【作者】 刘能辉；

【导师】 周昌菊；

【作者基本信息】 中南大学 ， 妇产科学， 2008， 博士

【摘要】 研究背景和目的:胚泡着床是指胚泡粘附并植入子宫内膜的一个连续复杂的生理过程,是胚泡与母体子宫内膜相互作用的过程。胚泡着床过程受到多种因素的调节,不仅受母体因素和胚泡本身因素的调控,而且还有许多生物大分子物质参与,如骨桥蛋白(osteopontin,OPN)和整合素ανβ3等。骨桥蛋白是一种含精氨酸一甘氨酸一天冬氨酸(Arg—Gly—Asp,RGD)的分泌型糖基化磷蛋白,归类于细胞外基质蛋白。OPN最先是从骨组织分离的,后来发现在分泌期子宫内膜、入侵内膜的细胞滋养层、内膜腺上皮、蜕膜化的基质细胞及胎盘表面上也有表达。OPN既是细胞因子,又是细胞粘附分子。虽然OPN能与各种整合素受体结合,但是整合素ανβ3是OPN的最主要的受体。OPN的主要生物学功能是通过其特异性序列RGD与整合素等多种受体结合,对细胞的粘附和迁移起重要作用。OPN和整合素ανβ3在胚泡着床中起重要作用。研究OPN和整合素ανβ3对胚泡着床的影响,阐明其作用的分子机制是非常重要的。本研究将从以下几方面进行研究:(1)建立成功的小鼠早期妊娠模型;(2)检测骨桥蛋白和整合素β3在围植入期小鼠子宫内膜中的表达,明确OPN和整合素β3表达规律;(3)研究骨桥蛋白抗体和整合素β3抗体对小鼠胚泡着床的影响。方法:1.小鼠早期妊娠模型的建立选择动情前期的雌性昆明小鼠20只,随机分为二组,每组10只,促排卵组腹腔注射孕马血清促性腺激素(pregnant mare serumgonadotropin,PMSG)和HCG促排卵治疗,对照组注射生理盐水,随后雌雄交配,次日检查阴栓,发现阴栓计为妊娠第1天(孕D1)。于妊娠第8天,用颈椎脱臼法处死,迅速剖腹,检查子宫,记录所有孕鼠数及每只孕鼠的着床胚泡数,计算妊娠率和平均胚泡着床数;采用化学发光法检测促排卵组和对照组的血清及子宫组织雌二醇、孕酮的含量,利用光镜观察子宫内膜及卵巢的形态结构。2.围植入期小鼠子宫内膜中OPN和整合素β3蛋白和基因检测将清洁级性成熟期昆明小鼠44只,雌性24只,雄性20只,雌性小鼠阴道涂片检查为动情前期,在此期注射PMSG和HCG,4只雌鼠不合笼,用颈椎脱臼法处死,剪取子宫组织(孕D0,对照组),直接液氮保存和福尔马林固定保存,其余的雌:雄小鼠1:1合笼,次日检查阴栓,发现阴栓计为妊娠第1天。分别于妊娠第4天,5天,6天,7天,8天用颈椎脱臼法处死,迅速剖腹,剪取子宫组织予以保存(实验组),采用免疫组化方法和Western-blotting技术分析围植入期小鼠子宫内膜中的骨桥蛋白和整合素β3表达规律,采用RT-PCR技术半定量分析围植入期小鼠子宫内膜骨桥蛋白和整合素β3 mRNA表达特征。3.OPN抗体和整合素β3抗体对小鼠胚泡着床的影响取孕4天小鼠,分别在两侧子宫角内注射骨桥蛋白抗体、整合素β3抗体及生理盐水,正常妊娠空白对照组未行任何处理,于孕8天处死小鼠,统计胚泡着床数及妊娠率,取子宫观察内膜的组织学变化。结果:1.用PMSG+HCG建立小鼠早期妊娠模型PMSG+HCG组妊娠率为90%,平均胚泡着床数9.5±3.6;对照组妊娠率为60%,平均胚泡着床数3.8±3.9,促排卵组妊娠率和平均胚泡着床数明显高于对照组(P＜0.05);促排卵组小鼠血清和子宫组织中的雌二醇和孕酮含量高于对照组,P＜0.05;两组小鼠子宫内膜的形态变化没有差异。2.围植入期小鼠子宫内膜中的OPN和整合素β3的表达小鼠整个围植入期子宫内膜中均有OPN和整合素β3表达,但表达的高峰在着床窗口期,即孕D5。孕D0(非孕)子宫内膜的OPN和整合素β3表达较弱,孕D4时表达增强,孕D5时表达最强,孕D6时表达突然回落,孕D7、孕D8时表达减弱,降至非孕水平。3.骨桥蛋白抗体和整合素β3抗体抑制胚胎着床结果显示骨桥蛋白抗体组小鼠妊娠率为60%,平均胚胎着床数为1.6±1.19,整合素β3抗体组妊娠率为70%,平均胚胎着床数为1.7±1.28,明显低于生理盐水组及空白对照组(90%,10.0±1.63及100%,10.5±1.58),提示骨桥蛋白抗体和整合素β3抗体明显抑制小鼠胚泡着床。结论:1.注射PMSG和HCG可有效介导小鼠早期妊娠模型的建立,妊娠率和胚泡着床数明显高于自然条件受孕,该模型的建立为进一步研究胚泡着床及子宫内膜容受性等提供理想的技术平台。2.骨桥蛋白和整合素β3在围植入期小鼠子宫内膜中的表达随着孕龄的增加逐渐增强,但表达的高峰在孕D5,与植入窗的开放相吻合,是子宫内膜接受性的客观标志,提示骨桥蛋白和整合素β3在胚泡着床过程中起重要作用,本研究为阐明胚泡着床机制提供实验依据。3.骨桥蛋白和整合素β3抗体明显抑制小鼠胚泡的着床并导致子宫内膜的形态学变化,这提示骨桥蛋白和整合素β3与子宫内膜容受性获得有关。骨桥蛋白和整合素β3参与了小鼠胚泡的着床。更多还原

【Abstract】 BACKGROUND AND OBJECTIVES:Embryo implantation is an successive complex physiological process,which the embryo adhere to and implant into uterine.Embryo implantation not only is regulated by many factors from uterus and embryo,but also by a lot of biological moleculars,such as osteopontin, integrinανβ3 and so on.Osteopontin(OPN) is a secreted glycosylated phosphoprotein,which contains an Arg-Gly-Asp(RGD)sequence.It belongs to extracellular matrix protein(ECM).The original observation shows that OPN is expressed by bone tissue,and the subsequent research found that OPN is expressed in uterine endometrium of secretion phase, the invade trophoblastic cell,the glandular epithelium of deciduas and placenta.OPN is both