体外受精中评价卵巢反应性的研究进展

自从世界上诞生了首例试管婴儿,体外受精与胚胎移植（IVF-ET）技术就成为了当今治疗不孕症的主要方法,其中控制性超排卵（controlled ovarian hyperstimulation,COH）是该技术中的关键步骤,在超排卵过程中,卵巢的反应性存在明显的个体差异,     

卵巢颗粒细胞凋亡率的比较

通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记发 (TUNEL)检测了卵巢颗粒细胞凋亡率。结果显示 ,妊娠组卵巢颗粒细胞凋亡率为 0 .17± 0 .0 2 ,显著低于非妊娠组 0 .3 6± 0 .0 3 ,P<0 .0 5     

顺铂对人卵巢黄素化颗粒细胞的毒性及凋亡的影响

目的研究顺铂(cisplatin,CDDP)对人卵巢黄素化颗粒细胞的毒性及凋亡的影响。方法收集体外受精—胚胎移植(IVF-ET)时的人黄素化颗粒细胞进行体外培养,采用MTT法检测不同浓度的CDDP(0、0.5、1、2.5、5mg.L-1)对人黄素化颗粒细胞生长的抑制作用;采用Hochest33258荧光染色检测细胞凋亡,流式细胞仪(flowcytometry,FCM)检测bcl-2和bax基因蛋白的表达。结果CDDP浓度≥1mg.L-1可抑制颗粒细胞的生长;1、2.5、5mg.L-1CDPP处理人黄素化颗粒细胞24h后,荧光染色可观察到明显的核浓缩、核碎裂形态。FCM检测bcl-2蛋白表达逐渐减少(P<0.05),各组间比较差别有统计学意义;bax蛋白表达逐渐增加(P<0.05),各组间比较差别有统计学意义。结论CD-DP可抑制人黄素化颗粒细胞生长,促进凋亡,其作用机制可能是通过上调bax和下调bcl-2基因表达实现的。     

多囊卵巢综合征患者卵巢颗粒细胞凋亡基因的研究

目的对多囊卵巢综合征患者进行卵巢颗粒细胞的探究,研究得出卵巢颗粒细胞凋亡的差异性并通过对该类患者卵巢的颗柱细胞凋亡的研究,为病理研究提供理论依据,同时也可以为临床治疗新方向、新途径。方法设置了实验组和对比组,将这一类的患者和对照组的进行比较。利用在研究凋亡基因上的先进的仪器和设备,将体外受精后的这一类患者以及取卵后的对照组的卵泡颗粒细胞作为研究对象,并且对它们两组分别作分析。结果试验纽中明显具有差异性表现（P〈0．05）。结论部分卵巢颗粒细胞凋亡基因呈现出紊乱状态,表现出颗粒细胞在多囊卵巢综合征病患中起十分重要的作用。     

丙戊酸钠对人卵巢黄素化颗粒细胞激素分泌及相关甾体合成酶mRNA影响

目的　丙戊酸钠 (VPA)为抗癫痫的有效药物 ,长期应用可引起类似于多囊卵巢综合征 (PCOS)生殖内分泌功能紊乱 ,本研究通过人黄素化卵巢颗粒细胞原代培养体系 ,研究VPA对黄素化颗粒细胞的激素生成及类固醇激素生成急性调节蛋白 (StAR)、胆固醇侧链裂解酶 (P4 5 0scc)和芳香化酶(P4 5 0arom)的mRNA影响。方法　收集体外受精 -胚胎移植 (IVF ET)时的人黄素化颗粒细胞进行体外培养 ,适时给于不同浓度的VPA(0、10 0、2 5 0mg·L-1)处理 ,2d后收集培养液、细胞 ,采用放射免疫方法测定颗粒细胞分泌孕酮及雌二醇的量 ,并以细胞蛋白浓度校正激素含量。采用荧光实时RT PCR (TaqMan assay)方法检测颗粒细胞中StAR、P4 5 0scc和P4 5 0arommRNA表达。结果　VPA刺激人黄素化颗粒细胞的孕酮 (P <0 0 5 )和雌二醇 (P <0 0 1)的分泌 ,且StARmRNA (P <0 0 1)、P4 5 0scc(P <0 0 5 )和P4 5 0arom(P <0 0 1)表达增加。结论　VPA可直接作用于黄素化颗粒细胞 ,通过改变StAR、P4 5 0scc和P4 5 0arommRNA的表达 ,而引起孕酮和雌二醇的分泌变化 ,而临床上长期服用VPA的癫痫妇女出现类似于PCOS患者的生殖内分泌功能紊乱可能与该药物本身作用有密切联系 ,同时提示了非遗传因素即环境因素在PCOS发病中也发挥一定作用。     

