本文選題：PDCD4 + 子宮內(nèi)膜異位癥�。� 參考：《山東大學(xué)》2017年碩士論文

【摘要】：實驗?zāi)康?子宮內(nèi)膜異位癥是指子宮內(nèi)膜上的腺上皮細胞和間質(zhì)細胞出現(xiàn)在除子宮腔以外的其他部位,例如盆腔、腎上腺、肺,甚至是中樞神經(jīng)系統(tǒng)。它是一種臨床常見的依賴雌激素的婦科疾病,發(fā)病率約為10%-15%,常見于育齡期女性。子宮內(nèi)膜異位癥主要發(fā)生在卵巢(96.4%),其次是軟組織(2.8%)、胃腸道(0.3%)還有泌尿道(0.2%)。子宮內(nèi)膜異位癥的臨床癥狀主要表現(xiàn)為慢性盆腔痛,發(fā)生在大約50%病人,特別是在月經(jīng)期。另外,還會導(dǎo)致不孕,發(fā)生率為30%-50%。盡管子宮內(nèi)膜異位癥是一種良性疾病,但它卻有類似于惡性腫瘤的惡性生物學(xué)行為,如遷移、侵襲以及非限制性惡性增殖。現(xiàn)發(fā)現(xiàn)子宮內(nèi)膜異位癥的發(fā)病機制與上皮向間質(zhì)轉(zhuǎn)化過程有關(guān),據(jù)報道大約有10%-15%的子宮內(nèi)膜異位癥患者的內(nèi)膜組織具有更強的侵襲力,極易侵入到相關(guān)的組織及器官上,從而產(chǎn)生一系列嚴重的臨床癥狀,而且深部浸潤型的子宮內(nèi)膜異位癥患者對激素的抵抗治療效果不佳�，F(xiàn)在被大家廣為接受的子宮內(nèi)膜異位癥的發(fā)病機制是"經(jīng)血逆流學(xué)說",但幾乎所有的育齡期女性在月經(jīng)時期都會有經(jīng)血逆流的現(xiàn)象,而發(fā)現(xiàn)子宮內(nèi)膜異位癥的發(fā)病率只有10%-15%左右。隨著越來越多的研究,我國郎景和教授又提出了"在位內(nèi)膜決定論"的假說,即由于子宮內(nèi)膜上某些基因的變化引起了子宮內(nèi)膜異位癥的發(fā)生。但是子宮內(nèi)膜異位癥的在位內(nèi)膜的細胞和分子方面的特性尚未完全闡明,需要進一步的研究,為子宮內(nèi)膜異位癥尋找有效的治療方法。PDCD4最初是作為一個促凋亡因子被發(fā)現(xiàn)的,隨著后來的研究發(fā)現(xiàn)PDCD4還可以通過抑制某些蛋白翻譯和基因轉(zhuǎn)錄來發(fā)揮抗腫瘤抑制作用。據(jù)報道在多種惡性腫瘤中PDCD4的mRNA和蛋白水平都是明顯下調(diào)的,例如肝癌、膠質(zhì)瘤、肺癌、胃癌和卵巢癌。PDCD4的過表達可以明顯誘導(dǎo)腫瘤細胞的凋亡、周期阻滯、增加腫瘤細胞對藥物的敏感性、抑制腫瘤細胞的遷移、侵襲及血管形成作用。另外,PDCD4還可作為炎性因子參與肥胖、脂肪組織炎癥、動脈粥樣硬化、LPS/D-GalN誘導(dǎo)肝損傷的發(fā)生。鑒于PDCD4在腫瘤及炎癥中的作用,我們推測PDCD4在子宮內(nèi)膜異位癥中發(fā)揮著一定的作用。在本研究中,我們檢測子宮內(nèi)膜異位癥患者的在位內(nèi)膜和異位內(nèi)膜以及對照子宮內(nèi)膜中PDCD4的表達,利用體外實驗研究PDCD4過表達或干擾后對子宮內(nèi)膜細胞HEC-1-A和Ishikawa增殖、遷移及侵襲的影響及其機制,同時在原代子宮內(nèi)膜腺上皮細胞和間質(zhì)細胞中進行驗證。本實驗的研究目的是探究PDCD4對子宮內(nèi)膜異位癥的作用,并對其機制進一步探討。通過本研究有利于解釋子宮內(nèi)膜異位癥的發(fā)病機制,尋找新的治療靶點。實驗方法:1.PDCD4在對照組子宮內(nèi)膜和子宮內(nèi)膜異位癥患者的在位內(nèi)膜及異位內(nèi)膜中的表達收集35例對照組子宮內(nèi)膜(control)及26例卵巢型子宮內(nèi)膜異位癥(EM)患者的在位及異位子宮內(nèi)膜,利用qRT-PCR、western blot及免疫組化檢測對照組子宮內(nèi)膜和子宮內(nèi)膜異位癥患者的在位及異位子宮內(nèi)膜中PDCD4 mRNA及蛋白表達水平。2.雌二醇、孕酮對子宮內(nèi)膜細胞中PDCD4表達水平的影響利用不同濃度的雌二醇(0.1nM、1nM、10nM、100nM和1000nM)刺激子宮內(nèi)膜細胞HEC-1-A和Ishikawa,然后檢測PDCD4表達水平。利用不同濃度的孕酮(0.01uM、0.1uM、1uM、10uM 和 20uM)刺激 HEC-1-A 和 Ishikawa 細胞,48h后收集蛋白,然后檢測PDCD4表達變化。然后,用10uM的孕酮刺激HEC-1-A和Ishikawa細胞不同的時間點(6h,12h,24h,36h,48h),檢測PDCD4表達水平的變化。3.PDCD4對子宮內(nèi)膜細胞的增殖、遷移及侵襲的影響我們首先在RNA水平和蛋白水平檢測子宮內(nèi)膜細胞HEC-1-A和Ishikawa細胞中PDCD4的表達水平,發(fā)現(xiàn)PDCD4在Ishikawa細胞中相對高表達,在HEC-1-A細胞中低表達。