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乙醇在HBV DNA轉(zhuǎn)染中對大鼠血睪屏障的影響及其機(jī)制探討

發(fā)布時間:2018-11-27 08:56
【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(xì)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(diǎn)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(jī)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(jī)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(xì)胞,并進(jìn)一步探討影響B(tài)TB完整性的因素和作用機(jī)制。 方法取20只Wistar成年雄性大鼠隨機(jī)分為兩組,每組10只。A組為實(shí)驗(yàn)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(shí)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(xué)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(xì)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(xì)胞與支持細(xì)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(xì)胞及支持細(xì)胞與相鄰細(xì)胞之間的間隙擴(kuò)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(xì)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?梢詫(dǎo)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(xì)胞的主要功能之一,它保護(hù)著處于分化狀態(tài)的生精細(xì)胞,防止自身免疫反應(yīng),并具有調(diào)控生精細(xì)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(hù)功能的重要基礎(chǔ),乙醇可以破壞其完整性,使全部生精細(xì)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(xué)位授予單位】:青島大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2010
【分類號】:R373

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