【作者】 张丹；

【导师】 黄荷凤；

【作者基本信息】 浙江大学 ， 妇产科学， 2011， 博士

【摘要】 第一部分FSH促排卵对人子宫内膜AQPs和容受因子的影响目的：验证FSHR在人子宫内膜组织和内膜细胞系Ishikawa细胞的表达,探讨FSH促排卵对人子宫内膜形态、FSHR、AQPs和内膜容受因子表达的影响。材料与方法：收集促排卵(COS)妇女着床窗期子宫内膜15例、同期对照内膜40例,采用化学发光法检测血清FSH和E2水平；采用RT-PCR产物纯化克隆测序、蛋白免疫印记、免疫组织化学、细胞免疫荧光等方法全面验证人子宫内膜组织和细胞FSHR的表达。电镜观察两组子宫内膜形态和超微结构；蛋白免疫印记方法检测两组子宫内膜FSHR、水通道蛋白(AQP1、AQP2、AQP8)、内膜容受因子(Integrinβ3、LIF)、细胞紧密连接相关蛋白(Claudin4)的表达与改变。结果：人子宫内膜组织和子宫内膜细胞系Ishikawa细胞表达FSHR mRNA和蛋白,RT-PCR产物序列与NCBI数据库BLAST比对吻合率100%；蛋白表达主要定位于细胞膜和细胞浆COS妇女血清FSH和E2水平显著高于对照组。扫描电镜下见对照组内膜腔上皮排列有序、胞饮突形成,COS组内膜腔上皮细胞排列分布紊乱、未见明显胞饮突；透视电镜见COS组内膜上皮细胞凋亡增加,细胞紧密连接未见明显改变。COS组内膜组织FSHR蛋白表达显著上调,AQP1、AQP2、AQP8、Integrinβ3、LIF蛋白表达显著下调,Claudin4表达无显著改变。结论：FSHR表达于人子宫内膜组织和子宫内膜细胞系Ishikawa细胞。促排卵妇女血清FSH和E显著升高,内膜FSHR表达上调、AQP1、AQP2、AQP8、Integrinβ3、LIF蛋白表达下调,并致内膜腔上皮形态改变、凋亡增加。FSH可能通过FSHR直接调节育龄期子宫内膜形态和功能。第二部分FSH影响育龄期子宫内膜容受性的调控机制目的：以动物模型和体外模型为基础,研究FSH对育龄期子宫内膜容受性、胚胎着床的影响及可能机制。材料与方法：建立FSH促排卵小鼠动物模型,检测外源性FSH刺激对小鼠血FSH/E2水平、子宫内膜AQPs和着床容受因子、妊娠率和活产率的影响,创新性建立小鼠囊胚粘附模型、检测FSH/E2、PCMB (AQP抑制剂)预处理子宫内膜细胞对囊胚粘附着床的影响；同时进行体外细胞实验,采用RT-Real time PCR检测FSH/E2对AQPs、integrinB3、LIF等容受因子目的基因表达水平；采用ssiRNA干扰技术和JAr滋养细胞球粘附模型,检测AQP2/AQP8siRNA干扰后内膜容受因子的表达改变和JAr滋养细胞球粘附率的改变。结果：FSH促排卵组小鼠血FSH/E2水平显著增高,内膜AQP3、AQP2蛋白表达呈下调趋势、FSHR呈微弱上调趋势,妊娠率和活产率显著低于对照组。小鼠囊胚粘附模型实验显示,体外FSH、FSH/E及PCMB预处理子宫内膜细胞均导致囊胚48h和72h粘附率显著下降。E2对IK细胞AQP2和AQP8 mRNA呈浓度依赖性上调作用,FSH (0、1、3、10、30 IU/L)对IK细胞AQP2、AQP8和LIF mRNA呈浓度依赖性和时间依赖性下调作用,其中以10IU/L和301U/L浓度FSH在作用48hr时的下调作用最为显著。AQP2siRNA干扰后IK细胞着床因子LIF和Integrinβ3表达显著下调,OLFM1表达显著上调；AQP8siRNA干扰后LIF表达显著下调,OLFM1表达显著上调。子宫内膜细胞AQP8siRNA和AQP2siRNA干扰后,JAr滋养细胞团粘附率均显著下降。结论：超生理水平FSH通过下调育龄期子宫内膜AQPs、干扰内膜容受因子表达,进而影响子宫内膜对胚胎的容受性、不利于胚胎着床。第三部分长期高FSH刺激对围绝经期子宫内膜萎缩的影响目的：通过在体组织、动物模型和体外细胞模型,探讨绝经后高FSH水平在子宫内膜功能及萎缩中所起的作用及其机制。材料与方法：采用化学发光法检测围绝经期和绝经后不同年龄段妇女血清FSH、LH、E2变化动态,免疫组织化学方法检测绝经后妇女和育龄期妇女子宫内膜FSHR、Ki67、AQP1、AQP2、AQP8蛋白表达。采用MTT法检测FSH对人原代培养子宫内膜腺上皮细胞和Ishikawa细胞增殖的影响,流式细胞仪检测FSH对Ishikawa细胞周期的影响,Real time PCR检测高浓度FSH对子宫内膜细胞AQP2和AQP8表达的影响、并检测AC激动剂和拮抗剂对FSH作用的影响。建立手术去势动物模型、药物去势动物模型和假手术(对照)动物模型,通过RIA方法检测血FSH和E2水平,采用透视电镜观察子宫内膜细胞形态和凋亡指征、Western blotting检测子宫内膜增殖和凋亡相关指标。结果：绝经后妇女血清FSH水平随年龄从41-50岁年龄段到61-70年龄段呈逐步上升,从61-70年龄段到71-80年龄段无显著上升。血清E2水平呈现与FSH同步的反向变化。与育龄期内膜相比,绝经后子宫内膜FSHR、AQP2和AQP1蛋白表达无显著改变,Ki67表达显著下降, AQP8蛋白间质表达较育龄期增高、腺体表达较育龄期降低。FSH对原代子宫内膜腺上皮细胞呈浓度依赖性的抑制增殖作用,但对Ishikawa细胞增殖和细胞周期无明显影响。高浓度FSH(50IU/L、100IU/L、200IU/L、400IU/L)呈浓度依赖性显著上调Ishikawa细胞AQP2 mRNA的表达,该上调作用可部分被SQ22536所拮抗。Forskolin可上调Ishikawa细胞AQP2 mRNA的表达。动物模型实验方面,成功建立了手术去势(OVX,高FSH、低E2)、药物去势(GnRHa,低FSH、低E2)和假手术对照组(SHAM, FSH和E2水平如常)小鼠。OVX组小鼠子宫萎缩明显、透视电镜下见大片凋亡细胞和散在凋亡小体,而GnRHa组小鼠子宫大小、萎缩度及细胞凋亡现象介于SHAM组和OVX组之间。与SHAM组相比,GnRHa组小鼠子宫C-FOS、C-JUN、Bcl-2蛋白表达量呈下降趋势。与SHAM组和GnRHa组相比,OVX组小鼠子宫C-FOS、C-JUN、Bcl-2蛋白表达显著下降,而Caspase3蛋白的表达显著增高。结论：绝经后循环高FSH水平可通过腺苷酸环化酶通路上调子宫内膜AQP2,抑制子宫内膜增殖、促进子宫内膜细胞凋亡,协同低E调节子宫内膜生理性萎缩。更多还原

【Abstract】 Part I The effects of FSH treatment on AQPs and receptive factors in human endometriumObjective:To identify whether FSHR expresses in human endometrium and Ishikawa cells, the endometrial adenocarcinoma cell line. To observe the human endometrial morphological and functional changes, as well as endometrial expression of FSHR, AQPs and endometrial receptive factors after controlled ovarian stimulation (COS) using FSH treatment.Patients and methods:We collected 15 endometrial samples in implantation window after COS, and 40 samples in implantation window of natural cycle as control group. We measured the serum FSH and E2 with chemoluminescence, detected FSHR expression in human endometrial tissue and Ishikawa cells with a few methods, including sequencing RT-PCR