载脂蛋白H在携带乙肝病毒不孕妇女中的表达及其与颗粒细胞凋亡和体外受精—胚胎移植结局相关性研究

发布时间：2018-04-04 08:06

  本文选题：载脂蛋白H　切入点：乙型肝炎病毒　出处：《浙江大学》2013年硕士论文

【摘要】：背景： 乙型肝炎病毒(hepatitis B virus,HBV)是一种造成全世界约3.5亿人慢性感染的嗜肝DNA病毒。其广泛存在于乙肝患者的体液中,包括唾液、泪液、汗液、乳汁、血液、精液和卵泡液等。HBV传播途径包括水平传播(如输血、性交、医源性及密切的生活接触等)和垂直传播(即父婴和母婴垂直传播)。经精液和精子传递的父婴垂直传播近年来受到较为广泛的关注。尽管通过主动和被动免疫,90%的母婴垂直传播已得到有效控制,但仍有10%的婴儿因阻断失败而感染HBV。近年来在卵泡液,颗粒细胞和女方HBV不孕妇女胚胎中HBV DNA的阳性检出,为HBV母婴垂直传播的学说提供了更多的依据和猜测。 载脂蛋白H(apoH),是血浆脂蛋白的一种,其生理作用尚不完全了解。主要作用是参与血液凝固和激活脂蛋白酶。与脂质代谢相关疾病、自身免疫性疾病如APS和SLE、细胞凋亡、细菌及病毒感染等也相关。apoH与包括HBV在内的多种病毒结合,从而促进病毒感染。国内外研究认为apoH可以促进HBV感染,但它们之间的真正关系尚未揭示,apoH促进HBV感染的机制更是未知。另外apoH可以与磷脂酰丝氨酸结合从而在识别和清除凋亡及衰老细胞中起着一定作用。 颗粒细胞凋亡常被用来评估体外受精-胚胎移植(IVF-ET)结局。颗粒细胞凋亡率增加使IVF周期卵子质量和胚胎质量下降而导致妊娠率下降,换言之,降低颗粒细胞凋亡率能提高IVF成功率。而HBV对颗粒细胞凋亡的影响尚未有研究报道。 HBV感染不但可以引起肝脏一系列病变,亦可影响人类生殖能力。鉴于HBV对女性生殖力影响的相关报道及HBV母婴垂直传播的可能性的不断证实,IVF的开展为研究HBV经生殖细胞传播研究提供了良好平台的同时增加了人们对于IVF治疗结局的关注。然而,关于HBV不孕妇女IVF结局的观点并不一致。基于上述理论我们推测卵泡液中apoH是否能通过促进HBV感染并介导了HBV对颗粒细胞凋亡的影响从而对IVF结局产生影响。 目的： 拟观察携带乙肝病毒的不孕妇女血清和卵泡液中apoH表达水平与HBV感染之间关系,以及HBV感染与颗粒细胞凋亡的关系,以探讨apoH是否介导了HBV对颗粒细胞凋亡的影响从而影响体外受精治疗结局,为研究阻断HBV母婴垂直传播和提高临床携带乙肝病毒不孕妇女IVF的成功率提供理论依据。 材料与方法： 收集2011年3月～2012年10月在浙江大学医学院附属妇产科医院接受常规体外受精/卵胞浆单精子注射-胚胎移植治疗的137例包括83例携带乙肝病毒(其中59例HBsAg阳性组,19例HBeAg阳性)和59例非乙肝(HBsAg阴性)不孕妇女的血清和卵泡液；137例中的55例同时收集颗粒细胞。采用蛋白免疫印迹方法(Western blot, WB)检测血清和卵泡液中apoH的表达水平,利用荧光定量PCR方法分别检测血清与卵泡液中的HBV DNA浓度,根据实时定量逆转录PCR (Real-Time RT PCR)方法检测颗粒细胞的凋亡相关基因(bcl-2, bax,p53,capase3)的mRNA的表达水平。同时采集所有样本病例的临床资料进行统计分析。 结果： 乙肝组血清apoH表达水平显著高于非乙肝组(p=0.000),其中组间比较HBeAg阳性组明显高与HBsAg阳性组和对照组。卵泡液apoH表达水平乙肝组同样显著高于非乙肝组(p=0.047),但组间比较仅HBeAg阳性组高于对照组。线性分析显示血清apoH表达水平与卵泡液apoH呈中等程度线性相关(r=0.414,p=0.000)。血清apoH表达水平与血清HBV DNA含量不一定相关,但卵泡液中apoH表达水平与卵泡液HBV DNA含量呈正相关。乙肝不孕妇女颗粒细胞capase3基因mRNA水平明显高于非乙肝妇女(p=0.025),不同HBV感染及复制状态下HBV感染妇女2PN受精率明显高于而优质胚胎率明显低于非乙肝对照组(p0.05)。血清和卵泡液apoH水平与颗粒细胞凋亡及IVF取卵周期妊娠结局无相关性(p0.05)。 结论： 1.携带乙肝病毒不孕妇女血清apoH表达水平高于非乙肝不孕妇女,血清HBeAg阳性不孕妇女卵泡液apoH表达水平明显高于非乙肝不孕妇女； 2.携带乙肝病毒不孕妇女血清apoH表达水平与血清HBV DNA含量无明显相关,但其卵泡液中apoH表达水平与卵泡液HBV DNA含量呈正相关； 3.HBV感染妇女颗粒细胞capase3基因mRNA水平明显高于非乙肝不孕妇女,提示HBV感染可以促进颗粒细胞凋亡。 4.HBV感染不孕妇女IVF周期2PN受精率增高和优质胚胎率下降。 5.血清和卵泡液apoH表达水平与颗粒细胞凋亡和IVF取卵周期妊娠结局无相关性。
[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锛，

本文编号：1709089