雄激素对卵巢颗粒细胞乳酸生成的作用

目的观察雄激素对小鼠卵巢颗粒细胞乳酸生成的作用,探讨多囊卵巢综合征高雄激素状态下的卵巢病理生理变化及其机制。方法体外培养小鼠颗粒细胞,分别用10-10、10-8、10-7和10-6 mol/L的睾酮刺激24h,测量培养上清液的乳酸含量。然后,加入10-8 mol/L胰岛素作用30min,检测胰岛素刺激后的乳酸生成量。结果 10-8、10-7和10-6 mol/L睾酮作用24h后,颗粒细胞乳酸生成显著下降(P<0.05);10-7 mol/L睾酮作用24h,再用胰岛素刺激30min后颗粒细胞的乳酸生成亦显著下降(P<0.05)。结论较高浓度的雄激素可抑制小鼠卵巢颗粒细胞的乳酸生成,影响其能量代谢。     

多囊卵巢综合征患者卵巢颗粒细胞COX-2和15-PGDH的表达研究

目的研究COX-2和15-PGDH在PCOS患者卵巢颗粒细胞中的表达,探讨PCOS患者排卵障碍的分子机制。方法应用荧光半定量Real—time PCR检测35例行IVF—ET的PCOS患者（实验组）及同期40例因输卵管因素和／或男方因素行IVF—ET的患者（对照组）的卵巢颗粒细胞中COX-2和15-PGDH的mRNA表达的差异。结果实验组COX-2 mRNA的相对表达7倍于对照组,差异有统计学意义（P〈0．001）;实验组15-PGDH mRNA的相对表达是对照组的0．60倍,差异有统计学意义（P〈0．05）。结论COX-2和15-PGDH的差异表达可能与PCOS发病机制相关。     

左侧卵巢颗粒细胞癌合并妊娠1例

患者，24岁，月丝周期规律，末次月经1998－03－07，妊娠18周自觉胎动，19周以后出现左倒腹痛，逐渐加重，来我院就诊，B超诊断妊娠合并卵巢囊肿。病理检查：送检组织呈卵圆形。大小16cm×12cm×10cm，包膜不清，切面有出血、坏死组织，质脆。镜下见：肿瘤主要由颗粒细胞组成，细胞有近似圆形，多角形，大小较一致，组织界限不清楚，核为卵圆或圆形，核膜薄而明显淡色，质呈细网状，细胞异型性较轻，核分裂较少，细胞周围无网织纤维包统．病理诊断：左侧卵巢颗粒细胞癌合并妊娠。讨论颗粒细胞癌属常见恶性肿瘤，但合并妊娠少见，由于颗粒细…     

颗粒细胞凋亡及线粒体膜电位与卵巢功能的相关性研究

目的探讨人卵巢黄素化颗粒细胞的凋亡率及线粒体膜电位的变化与卵巢功能及妊娠结局的相关性。方法应用流式细胞仪AnnexinV-PI双染法检测颗粒细胞凋亡,同时利用新型荧光探针JC-1测定细胞线粒体跨膜电位,分析其与卵巢功能的关系。结果黄素化颗粒细胞的凋亡率与年龄、基础FSH水平呈正相关（P〈0.01）,与获卵数,HCG日雌二醇水平及优质胚胎率呈负相关（P〈0.01）,颗粒细胞的凋亡率与Gn支数差异无统计学意义（P〉0.05）。妊娠组颗粒细胞的凋亡率低于未妊娠组（P〈0.05）。黄素化颗粒细胞线粒体膜电位与颗粒细胞凋亡率呈显著负相关（r=0.634,P〈0.01）,与优质胚胎率及妊娠率呈正相关关系（r=0.460,P〈0.05）。结论颗粒细胞的凋亡和线粒体膜电位可能影响卵巢功能及胚胎的体外发育潜能,从而影响妊娠结局。     