利用PDCD4過表達載體(pEGFP-C1-PDCD4)轉(zhuǎn)染HEC-1-A和Ishikawa細胞,轉(zhuǎn)染24h后利用CCK8試劑盒、克隆形成及Transwell實驗檢測PDCD4對細胞增殖、遷移及侵襲的影響,在Ishikawa細胞中用兩種siRNA轉(zhuǎn)染干擾PDCD4以后對細胞增殖、遷移及侵襲的的影響。4.PDCD4在子宮內(nèi)膜細胞中對細胞周期及凋亡的影響利用流式細胞術(shù)檢測轉(zhuǎn)染HEC-1-A和Ishikawa細胞(過表達或干擾PDCD4)48h后對細胞周期的影響及轉(zhuǎn)染24h對細胞凋亡的作用。5.PDCD4對子宮內(nèi)膜細胞自噬水平的影響利用western blot方法分別檢測過表達HEC-1-A細胞24h后的蛋白和干擾Ishikawa細胞48h后的蛋白中自噬標志物L(fēng)C3B的表達變化。6.PDCD4抑制子宮內(nèi)膜細胞的遷移和侵襲相關(guān)機制利用PDCD4過表達載體轉(zhuǎn)染HEC-1-A和Ishikawa細胞,或利用特異性針對PDCD4的兩種siRNA轉(zhuǎn)染Ishikawa細胞,轉(zhuǎn)染24h后分別收蛋白,利用western blot實驗檢測p-P65、MMP2和MMP9的表達情況,探討PDCD4對遷移及侵襲作用的機制。7.PDCD4對原代子宮內(nèi)膜腺上皮細胞及間質(zhì)細胞增殖和遷移的作用及其機制分離培養(yǎng)處于分泌期子宮內(nèi)膜的腺上皮細胞及間質(zhì)細胞。利用免疫細胞化學(xué)檢測腺上皮細胞標志物(角蛋白)及間質(zhì)細胞標志物(波形蛋白)的表達鑒別細胞類別,用CCK8試劑盒檢測PDCD4對原代腺上皮細胞及間質(zhì)細胞增殖能力的影響。然后又利用Transwell實驗檢測PDCD4對原代腺上皮細胞及間質(zhì)細胞遷移的影響。進一步利用western blot實驗檢測在原代腺上皮細胞和間質(zhì)細胞中過表達PDCD4或干擾PDCD4后p-P65、MMP2和MMP9的表達情況,探討PDCD4抑制細胞遷移的機制。實驗結(jié)果:1.在子宮內(nèi)膜異位癥患者在位內(nèi)膜及異位內(nèi)膜中PDCD4表達明顯降低利用qRT-PCR和western blot檢測結(jié)果顯示子宮內(nèi)膜異位癥患者在位子宮內(nèi)膜中PDCD4的表達低于對照子宮內(nèi)膜。免疫組化的結(jié)果顯示PDCD4表達主要位于子宮內(nèi)膜腺上皮細胞的胞漿中,而在間質(zhì)細胞中幾乎不表達。與對照組子宮內(nèi)膜相比,子宮內(nèi)膜異位癥患者在位內(nèi)膜及異位內(nèi)膜中PDCD4的表達明顯降低。同時發(fā)現(xiàn)對照組子宮內(nèi)膜中位于增生期的內(nèi)膜PDCD4表達明顯高于分泌期的內(nèi)膜,另外,與對照組處于增生期的子宮內(nèi)膜相比,子宮內(nèi)膜異位癥患者增生期的在位內(nèi)膜及異位內(nèi)膜中PDCD4的表達明顯降低。2.孕酮在子宮內(nèi)膜細胞中可以下調(diào)PDCD4表達,雌二醇對PDCD4的表達沒有明顯的影響分別用不同濃度的孕酮、雌二醇刺激子宮內(nèi)膜上皮細胞后收集蛋白,發(fā)現(xiàn)孕酮能夠明顯下調(diào)子宮內(nèi)膜細胞PDCD4蛋白的表達,但雌二醇對PDCD4的表達沒有明顯的影響;利用濃度 10uM的孕酮分別刺激細胞不同的時間也可以明顯下調(diào)PDCD4蛋白的表達。3.PDCD4抑制子宮內(nèi)膜細胞的增殖及克隆形成能力利用PDCD4過表達載體轉(zhuǎn)染HEC-1-A和Ishikawa細胞,轉(zhuǎn)染24h后檢測PDCD4對細胞增殖的影響,結(jié)果顯示轉(zhuǎn)染PDCD4過表達載體后可明顯抑制細胞增殖能力,在Ishikawa細胞中用兩種siRNA轉(zhuǎn)染細胞干擾PDCD4以后結(jié)果顯示PDCD4 siRNA轉(zhuǎn)染組細胞活力明顯高于對照siRNA轉(zhuǎn)染組。利用克隆形成實驗檢測PDCD4對細胞克隆形成能力的影響,結(jié)果顯示過表達PDCD4后可明顯抑制HEC-1-A和Ishikawa細胞的克隆形成能力,在Ishikawa細胞中用兩種siRNA轉(zhuǎn)染干擾PDCD4以后細胞的克隆形成能力明顯上調(diào)。4.PDCD4在子宮內(nèi)膜細胞中對細胞周期及凋亡沒有明顯的作用利用流式細胞術(shù)檢測PDCD4對細胞周期的影響,結(jié)果顯示無論過表達或干擾PDCD4對細胞周期都沒有明顯的影響。同時也利用流式細胞術(shù)檢測PDCD4對細胞凋亡的影響,結(jié)果顯示無論過表達或干擾PDCD4對細胞凋亡都沒有明顯的影響。5.PDCD4能夠抑制子宮內(nèi)膜細胞的自噬水平利用PDCD4過表達載體或siRNA轉(zhuǎn)染細胞,轉(zhuǎn)染48h后收蛋白,結(jié)果顯示過表達PDCD4后,LC3B表達水平明顯降低;下調(diào)細胞PDCD4后,LC3B表達水平明顯升高。