護功能的重要基礎(chǔ),乙醇可以破壞其完整性,使全部生精細胞易受HBV的感染。
[Abstract]:Objective the vertical transmission of hepatitis B virus (hepatitis B virus, HBV) means that HBV takes germ cells as the carrier, brings HBV DNA into the embryo, and causes the offspring to develop or become a carrier of HBV. The key point of vertical transmission of HBV is to elucidate the pathway of HBV transfection through the blood-testis barrier. The purpose of this study was to investigate the changes of the structural integrity and physiological function of the blood testis barrier (blood-testis barrier,BTB) after a large amount of alcohol intake into the testis of different groups of rats in vivo. And whether the plasmid containing HBV DNA can effectively prevent the transfection of seminiferous tubule and seminiferous epithelial cells, and further explore the factors and mechanism of affecting the integrity of BTB. Methods Twenty adult Wistar male rats were randomly divided into two groups, 10 rats in each group. Group B was treated with normal saline (1ml 100g-1 d-1) for 40 days. The plasmid containing HBV DNA was transfected into testis. Polymerase chain reaction (polymerase chain reaction, PCR),) real-time fluorescence quantitative PCR (Real-time quantitative PCR, Real-time PCR), in situ hybridization (in situ hybridization, ISH) and transmission electron microscope (transmission electron microscopy,) was used. TEM was used to detect the transfection of HBV DNA in testis. Results the specific HBV DNA positive bands were found in the samples of 1PCR:A group. (2) the average content of HBV DNA in group A was higher than that in group B by Real-time PCR detection, and the difference was statistically significant. (3) in situ hybridization: in group A, positive hybridization signals were diffused, which could be widely found in spermatogenic cells in basal and proximal chambers of spermatogenic epithelium. In 4TEM:A group, the thickness of seminiferous tubules was uneven, the basal membrane tissue was loosely thickened, the basal membrane was creased, the basement membrane was broken, and there were more vacuoles between spermatogonium and Sertoli cells and seminiferous tubules, and seminiferous tubules. The gap between spermatogenic epithelium, spermatogenic cells and Sertoli cells and adjacent cells is enlarged. Conclusion (1) ethanol intake can destroy the integrity of the blood-testis barrier in rats and cause the basement membrane of seminiferous tubules, Sertoli cells and other ultrastructural abnormalities. 2 the damage of blood-testis barrier and the abnormal structure of seminiferous tubules can make the whole spermatogenic epithelium of rat testis vulnerable to HBV DNA transfection, resulting in the increase of transfection efficiency. The blood-testis barrier is one of the main functions of Sertoli cells. It protects spermatogenic cells from differentiation, prevents autoimmune reaction, and regulates the division and differentiation of spermatogenic cells. It is important to maintain a special microenvironment in seminiferous tubules that is conducive to spermatogenesis and maturation. The integrity of blood-testis barrier is an important basis for its protective function. Ethanol can destroy its integrity and make all spermatogenic cells susceptible to HBV infection.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前10條

1 張清健,謝慶東;乙型肝炎病毒垂直傳播新途徑的研究[J];癌變.畸變.突變;2003年02期

2 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制與表達[J];癌變.畸變.突變;2005年03期

3 黃天華,謝慶東,陳霓彤,曾曦凡,吳叢梅,王曉梅;精子攜帶的乙肝病毒基因在金黃地鼠胚胎中的復制與表達[J];癌變.畸變.突變;2005年04期

4 趙松,郭芳,解麗君,胡文媛;酒精對大鼠睪丸的亞慢性氧化損傷及維生素A的保護作用[J];癌變.畸變.突變;2005年04期

5 熊俊,訾曉淵,姚玉成,金艷花,李建秀,朱海英,王新民,胡以平,余宏宇;乙型肝炎病毒x基因轉(zhuǎn)基因小鼠模型的建立及培育[J];第二軍醫(yī)大學學報;2002年11期

6 劉紅,姚玉成,何金,李文林,李建秀,蘇小平,張欽憲,胡以平;乙型肝炎病毒核心抗原轉(zhuǎn)基因小鼠的建立[J];第二軍醫(yī)大學學報;2003年02期

7 胡衛(wèi)江,李建秀,余宏宇,戴德順,王新民,孫偉,郝光榮,王肖鵬,胡以平;乙型肝炎病毒(adr亞型)轉(zhuǎn)基因小鼠的研究[J];第二軍醫(yī)大學學報;1999年04期

8 朱煥章,史景泉,成國祥,陳建泉,曠雙運,孫強,成勇,張新立,李厚達,徐少甫,錢耕蓀,顧建人;乙型肝炎病毒X基因轉(zhuǎn)基因小鼠的初步建立[J];第三軍醫(yī)大學學報;1999年02期

9 宋春風,尹桂山,馬洪駿,楊長春;糖皮質(zhì)激素對大鼠睪丸血睪屏障的影響[J];電子顯微學報;2000年03期

10 侯武剛;趙潔;張遠強;曾麗華;郭國楨;;電磁脈沖輻射對小鼠睪丸血-睪屏障的影響[J];輻射研究與輻射工藝學報;2005年06期

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