生长分化因子-9与超排卵周期卵巢低反应的相关性研究

目的:研究超排卵周期卵巢低反应与颗粒细胞中生长分化因子(GDF)-9表达的相关性。方法:选取行体外受精-胚胎移植(IVF-ET)的患者70例,采用本中心常规方案进行超排卵,依据取卵时获得平均直径>14mm的卵泡数分为卵巢低反应组21例,中反应组25例,高反应组24例。应用实时荧光定量聚合酶链反应(Real-timePCR)技术检测卵巢不同反应组患者卵泡壁颗粒细胞和卵丘颗粒细胞GDF-9mRNA的表达,分析其与卵巢低反应的关系。结果:卵巢不同反应组患者年龄、不孕年限、基础卵泡刺激素(FSH)、雌二醇(E2)水平及促性腺激素(Gn)用量差异均无统计学意义(P>0.05)。卵巢低反应组卵泡壁颗粒细胞和卵丘颗粒细胞的GDF-9mRNA表达均低于中反应组和高反应组(P<0.05),GDF-9在卵泡壁颗粒细胞和卵丘颗粒细胞中的表达与获卵数呈正相关(r分别为0.508、0.632,均P<0.01)。结论:超排卵周期卵巢低反应与颗粒细胞GDF-9表达有关。     

梓醇对顺铂诱导大鼠卵巢颗粒细胞损伤的保护作用

目的探讨梓醇对顺铂诱导大鼠卵巢颗粒细胞损伤的保护作用及作用机制。方法收集大鼠卵巢颗粒细胞,经原代培养后设为对照组、模型组(顺铂50 nmol/L)和梓醇50、100、200μmol/L组,各组细胞按相应方式进行处理。采用MTT法测定细胞活力;流式细胞术Annexin V/PI双染法考察细胞凋亡情况;免疫组化法观察细胞增殖能力,WB法定量细胞凋亡蛋白的分子水平。结果顺铂可显著抑制卵巢颗粒细胞活力、增殖,诱导细胞凋亡;在给予梓醇进行干预后,大鼠卵巢颗粒细胞活力增强、增殖上调,细胞凋亡减弱;经顺铂进行刺激损伤,卵巢颗粒细胞中caspase-3、caspase-9的活化蛋白表达量显著上升,而不同浓度的梓醇可显著下调细胞中cleaved caspase-3、cleaved caspase-9的蛋白表达量。结论梓醇可以改善大鼠细胞活力、提高细胞增殖和抗凋亡能力,对顺铂诱导的卵巢颗粒细胞损伤产生明显的保护作用。     

Apoptosis of rat ovarian granulosa cells by 2,5‐hexanedione in vitro and its relevant gene expression

Although n‐hexane is toxic to the ovary, the related mechanism remains unknown. Apoptosis of rat ovarian granulosa cells is thought to be one of the important reasons for ovarian toxicity. Rat ovarian granulosa cells were exposed in vitro to different concentrations of 2,5‐hexanedione (HD, a major metabolite of n‐hexane; 0, 20, 40 and 60 mmol l^?1) to observe the apoptosis, and its relevant gene expression was investigated. It was found by MTT assay that different exposure doses and different exposure time could inhibit the viability of ovarian granulosa cells. After a 12 h exposure, we found the apoptosis rate of the ovarian granulosa cells increased notably with the increase of HD dose. The HD concentration of each exposure dose could inhibit the message RNA (mRNA) expression of both bcl‐2 and xiap, and increase the mRNA expression of fasl without any influence on that of bcl‐xl, while only an HD concentration of 40 mol l^?1 could raise the mRNA expression of bax. Concentrations of HD of 40 and 60 mol l^?1 could inhibit the protein expression of NF‐κB, but only an HD concentration of 60 mol l^?1 could improve theactivation of NF‐κB. In summary, our results indicate that HD can induce the apoptosis of ovarian granulosa cell, and the occurrence of apoptosis is related to inhibition of protein expression of NF‐κB, activation of NF‐κB, upregulation of mRNA levels of fasl and bax, and downregulation of mRNA levels of xiap and bcl‐2. Copyright © 2012 John Wiley & Sons, Ltd.     

