本文選題：載脂蛋白H　切入點：乙型肝炎病毒　出處：《浙江大學(xué)》2013年碩士論文

【摘要】：背景： 乙型肝炎病毒(hepatitis B virus,HBV)是一種造成全世界約3.5億人慢性感染的嗜肝DNA病毒。其廣泛存在于乙肝患者的體液中,包括唾液、淚液、汗液、乳汁、血液、精液和卵泡液等。HBV傳播途徑包括水平傳播(如輸血、性交、醫(yī)源性及密切的生活接觸等)和垂直傳播(即父嬰和母嬰垂直傳播)。經(jīng)精液和精子傳遞的父嬰垂直傳播近年來受到較為廣泛的關(guān)注。盡管通過主動和被動免疫,90%的母嬰垂直傳播已得到有效控制,但仍有10%的嬰兒因阻斷失敗而感染HBV。近年來在卵泡液,顆粒細(xì)胞和女方HBV不孕婦女胚胎中HBV DNA的陽性檢出,為HBV母嬰垂直傳播的學(xué)說提供了更多的依據(jù)和猜測。 載脂蛋白H(apoH),是血漿脂蛋白的一種,其生理作用尚不完全了解。主要作用是參與血液凝固和激活脂蛋白酶。與脂質(zhì)代謝相關(guān)疾病、自身免疫性疾病如APS和SLE、細(xì)胞凋亡、細(xì)菌及病毒感染等也相關(guān)。apoH與包括HBV在內(nèi)的多種病毒結(jié)合,從而促進病毒感染。國內(nèi)外研究認(rèn)為apoH可以促進HBV感染,但它們之間的真正關(guān)系尚未揭示,apoH促進HBV感染的機制更是未知。另外apoH可以與磷脂酰絲氨酸結(jié)合從而在識別和清除凋亡及衰老細(xì)胞中起著一定作用。 顆粒細(xì)胞凋亡常被用來評估體外受精-胚胎移植(IVF-ET)結(jié)局。顆粒細(xì)胞凋亡率增加使IVF周期卵子質(zhì)量和胚胎質(zhì)量下降而導(dǎo)致妊娠率下降,換言之,降低顆粒細(xì)胞凋亡率能提高IVF成功率。而HBV對顆粒細(xì)胞凋亡的影響尚未有研究報道。 HBV感染不但可以引起肝臟一系列病變,亦可影響人類生殖能力。鑒于HBV對女性生殖力影響的相關(guān)報道及HBV母嬰垂直傳播的可能性的不斷證實,IVF的開展為研究HBV經(jīng)生殖細(xì)胞傳播研究提供了良好平臺的同時增加了人們對于IVF治療結(jié)局的關(guān)注。然而,關(guān)于HBV不孕婦女IVF結(jié)局的觀點并不一致。基于上述理論我們推測卵泡液中apoH是否能通過促進HBV感染并介導(dǎo)了HBV對顆粒細(xì)胞凋亡的影響從而對IVF結(jié)局產(chǎn)生影響。 目的： 擬觀察攜帶乙肝病毒的不孕婦女血清和卵泡液中apoH表達水平與HBV感染之間關(guān)系,以及HBV感染與顆粒細(xì)胞凋亡的關(guān)系,以探討apoH是否介導(dǎo)了HBV對顆粒細(xì)胞凋亡的影響從而影響體外受精治療結(jié)局,為研究阻斷HBV母嬰垂直傳播和提高臨床攜帶乙肝病毒不孕婦女IVF的成功率提供理論依據(jù)。 材料與方法： 收集2011年3月～2012年10月在浙江大學(xué)醫(yī)學(xué)院附屬婦產(chǎn)科醫(yī)院接受常規(guī)體外受精/卵胞漿單精子注射-胚胎移植治療的137例包括83例攜帶乙肝病毒(其中59例HBsAg陽性組,19例HBeAg陽性)和59例非乙肝(HBsAg陰性)不孕婦女的血清和卵泡液；137例中的55例同時收集顆粒細(xì)胞。采用蛋白免疫印跡方法(Western blot, WB)檢測血清和卵泡液中apoH的表達水平,利用熒光定量PCR方法分別檢測血清與卵泡液中的HBV DNA濃度,根據(jù)實時定量逆轉(zhuǎn)錄PCR (Real-Time RT PCR)方法檢測顆粒細(xì)胞的凋亡相關(guān)基因(bcl-2, bax,p53,capase3)的mRNA的表達水平。同時采集所有樣本病例的臨床資料進行統(tǒng)計分析。 結(jié)果： 乙肝組血清apoH表達水平顯著高于非乙肝組(p=0.000),其中組間比較HBeAg陽性組明顯高與HBsAg陽性組和對照組。卵泡液apoH表達水平乙肝組同樣顯著高于非乙肝組(p=0.047),但組間比較僅HBeAg陽性組高于對照組。線性分析顯示血清apoH表達水平與卵泡液apoH呈中等程度線性相關(guān)(r=0.414,p=0.000)。血清apoH表達水平與血清HBV DNA含量不一定相關(guān),但卵泡液中apoH表達水平與卵泡液HBV DNA含量呈正相關(guān)。乙肝不孕婦女顆粒細(xì)胞capase3基因mRNA水平明顯高于非乙肝婦女(p=0.025),不同HBV感染及復(fù)制狀態(tài)下HBV感染婦女2PN受精率明顯高于而優(yōu)質(zhì)胚胎率明顯低于非乙肝對照組(p0.05)。血清和卵泡液apoH水平與顆粒細(xì)胞凋亡及IVF取卵周期妊娠結(jié)局無相關(guān)性(p0.05)。 結(jié)論： 1.攜帶乙肝病毒不孕婦女血清apoH表達水平高于非乙肝不孕婦女,血清HBeAg陽性不孕婦女卵泡液apoH表達水平明顯高于非乙肝不孕婦女； 2.攜帶乙肝病毒不孕婦女血清apoH表達水平與血清HBV DNA含量無明顯相關(guān),但其卵泡液中apoH表達水平與卵泡液HBV DNA含量呈正相關(guān)； 3.HBV感染婦女顆粒細(xì)胞capase3基因mRNA水平明顯高于非乙肝不孕婦女,提示HBV感染可以促進顆粒細(xì)胞凋亡。 4.HBV感染不孕婦女IVF周期2PN受精率增高和優(yōu)質(zhì)胚胎率下降。 5.血清和卵泡液apoH表達水平與顆粒細(xì)胞凋亡和IVF取卵周期妊娠結(jié)局無相關(guān)性。
[Abstract]:Background:
Hepatitis B virus (hepatitis B, virus, HBV) is a kind of caused about 350 million people worldwide chronic infection with hepatotropic DNA virus. It exists extensively in hepatitis B patients with body fluids, including saliva, tears, sweat, milk, blood, semen and follicular fluid.HBV transmission including horizontal transmission (such as blood transfusion, sexual intercourse, iatrogenic and close life contact etc.) and vertical transmission (i.e., paternal and maternal transmission). The paternal fetal vertical transmission of semen and sperm transfer in recent years has been widespread concern. Although through active and passive immunization, maternal infant vertical transmission 90% has been effectively controlled, but there are still 10% the baby due to blocking failure and HBV. infection in recent years in the follicular fluid, HBV DNA and HBV the granulosa cells in embryos of infertile women positive, provides more evidence and speculation for the vertical transmission of HBV theory.