6.PDCD4可以抑制子宮內(nèi)膜細胞的遷移和侵襲作用利用PDCD4過表達載體轉(zhuǎn)染細胞,轉(zhuǎn)染24h后做Transwell實驗,結(jié)果顯示在轉(zhuǎn)染PDCD4過表達載體的Ishikawa細胞遷移的細胞數(shù)明顯低于空載體轉(zhuǎn)染組,在HEC-1-A細胞中也發(fā)現(xiàn)了同樣的現(xiàn)象。利用特異性針對PDCD4的兩種siRNA轉(zhuǎn)染Ishikawa細胞,結(jié)果顯示PDCD4 siRNA轉(zhuǎn)染組細胞中遷移的細胞數(shù)目明顯的多于對照siRNA轉(zhuǎn)染組。同時利用Transwell實驗(鋪基質(zhì)膠)檢測過表達PDCD4后對細胞侵襲的影響,結(jié)果顯示PDCD4過表達后可明顯抑制HEC-1-A和Ishikawa細胞的侵襲能力,干擾PDCD4后則明顯的促進Ishikawa細胞的侵襲能力。7.PDCD4通過抑制NF-κB/MMP2/MMP9通路抑制子宮內(nèi)膜細胞的遷移和侵襲利用western blot實驗探討PDCD4抑制遷移及侵襲的機制,結(jié)果顯示在HEC-1-A細胞過表達PDCD4以后,p-P65、MMP2和MMP9的表達明顯的降低。對Ishikawa細胞干擾PDCD4以后發(fā)現(xiàn)p-P65、MMP2和MMP9的表達明顯的升高。8.PDCD4抑制原代子宮內(nèi)膜腺上皮細胞、間質(zhì)細胞的遷移和增殖為了進一步的驗證PDCD4在子宮內(nèi)膜異位癥中的作用,分離培養(yǎng)正常的位于分泌期的子宮內(nèi)膜的腺上皮細胞及間質(zhì)細胞。用CCK8試劑盒檢測PDCD4對原代腺上皮細胞及間質(zhì)細胞增殖能力的影響,結(jié)果顯示在原代細胞中過表達PDCD4后明顯抑制細胞的增殖,干擾PDCD4后明顯的促進細胞的增殖。然后又利用Transwell實驗檢測PDCD4對原代腺上皮細胞及間質(zhì)細胞遷移作用的影響,結(jié)果顯示在原代細胞中過表達PDCD4后明顯抑制細胞的遷移,干擾PDCD4后明顯的促進細胞的遷移。9.PDCD4通過抑制NF-κB/MMP2/MMP9通路抑制原代子宮內(nèi)膜腺上皮細胞、間質(zhì)細胞的遷移進一步利用western blot實驗探討在原代腺上皮細胞及間質(zhì)細胞中PDCD4抑制遷移的機制,結(jié)果顯示在原代細胞過表達PDCD4以后,p-P65、MMP2和MMP9的表達明顯的降低。在原代細胞中干擾PDCD4以后發(fā)現(xiàn)p-P65、MMP2和MMP9的表達明顯的升高。實驗結(jié)論:1.PDCD4在對照子宮內(nèi)膜的表達明顯高于子宮內(nèi)膜異位癥患者的在位內(nèi)膜和異位內(nèi)膜中的表達。2.孕酮可以下調(diào)子宮內(nèi)膜細胞中PDCD4表達水平,雌二醇對子宮內(nèi)膜細胞中PDCD4的表達沒有明顯的作用。3.PDCD4能夠明顯抑制子宮內(nèi)膜細胞的增殖及克隆形成能力,但對細胞周期及凋亡沒有明顯的作用。4.PDCD4可以抑制子宮內(nèi)膜細胞的自噬水平。5.PDCD4可以通過NF-κB/MMP2/MMP9通路抑制子宮內(nèi)膜細胞的遷移和侵襲作用。6.PDCD4可以抑制原代子宮內(nèi)膜腺上皮細胞及間質(zhì)細胞的遷移和增殖,抑制遷移的機制與NF-κB/MMP2/MMP9通路有關(guān)。創(chuàng)新性及意義:1.首次探討了PDCD4與子宮內(nèi)膜異位癥之間的關(guān)系,發(fā)現(xiàn)PDCD4在子宮內(nèi)膜異位癥患者在位內(nèi)膜及異位內(nèi)膜中的表達明顯降低。2.在體外,PDCD4可以明顯抑制子宮內(nèi)膜細胞的增殖、克隆形成能力、遷移及侵襲作用。3.我們的研究發(fā)現(xiàn)可能為子宮內(nèi)膜異位癥的治療提供一個新的治療靶點。
[Abstract]:Objective: Endometriosis is an endometriosis that refers to the appearance of epithelial cells and interstitial cells on the endometrium other than the uterine cavity, such as pelvic cavity, adrenal gland, lung, and even the central nervous system. It is a common clinical gynecologic disease dependent on estrogen, the incidence of which is about 10%-15%, common in women of childbearing age. Endometriosis occurs mainly in the ovary (96.4%), followed by soft tissue (2.8%), gastrointestinal tract (0.3%) and urinary tract (0.2%). The main clinical symptoms of endometriosis are chronic pelvic