乙醇在HBV DNA转染中对大鼠血睾屏障的影响及其机制探讨

发布时间：2018-11-27 08:56

【摘要】： 目的乙型肝炎病毒(hepatitis B virus, HBV)垂直传播是指HBV以生殖细胞作为载体,把HBV DNA带入胚胎,使子代发病或成为HBV携带者的一种新的、尚待证明的传播方式。HBV父婴垂直传播研究的关键点在于阐明HBV通过血睾屏障转染精子的途径。本课题通过活体转染不同组的大鼠睾丸,旨在探讨机体大量摄入乙醇后,血睾屏障(blood-testis barrier,BTB)的结构完整性和生理机能的改变,以及能否有效阻止含HBV DNA的质粒转染生精小管生精上皮细胞,并进一步探讨影响BTB完整性的因素和作用机制。方法取20只Wistar成年雄性大鼠随机分为两组,每组10只。A组为实验乙醇组,每只按1ml·100g-1·d-1灌胃50%乙醇,连续40天；B组为空白组,每只按1ml·100g-1·d-1灌胃生理盐水,连续40天。向睾丸转染含HBV DNA的质粒,应用聚合酶链反应(polymerase chain reaction, PCR)、实时荧光定量PCR(Real-time quantitative PCR, Real-time PCR)、原位杂交(in situ hybridization, ISH)和透射电子显微镜(transmission electron microscopy, TEM)检测睾丸组织HBV DNA的转染情况。结果①PCR:A组样本可见特异的HBV DNA阳性条带；②通过Real-time PCR的检测发现A组HBV DNA平均含量高于B组,差异有统计学意义；③原位杂交：A组发现阳性杂交信号弥散,可被广泛发现于生精上皮基底室和近腔室的生精细胞上；④TEM：A组大鼠睾丸的生精小管基膜厚薄不均,基膜组织疏松增厚,成波浪式皱褶,可见基膜断裂,精原细胞与支持细胞及生精小管的基膜之间出现较多空泡,生精小管、生精上皮、生精细胞及支持细胞与相邻细胞之间的间隙扩大。结论①乙醇的摄入会破坏大鼠睾丸血睾屏障的完整性,引起生精小管的基膜、支持细胞等超微结构异常；②血睾屏障的损伤及生精小管的结构异常可以导致大鼠睾丸的整个生精上皮易受到HBV DNA的转染,造成转染率的升高。血睾屏障是支持细胞的主要功能之一,它保护着处于分化状态的生精细胞,防止自身免疫反应,并具有调控生精细胞分裂和分化,维持曲精小管内有利于精子发生和成熟排放的特殊微环境等重要作用。血睾屏障的完整性是其起保护功能的重要基础,乙醇可以破坏其完整性,使全部生精细胞易受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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

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