Apolipoprotein H (apoH), is a kind of plasma lipoproteins, its physiological function is still not fully understood. The main role is to participate in blood coagulation and activation of lipoprotein lipase. Associated with lipid metabolism disorders, autoimmune diseases such as APS and SLE, apoptosis, bacterial and viral infection is also associated with a variety of.ApoH virus including the HBV combination, so as to promote viral infection. Research shows that apoH can promote HBV infection, but the real relationship between them has not yet been revealed that the mechanism of apoH promoting HBV infection is unknown. In addition apoH can be combined in order to identify and remove plays a role in cell apoptosis and senescence and phosphatidylserine.
Granulosa cell apoptosis is often used to evaluate the in vitro fertilization and embryo transfer (IVF-ET) outcome. The rate of apoptosis in granulosa cells increased IVF cycle oocyte quality and embryo quality decline due to pregnancy rate decreased, in other words, reduce the rate of apoptosis in granulosa cells can improve the success rate of IVF. The effect of HBV on the apoptosis of granulosa cells has not yet been reported.
HBV infection can not only cause a series of liver disease, can affect human reproductive ability. In view of the possibility of vertical transmission related effects of HBV on female fecundity and HBV has confirmed that the development of IVF for the study of HBV by germ cell communication research provides a good platform for people and increase the treatment outcome of IVF attention. However, about HBV of infertile women IVF outcome opinion is not consistent. Based on the above theory we speculate on whether apoH in follicular fluid by promoting HBV infection and mediates the effects of HBV on apoptosis of granulosa cells which have an impact on the IVF node.
Objective:
The relationship between the level of expression of apoH and HBV infection in infertile women carry the hepatitis B virus in serum and follicular fluid were observed, and the relationship between HBV infection and apoptosis of granulosa cells, to investigate whether apoH mediates the effects of HBV on apoptosis of granulosa cells that can affect the outcome of in vitro fertilization treatment, to study the blocking of HBV vertical transmission and improve the clinical carry hepatitis B virus in infertile women with the success rate of IVF and provide a theoretical basis.
Materials and methods:
From March 2011 to October 2012 in the affiliated obstetrics and Gynecology Hospital of Zhejiang University hospital received conventional IVF / intracytoplasmic sperm injection embryo transplantation in the treatment of 137 cases including 83 cases of hepatitis B virus (including 59 cases of HBsAg positive group, HBeAg was positive in 19 cases) and 59 cases of non hepatitis B (HBsAg negative) serum and follicular in infertile women liquid; 55 cases of the 137 cases were collected at the same time granulosa cells. Western blot method (Western blot WB) to detect the expression of apoH in serum and follicular fluid were detected by HBV, the concentration of DNA in serum and follicular fluid by fluorescent quantitative PCR method, according to the real-time quantitative reverse transcription PCR (Real-Time RT PCR) granulosa cells detection of apoptosis related genes (Bcl-2, Bax, p53, capase3) the expression level of mRNA. The clinical data collected at the same time all the sample cases were analyzed.
Result錛，

本文編號：1709089