pain, occurring in about 50% patients, especially during the menstrual period. In addition, the incidence of endometriosis is 30%-50%. although endometriosis is one. It is a benign disease, but it has malignant biological behavior similar to malignant tumor, such as migration, invasion, and non restrictive malignant proliferation. It is found that the pathogenesis of endometriosis is related to the process of epithelial mesenchymal transition. It is reported that the endometrium of the patients with endometriosis of 10%-15% is more aggressive, It is very easy to intrude into related tissues and organs to produce a series of serious clinical symptoms, and the effect of deep infiltrating endometriosis in the treatment of hormone resistance is not good. The pathogenesis of endometriosis widely accepted is "blood flow theory", but almost all women of childbearing age In the period of menstruation, there is a phenomenon of reverse flow of blood, and the incidence of endometriosis is only about 10%-15%. With more and more research, Professor Lang Jinghe in our country has put forward the hypothesis of "the determinism of eutopic endometrium", that is, the occurrence of endometriosis caused by the alteration of some genes on the endometrium. But the uterus is the uterus. The cellular and molecular characteristics of intima intima in endometriosis have not yet been fully elucidated. Further research is needed. The search for effective treatment for endometriosis,.PDCD4 was initially found as a factor promoting apoptosis. As later research found that PDCD4 could also inhibit some protein translation and gene transcription. It is reported that the mRNA and protein levels of PDCD4 are obviously downregulated in various malignant tumors, such as liver cancer, glioma, lung cancer, gastric cancer and ovarian cancer.PDCD4, which can obviously induce apoptosis of tumor cells, cycle block, increase the sensitivity of tumor cells to drugs, and inhibit the migration of tumor cells. In addition, PDCD4 can also act as an inflammatory factor in obesity, adipose tissue inflammation, atherosclerosis, and LPS/D-GalN induced liver injury. In view of the role of PDCD4 in tumor and inflammation, we speculate that