Mechanism of toxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (tcdd) in cultured human luteinized granulosa cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a significant decrease in estradiol (E₂) production when it was administered to human luteinized granulosa cells (hLGCs) in culture. We investigated the involvement of the epidermal growth factor receptor (EGFR) and protein tyrosine kinase (PTK) in this TCDD-induced toxicity. Upregulation in ¹²⁵I-EGF binding to EGFR was measured after 24 h of TCDD treatment, while downregulation in EGFR binding was measured after 72 h of TCDD treatment. Upregulation of EGFR binding was associated with a significant decrease in postnuclear (7000 × g supernatant) PTK activity, but this activity was stimulated after 72 h of TCDD treatment. TCDD altered the level of tyrosine phosphorylation in proteins with molecular weights 35, 40, 43, 45, 60, and > 205 kDa. TCDD caused a significant increase in postnuclear cAMP-dependent protein kinase (PKA) after 24 h of treatment. The actions of TCDD on protein kinases were partially blocked by the protein synthesis inhibitor, cycloheximide. On the other hand, TCDD increased nuclear PTK and decreased nuclear PKA activity. E₂ inhibited the postnuclear and nuclear activity of both PTK and PKA in control samples, but did not affect TCDD actions on either postnuclear or nuclear PTK activity. However, E₂ abolished the stimulatory effect of TCDD on PKA activity in postnuclear protein. In the presence of insulin, TCDD did not induce any additional changes in postnuclear or nuclear PTK. Forskolin (FK) alone inhibited postnuclear PTK activity and stimulated its nuclear activity. The addition of TCDD 20 min after FK resulted in an increase in postnuclear PTK, but there was little change in nuclear PTK as compared to the effect of FK alone. The stimulatory effect of TCDD on postnuclear PKA activity was enhanced by insulin and TCDD reversed the negative effect of FK, but there was no effect of either insulin or FK on the inhibition by TCDD of nuclear PKA activity. TCDD decreased the activity of MAP2 kinase and reduced the binding activity of AP-1 DNA when given alone, and also blocked the E₂ stimulation of MAP2K. These findings suggest that TCDD may interrupt the endocrine function of hLGCs through the blockage of the mitotic signal directly or indirectly through the interaction of PTK/MAP2K and PKA signaling.     

Survivin、PKA在上皮性卵巢癌组织中的表达及意义

目的探讨Survivin、蛋白激酶A（PKA）在上皮性卵巢癌组织中的表达及其与临床相关指标之间的联系。方法 48例上皮性卵巢癌组织（卵巢癌组）采用免疫组织化学SP法测定Survivin、PKA的表达强度,结合临床指标进行分析,43例上皮性卵巢良性肿瘤作为对照组。结果 Survivin在上皮性卵巢良性肿瘤组织中的阳性表达率为14.0%（6/43）,在上皮性卵巢癌组织中的阳性表达率为83.3%（40/48）;PKA在上皮性卵巢良性肿瘤组织中的阳性表达率为90.7%（39/43）,在上皮性卵巢癌组织中的阳性表达率为52.1%（25/48）,两组比较,差异有统计学意义（P〈0.01）。不同癌细胞分化程度及TNM临床分期Survivin和PKA的表达阳性率比较,差异有统计学意义（P〈0.01）。结论 Survivin在上皮性卵巢癌组织中呈高表达,PKA在上皮性卵巢癌组织中呈低表达,且与临床指标相关,提示两者不仅参与了上皮性卵巢癌的发生,且可能与肿瘤的恶性进展有关。     

Survivin基因启动子区-31C>G功能性多态与卵巢癌遗传易感性的关联研究

目的探讨Survivin基因启动子区域-31C>G多态(single nucleotide polymorphism,SNP)与中国北方汉族人群卵巢上皮性癌(epithelial ovarian cancer,EOC)发病易感性的关系。方法采用聚合酶链式反应-连接酶检测反应(polymerase chain reaction-ligase detection reac-tion,PCR-LDR)技术检测了138例EOC患者和138例健康对照个体survivin基因启动子区-31C>G多态的基因型。结果病例组三种基因型频率(28.3%、47.1%和24.6%)与对照组(17.4%、46.4%和36.2%)比较,差异有统计学意义(χ2=6.63,P=0.04),病例组C/C基因型的频率明显高于对照人群(P<0.01);与G/G基因型相比,携带C/C基因型能显著增加EOC的发病风险(OR=2.39,95%CI=1.22～4.67)。结论 Survivin基因-31C>G多态与EOC发病风险有关,-31G变异基因型可能成为预测中国北方汉族人群卵巢上皮性癌发病风险的独立危险因素。     