cytokine and adhesion molecule.Although OPN binds to various integrin receptors,theανβ3 integrin has been recognized as a primary receptor for OPN.OPN binds primarily to integrin receptors via its RGD sequence,and mediates cell adhesion and migration.Theανβ3 integrin and osteopontin play an important role in embryo implantation.So it is necessary to establish an animal model and elucidate their mechanism in embryo implantation.In this project,(1) Mice early pregnancy model was established using PMSG and HCG.(2) OPN and integrinβ3 expression were detected in the peri-implantation mouse endometrium,and the expression profile of OPN and integrinβ3 were determined.(3) Inhibitory effect on mice blastocyst implantation was observed after intra-uterus injection of antibodies against osteopontin and integrinβ3. METHODS:1.Establishment of mice early pregnancy model20 mice at proestrus stage were equally divided into two groups:the induced ovulation group(PMSG+HCG) were injected with PMSG and HCG,and the control group was injected with saline solution.Mice were sacrificed at 8 days after pregnancy(n=10 per group;day 1 is served as the day of vaginal plug),and anatomized.The implantation number of mice blastocyst was counted.The pregnancy rates were got.According, estradiol and progesterone concentrations were detected in serum and endometrium tissue homogenate of mice.The morphology of endometrium and ovary was observed under microscopy.2.OPN and integrinβ3 expression were detected in the peri-implantation mouse endometrium.Of 44 mice,24 female mice at proestrus,20 male mice at sexual maturity.Female mice were injected with PMSG and HCG.Four female mice were sacrificed at estrus(Day0).20 female mice assigned to pregnant status were mated to intact fertile 20 males of the same strain. Uteri were then collected on Day 4,5,6,7,and 8 of pregnancy(n=4 per group;Day 1 defined as the day of vaginal plug).The implantation number of mice blastocyst was counted.Endmetrium tissues were stored at liquid nitrogen or 4%paraformaldehyde.OPN and integrinβ3 expression were detected using immunohistochemistry and western-blotting.OPN and integrinβ3 gene expression was analyzed by RT-PCR.3.Effect of intra-uterus injection of antibody against osteopontin and antibody against integrinβ3.20 mice were respectively injected with antibodies against osteopontin or integrinβ3.The control group was injected saline solution. No treatment served as the blank control group.Mice were sacrificed at Day 8 of pregnancy,and anatomized.The implantation numbers of mice blastocysts were counted.The endometrium morphology was observed under microscopy.RESULTS:1.Establishment of mice early pregnancy modelThe results showed that the pregnancy rate in PMSG+HCG group was 90%,and implantation number of mice blastocysts