PDCD4 plays a role in endometriosis. In this study, we detect the intrauterine The expression of PDCD4 in endometrium and ectopic endometrium and endometrium in endometriosis patients, and the effects of PDCD4 over expression or interference on the proliferation, migration and invasion of HEC-1-A and Ishikawa in endometrium cells and its mechanism in vitro, are examined in the primary endometrium epithelial cells and interstitial cells. The purpose of the study is to explore the effect of PDCD4 on endometriosis and to further explore its mechanism. Through this study it is beneficial to explain the pathogenesis of endometriosis and find new therapeutic targets. Experimental methods: 1.PDCD4 in the eutopic and ectopic endometrium of endometriosis and endometriosis patients in the control group. The expression of eutopic and ectopic endometrium in 35 cases of control group endometrium (control) and 26 cases of ovarian endometriosis (EM) were collected, and PDCD4 mRNA and protein expression level.2. in endometriosis and endometrium endometrium in control group were detected by qRT-PCR, Western blot and immunohistochemistry. The effects of estradiol and progesterone on the expression of PDCD4 in endometrium cells were stimulated by different concentrations of estradiol (0.1nM, 1nM, 10nM, 100nM and 1000nM) to stimulate the HEC-1-A and Ishikawa in endometrium cells and then to detect the level of PDCD4 expression. Collect protein, then detect changes in PDCD4 expression. Then, 10uM progesterone is used to stimulate HEC-1-A and Ishikawa cells at different time points (6h, 12h, 24h, 36h, 48h), and the effects of.3.PDCD4 on the proliferation, migration and invasion of endometrial cells are detected by.3.PDCD4 on the endometrial cells. The expression level of PDCD4 in A and Ishikawa cells showed that PDCD4 was relatively high expression in Ishikawa cells and low expression in HEC-1-A cells. HEC-1-A and Ishikawa cells were transfected with PDCD4 overexpression vector (pEGFP-C1-PDCD4), and CCK8 reagent box was used for 24h after transfection, and the proliferation, migration and invasion of cells were cloned and tested. Effects of two kinds of siRNA transfected in Ishikawa cells on cell