| Endometriosis?EMS?is a chronic inflammatory disease of multifactorial etiology characterized by implantation and growth of endometrialglandsandstromaoutsidetheuterine cavity,with unknown causes and pathogenesis.Compared with the normal women endometrium,the genes of ectopic lesions were significantly changed.In previous studies,we found some new transcription factors that play an important role in EMS by using bioinformatics methods,which regulated the other key transcription factor,was also regulated by the other important transcription factors.Our preliminary experiments had verified that a key transcription factor Foxa2 was differentially expressed in EMs.Foxa2 is a member of the FOX family,which encodes a transcription factor that plays an important role in cell growth,proliferation,and differentiation.Studies have shown that Foxa2 has a close relationship with many sex hormone dependent tumors.Estrogen receptor 1?ERa?and androgen receptor?AR?binding to their target genes must rely on Foxa2.Foxa2 as a co-regulator of ERa and AR plays an important role in estrogen and androgen signaling pathway in sex hormone dependent diseases.Endometriosis is an estrogen dependent disease,the growth and invasion ofectopic endometrium depends on sex hormones,and sex hormones depend on its corresponding receptor binding.The level of sex hormone receptors upregulated or downregulated has a close relationship with sex hormone and cytokine.Based on the close relationship between the Foxa2 and estrogen receptor?ER?,we will investigate the expression of Foxa2 in endometriosis by q PCR westren-blot immunohistochemistry,to study the effects of Foxa2in endometrial stromal cells and its mechanisms by estrogen and progesterone stimulation and sh-RNA technology.Paper is divided into the following four parts:Part?:The expression of transcription factor Foxa2 on endometriosisObjective:To investigate the mRNA and protein expression of the Foxa2 in normal endometrium and eutopic endometrium and ectopic endometrium,decidua and villus.Methods:Endometrial samples were obtained from women with endometriosis or undergoing hysterectomy for myoma of uterus,Deciduas were obtained at the time of elective first trimester pregnancy termination of pregnancies.Using q PCR,Western blot,immunohistochemistry,immunofluorescence,the expression and cellular localization of transcription factor Foxa2 in normal endometrium and eutopic endometrium and endometriosis endometriosis,decidua,villus was analyzed.Results:Immunofluorescence and immunohistochemistry showed that the expression of Foxa2 in endometrium from normal women was higher than ectopic endometrium that from patients with endometriosis.q-PCR and western blotting analysis showed that the expression of Foxa2in endometrium>eutopic endometrium>ectopic endometrium?P<0.05?,in secretory phase endometrium from normal women was significantly higher than that from proliferative phase?P<0.05?,no significant difference between the proliferative phase and the secretory phase of eutopic endometrium and ectopic endometrium from endometriosis?P>0.05?.With the increase of the time of pregnancy,the expression of Foxa2 in the decidua and villus of the first trimester of pregnancy increased significantly.Conclusion:1.The expression of Foxa2 in eutopic endometrium and ectopic endometrium from endometriosis patients was significantly lower than endometrium that from normal women.2.The expression of Foxa2 in the secretory phase endometrium was higher than the proliferative phase endometrium from the normal women.But there were no significantly difference between secretion phase and proliferation phase of eutopic and ectopic endometrium from endometriosis patents.3.The expression of Foxa2 in the decidua and villus from the first trimester of pregnancy were significantly increased with the increase of the days of gestation.Part?:The effects of estrogen and progesterone on the expression of Foxa2 in human endometrial stromal cellsObjective:To investigate the effects of estrogen and progesterone on the expression of Foxa2 in human endometrial stromal cells.Methods:Cultured human endometrial stromal cells in vitro.Using different concentrations of estrogen,progesterone,estrogen and progesterone combined to stimulate endometrial stromal cells,detected the expressions of mRNA Foxa2 in the cells of each group.Results:1.Cell culture and identification:immunocytochemical staining showed that human endometrial stromal cells vimentin was positive and cytokeratin was negative.Numeration vimentin staining positive cells number showed that the purity of endometrial stromal cell was 90%-95%.2.QPCR showed that E2 up-regulated the expression of Foxa2.The expression of Foxa2 was up-regulated when the estrogen was increased from 10-9mol/L to 10-8mol/L,but continued to increase the estrogen concentration(10-7mol/L),the expression of Foxa2 was down regulated.High concentration of progesterone down-regulated the expression of Foxa2,but at low concentrations,the expression of Foxa2was slightly higher than the control group.When the estrogen and progesterone were combined to stimulate the cells,the expression of Foxa2was higher than that of the control group,but lower than that of the estrogen group.Conclusion:Estrogen and progesterone both affect the expression of Foxa2 in endometrial stromal cells,estrogen can up regulate the expression of Foxa2,and its expression varies with the change of E2 concentration.High concentration of progesterone down-regulate the expression of Foxa2,and the inhibitory effect was not obvious with the low concentration of progesterone.Part?:The effect of Foxa2 on the cell biological behavior of endometrial stromal cellsObjective:To investigate the role of Foxa2 in the cell proliferation,cell adhesion,cell migration and cell invasion of endometrial stromal cells.Methods:SiRNA Foxa2 was synthesized and screened,and transfected into human endometrial stromal cells.The effects of Foxa2 on proliferation,apoptosis,migration and invasion of endometrial stromal cells were detected by MTT,flow cytometry and Transwell.With estrogen and progesterone stimulation,once again through the MTT,flow cytometry,scratches,Transwell to detect the function of Foxa2.Results:After the transfection of plasmid,QPCR and Western-blot showed that the expression of Foxa2 was significantly down regulated.The down-regulation of Foxa2 can increase cell proliferation,migration,invasion,reduce cell apoptosis.The effects of siRNA Foxa2 combined with estrogen to enhance cell proliferation,migration,invasion and reduce apoptosis was more significantly than that estrogen alone group.With the treated by progesterone,cell proliferation,migration,invasion and apoptosis were not significantly different from the control group.Conclusion:Down-regulation of Foxa2,endometrial stromal cell proliferation,migration,invasion was significantly enhanced,while the apoptosis was decreased.Estrogen and Foxa2 si-RNA have synergistic effect,can further enhance the endometrial stromal cell proliferation,migration,invasion ability.Part?:The mechanism of the down-regulation of Foxa2 in endometriosisObjective:To explore the mechanism of the down-regulation of Foxa2in endometriosis.Methods:Cultured human endometrial stromal cells and treated with TNF-?,Using immunofluorescence to examinate the expression of Foxa2and it's intracellular localization.Western Blot detected the expression of IKK-?and Foxa2 in endometrial stromal cells which treated with TNF-?.The expression of Foxa2 in endometrial stromal cells which treated with TNF-?and IKK-?specific blocking agents was analyzed by western blot.Results:In the control group,Foxa2 was expressed in the nucleus,and the expression was less in the cytoplasm.After 24 hours of treatment with TNF-?,Foxa2 protein mainly located in cytoplasm and cell membrane surrounding.The expression of Foxa2 in endometrial stromal cells that treated with TNF-?decreased,while the expression of IKK-?increased.After 24 hours treated with TNF-?and IKK-?protein specific blocking agent,the expression of Foxa2 in endometrial stromal cells was no significant difference compared with the control group.Conclusion:After treated with TNF-?,Foxa2 lost its nuclear localization ability and transferred into the cytoplasm,and the expression of Foxa2 in cytoplasm was down regulated.TNF-?down-regulate the expression of Foxa2 must rely on IKK-?. |
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