product, Western blotting, immunohistochemistry and immunofluorescence. Endometrial ultrastructure was investigated with electron microscopy. We observe the changes of endometrial FSHR, AQPs(AQPl, AQP2, AQP 8), endometrial receptive factors (Integrinβ3、LIF) and Claudin4, a tight-junction associated protein with Western blotting.Results:We identified FSHR mRNA and protein in endometrial tissue and Ishikawa cells, and make 100% matchup between FSHR RT-PCR product sequence and BLAST results from NCBI. Protein mainly expressed on cellular membrane and cytoplasma. The serum FSH and E2 level from COS group is much higher than those of control group. With scanning electron microscope, we observed that endometrial luminal epithelium were arranged well, with pinopode seen in the control group, in contrast, endometrial luminal epithelium was deranged, without any pinopode in the COS group. With the transmission electron microscope, apoptosis of endometrial epithelium increased. No significant difference of tight-junction was observed. The Western blotting result shows that FSHR expression was up-regulated significantly while expression of AQP1, AQP2, AQP8, Integrinβ3, LIF were down-regulated significantly in the COS group, and Claudin4 did not change much in the COS group.Conclusion:FSHR is expressed in human endometrial tissue and Ishikawa cells. Compared with control group, women of COS group exhibited elevated serum FSH and E2 level, up-regulation of FSHR expression in endometrium, down-regulation of AQP1, AQP2, AQP8, Integrinβ3 and LIF expression, as well as alternation of endometrial luminal epithelium morphology and apoptosis. FSH may regulate the morphology aspect and function of child-bearing period endometrium directly through FSHR.Part II The mechanism of FSH affecting child-bearing period endometrium receptivityObjective:To investigate the effect and mechanism of FSH on child-bearing period endometrium receptivity.Patients and methods:We set up the OS mouse model by injecting exogenous rFSH, and assessed the influence of exogenous rFSH on serum FSH/E2 level, endometrial AQPs, receptive factors, pregnancy rate and live birth rate. We constructed the mouse blastocyst-adhesion model, and assessed the effect of FSH/E2, PCMB (AQP inhibitor) pre-treatment on blastocyst adhesion and implantation. We measured AQPs, receptive factors (integrinβ3, LIF) gene expression level after FSH/E2 treatment by RT-Real time quantitative PCR based on in vitro cell experiment. Assessing expression of endometrial receptive factors and JAr adhesion rate after AQP2/AQP8siRNA interfering.Results:In the OS group, serum FSH/E2 level elevated significantly, endometrial expression of AQP2, AQP3 protein expression was down-regulated and FSHR was slightly up-regulated, the pregnant rate and live birth rate decreased drastically. The results from the mouse blastocyst adhesion model showed that in vitro FSH, FSH/E2 and PCMB treatment on endometrium cells lead to decreased blastocyst adhesion rate at 48hr and 72hr. In Ishikawa cells, E2 display a concentration-dependent up-regulation on expression of AQP2 and AQP8 mRNA, while FSH (0,1,3,10,30 IU/L) display a concentration-dependent and time-dependent down-regulation on AQP2, AQP8 and LIF mRNA, especially 10IU/L and 30IU/L at 48hr. After AQP2siRNA interfering, the expression of LIF and IntegrinB3 were up-regulated and OLFM1 was up-regulated. After AQP8siRNA interfering, the expression of LIF was down-regulated and OLFM1 was up-regulated. The