MicroRNA-141通过PI3K/Akt/mTOR信号通路促进PCOS卵巢颗粒细胞增殖的机制研究

目的　研究microRNA-141（miR-141）对卵巢颗粒细胞增殖的影响,并探讨其在多囊卵巢综合征（PCOS）发生发展中的作用机制。方法　采用实时荧光定量PCR检测20例PCOS患者的卵巢组织、20例对照组的正常卵巢组织、人卵巢颗粒细胞KGN及人正常卵巢上皮细胞IOSE80中miR-141 mRNA表达水平;KGN细胞分为miR-141 minics组、LY294002+miR-141 minics组、雷帕霉素（rapamycin）+miR-141 minics组、NC组、对照组。采用MTT法和平板克隆实验检测各组细胞增殖和克隆形成能力,采用Western blot法检测各组细胞磷脂酰肌醇3-激酶（PI3K）/蛋白激酶（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路相关蛋白表达水平。结果　PCOS卵巢组织miR-141 mRNA表达水平显著高于正常卵巢组织,人卵巢颗粒细胞KGN miR-141 mRNA表达水平高于人正常卵巢上皮细胞IOSE80（P＜0.05）;miR-141 minics组、LY294002+miR-141 minics组及rapamycin+miR-141 minics组miR-141 mRNA表达水平均高于NC组和对照组（P＜0.05）;LY294002+miR-141 minics组及rapamycin+miR-141 minics组转染后24、48、72、96 h OD值和克隆形成率均低于miR-141 minics组,但高于NC组和对照组（P＜0.05）;LY294002+miR-141 minics组转染后p-Akt、p-mTOR蛋白表达水平低于miR-141 minics组,但高于NC组、对照组（P＜0.05）。rapamycin+miR-141 minics组转染后p-mTOR蛋白表达水平低于miR-141 minics组,高于NC组、对照组（P＜0.05）。结论　miR-141可通过激活PI3K/Akt/mTOR信号通路促进卵巢颗粒细胞的增殖。     

Kurarinone attenuates hydrogen peroxide-induced oxidative stress and apoptosis through activating the PI3K/Akt signaling by upregulating IGF1 expression in human ovarian granulosa cells

Dysregulated follicular development may lead to follicular atresia, and this is associated with oxidative stress in granulosa cells. Kurarinone is a natural compound possessing multiple activities, including antioxidative ability. However, the role of kurarinone in granulosa cell damage during follicular atresia remains unknown. Human ovarian granulosa KGN cells were treated with hydrogen peroxide (H₂O₂) to induce cellular damage. Cytotoxicity was investigated by lactate dehydrogenase (LDH) release assay. Oxidative stress was evaluated by detection of reactive oxygen species (ROS) generation and oxidative biomarker levels. Cell apoptosis was evaluated by flow cytometry, a Cell Death Detection ELISA Kit, and a Caspase-3 Assay Kit. The downstream target and related signaling pathway were analyzed by western blotting. Kurarinone attenuated H₂O₂-induced LDH release in KGN cells. Kurarinone relieved H₂O₂-induced increase in ROS generation and malondialdehyde level as well as decrease in superoxide dismutase-1 activity and heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 mRNA levels. Kurarinone inhibited H₂O₂-induced apoptosis in KGN cells. Kurarinone targeted insulin-like growth factor 1 (IGF1) and upregulated IGF1 expression to activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling. IGF1 silencing attenuated the suppressive effects of kurarinone on H₂O₂-induced oxidative stress and apoptosis in KGN cells. In conclusion, kurarinone attenuates H₂O₂-induced oxidative stress and apoptosis in KGN cells through activating the PI3K/Akt signaling by upregulating IGF1 expression, indicating the therapeutic potential of kurarinone in follicular atresia.