was 9.5±3.6.In the control group,the pregnancy rate was 60%,and implantation number of mice blastocyst was 3.8±3.9.Both pregnancy rate and the numbers of implantation sites were higher in PMSG+HCG group than the control group.In PMSG+HCG group,estrodiol and progesterone concentrations were also higher than the control group.2.OPN and integrinβ3 expression were detected in the peri-implantation mouse endometriumThe result of our study showed that the expression of OPN and integrin in mouse endometrium luminal epithelium on Day0 of pregnancy was very weak,OPN and integrinβ3 protein expression gradually increased along pregnancy process.OPN and integrinβ3 gene expression also increased along pregnancy process,the expression of OPN and integrinβ3 on Day5 was increased to the extreme,and but on Day6 was decreased,and on Day7or Day8 was decreased dramatically.3.To study the effect s of osteopontin and integrinβ3 antibodies on mice blastocyst implantation.The results showed that the pregnancy rate was 60%in osteopontin antiserum group,implantation number of mice blastocyst was 1.6±1.19. The pregnancy rate was 70%in integrinβ3-antiserum group,implantation number of mice blastocyst was 1.7±1.28.All of pregnancy rate and implantation number of mice blastocyst were significantly low in the osteopontin-antiserum and integrinβ3-antiserum group compared with N.S group(90%,10.0±1.63) and the blank control group(100%, 10.5±1.58).These suggest that osteopontin-antibody and integrinβ3-antibody block mice blastocyst attachment and implantation.CONCLUSIONS:1.Early pregnancy model of mice was established using PMSG and HCG injection.The mice pregnancy model using PMSG and HCG model is better than nature pregnancy one.Both pregnancy rate and the numbers of implantation sites were higher in PMSG+HCG group than the control.This model provides a novel technology for investigating mechanism of embryo implantation and uterine endometrial receptivity.2.Both gene and protein expression profile of osteopontin and integrinβ3 show that osteopontin and integrinβ3 increase along pregnancy process,peaked on Day 5.The expression of osteopontin and integrinβ3 in mouse endometrium frames the "implantation window", which is a candidate biochemical marker of uterine receptivity.This indicates that osteopontin and integrinβ3 involve in embryo implantation. This provides a novel clue for elucidating mechanism of embryo implantation.3.Osteopontin-antibody and integrinβ3-antibody could block mice blastocyst attachment and implantation,and induced the morphological changes of uterine endometrial.This indicates that osteopontin and integrinβ3 are associated with uterine endometrial receptivity at embryo implantation.Osteopontin and integfinβ3 may participate in mice blastocyst anchor into uterine.更多还原