JAr adhesion rate decreased after either AQP8 siRNA or AQP2siRNA interfering.Conclusion:Superphysiological FSH level disturb endometrium receptivity and embryo implantation through the down-regulation expression of endometrium AQPs and receptive factors.Part III The effects of high serum FSH on menopausal uterine endometrial atropy.Objective:To investigate the effect and mechanism of high FSH level during the post-menopause period on endometrial function and atrophy.Patients and methods:We measured serum FSH, LH and E2 dynamic level with chemoluminescence, and detect endometrial protein expression of FSHR, Ki67, AQP1, AQP2 and AQP8 with immunohistochemistry of pre-menopausal group and post-menopausal group. We assessed the effect of FSH on proliferation of human primary cultured endometrial glandular epithelium and Ishikawa cells with MTT assay, on cell cycles of Ishikawa cells with flow cytometry, and on endometrial expression of AQP2 and AQP8 mRNA with Real time quantitative PCR, and also assessed the influence of AC activator and inhibitor on FSH-associated effect. We constructed surgery castrated (OVX), drug castrated (GnRHa) animal model as well as SHAM group, measured their serum FSH and E2 level using RIA assay, and observed morphology aspect and apoptosis signs of endometrial cells via transmission electron microscope. We also detected the expression level of factors related to endometrial proliferation and apoptosis using Real time PCR and Western blotting.Results:The serum FSH level in post-menopausal women increased with aging from 41-50yrs to 61-70yrs, but 71-80 yrs group did not display a higher FSH level than 61-70yrs group, however, the serum E2 showed the exact opposite trend. Compared with FSHR, AQP2, and AQP1 protein expression of child-bearing period endometrium, those of post-menopausal endometrium did not change significantly, while expression of Ki67 decreased in post-menopausal group. AQP8 protein expression increased in endometrial interstitium, but decreased in endometrial gland in post-menopausal group. FSH displayed a concentration-dependent inhibition on the proliferation of primary endometrial gland epithelium, but not on that of Ishikawa cells. High concentration FSH (50IU/L,100IU/L,200IU/L,400IU/L) up-regulated the expression of AQP2 mRNA in Ishikawa cells as a concentration-dependent way, however, it can be partly antagonized by SQ22536, an AC inhibitor. Forskolin, an AC activator, treatment led to increased expression of AQP2 mRNA in Ishikawa cells. We constructed surgery castrated group(OVX, high FSH, low E2), drug castrated (GnRHa, low FSH、low E2) and sham group(SHAM, normal FSH and E2). The uterus of Mice in OVX group developed atrophy, and was observed plenty of apoptosis cells and few apoptosis body in the transmission electron scope. The extent of morphological and ultrastructural changes of GnRHa group lay between OVX group and SHAM group. The protein expression of C-FOS, C-JUN and Bcl-2 decreased in the GnRHa group, compared those in SHAM group. The protein expression of C-FOS, C-JUN and Bcl-2 decreased drastically while Caspase3 increased in OVX group, compared with SHAM group and GnRHa group.Conclusion:High serum FSH after menopause can up-regulate uterine endometrial AQP2 partly through AC pathway, inhibit the endometrial proliferation, and promote apoptosis of endometrial cells, and eventurally regulate the physiological atrophy of uterine endometrium, directly or coordinating with low serum E2.更多还原