【摘要】 目的:研究IKKα下调时cyclin D1的表达变化及对体内-外蜕膜化过程和胚胎着床的影响。方法:利用8-Br-cAMP和MPA诱导hESCs体外蜕膜化。构建IKKαsiRNA转染至体外培养的hESCs中,以NC siRNA转染的hESCs作为对照组,并用8-Br-cAMP和MPA对两组进行蜕膜化干预,观察细胞形态变化。Real-time PCR、Western blotting方法分别检测两组中IKKα、特异性周期蛋白-D1(cyclin D1)、泌乳素(PRL)和胰岛素样生长因子结合蛋白-1(IGFBP1) mRNA和蛋白表达,采用流式细胞分析仪分析细胞周期。构建假孕子宫内膜蜕膜化小鼠模型并于一侧宫角注射IKKαsiRNA干预,另一侧注射NC siRNA作为对照。HE染色观察两侧宫角子宫内膜基质细胞形态和腺管变化,免疫组织化学观察IGFBP1的表达,Real-time PCR和Western blotting分别检测小鼠两侧宫角子宫内膜细胞中IKKα、cyclin D1、PRL和IGFBP1水平。将性成熟雌性昆明鼠与正常雄性昆明鼠合笼后宫角注射IKKα(CHUK)/NC siRNA,记录双侧子宫的胚胎着床位点。结果:8-Br-cAMP和MPA可在体外诱导hESCs蜕膜化。蜕膜化干预组PRL mRNA(339.00±52.25 fold)、IGFBP1mRNA(199.6±9.36 fold)、蛋白(4.86±0.30)均高于对照组。hESCs发生了G0-1到S期的阻滞。体外下调IKKα使Cyclin D1过表达从而阻碍hESCs体外蜕膜化过程。对照组hESCs发生了G0-1到S期的阻滞。假孕小鼠宫角注射蓖麻油可诱导子宫内膜蜕膜化过程。小鼠体内下调IKKα表达并影响子宫内膜蜕膜化和胚胎着床过程。结论:下调IKKα可导致cyclin D1过表达而阻碍体内-外蜕膜化过程,影响胚胎着床。更多还原

【Abstract】 Objective: To study the change of cyclin D1 expression of endometrial stromal cells by down-regulating IKKα, and investigate its influences on the process of endometrial decidualization and the embryo implantation. In vitro decidualization of hESCs was induced by 8-Br-cAMP and IKKαsiRNA were transfected into hESC vitro cultured. While the cells in negtive control group was transfected by NC siRNA. In vitro decidualization of the hESC was induced by 8-Br-cAMP and MPA, and the morphology of cells was observed. The mRNA and protein expressions of IKKα, cyclin D1, prolactin(PRL), and insulin like growth factor binding protein 1(IGFBP-1) of the cells in the two groups were measured by realtime-PCR and Western blotting. The cell cycle was tested by flow cytometer. The mice model with pseudopregnancy endometrium decidualization was constructed, and IKKα siRNA was injected in one side of uterus cornu of the mice, while NC siRNA was injected in other one side of uterine cornu as the negative control. The changes of endometrial stromal cell morphology and glandular ducts in bilateral uterine cornu of the mice were observed after HE staining. The expression of IGFBP-1 was measured by immunohistochemistry. Realtime PCR and Western blotting was used for detecting the expressions of IKKα,cyclin D1, PRL, and IGFBP-1. IKKα(CHUK)/NC siRNA was injected into the uterus cornu of the sexually mature female kunming mice after feed together with the normal male kunming mice. The embryo implantation sites in bilateral uteri of the mice were recorded. Results: 8-Br-cAMP and MPA can induce hESCs decidualization in vitro. The levels of PRL mRNA(339.00±52.25 fold), IGFBP-1 mRNA(199.6±9.36 fold),and IGFBP-1 protein(4.86±0.30)of the mice endometrial cells in decidual intervention group were significant higher than those of the mice endometrial cells in the control group.There was a blocking from G0-1 phase to S phase of hESCs of the cells.Downregulation of IKKαin vitro leaded to the overexpression of Cyclin D1 and thus hindered the decidualization of hESCs in vitro.There was a blocking from G0-1 phase to S phase of hESCs of the cells in the control group.Injection of castor oil into the uterine cornu of pseudopregnant mice had induced the decidualization of endometrium.Down-regulated IKKαexpression of cells in mice could affect endometrial decidualization and embryo implantation.Conclusion:Down regulation of IKKαexpression of endomentrial stromal cells may lead to overexpression of cyclin D1,and then inhibit the decidualization process in vivo and in vitro,which will affect embryo implantation.更多还原