proliferation, migration and invasion after PDCD4 transfection, the effect of.4.PDCD4 on cell cycle and apoptosis in endometrium cells, the effect of transfection of HEC-1-A and Ishikawa cells on cell cycle after transfection of HEC-1-A and Ishikawa cells (overexpressed or interfered with PDCD4) and transfection of 24h pairs The effect of.5.PDCD4 on the autophagy level of endometrium cells using the Western blot method to detect the protein of 24h after 24h and the expression of autophagy marker LC3B in the protein that interferes with Ishikawa cells 48h, and.6.PDCD4 inhibits the migration and invasion of endometrial cells by PDCD4 over the mechanism. Transfection of HEC-1-A and Ishikawa cells by the vector or transfection of Ishikawa cells with two kinds of siRNA specific to PDCD4, and after transfection of 24h to 24h respectively. The expression of p-P65, MMP2 and MMP9 was detected by Western blot test. The mechanism of the migration and invasion of PDCD4 on the primary endometrium epithelial cells and interstitial cells was discussed. The role of proliferation and migration and the mechanism of isolation and culture of glandular epithelial cells and interstitial cells in the secretory endometrium. Using immunocytochemistry to detect the expression of glandular epithelial markers (keratin) and stromal cell markers (vimentin) to identify the cell categories, and to detect PDCD4 to the primary epithelial cells and between the epithelial cells by CCK8 kit. The effect of PDCD4 on the migration of primary glandular epithelial cells and interstitial cells was detected by Transwell experiment. The expression of p-P65, MMP2 and MMP9 in the primary glandular epithelial cells and interstitial cells was detected by Western blot test, and the inhibition of cell migration by PDCD4 was discussed. The mechanism of migration. Experimental results: 1. the expression of PDCD4 in the eutopic and ectopic endometrium of endometriosis patients was significantly reduced by qRT-PCR and Western blot detection results showed that the expression of PDCD4 in endometriosis of endometriosis patients was lower than that of the control endometrium. The immunohistochemical results showed that the expression of PDCD4 was mainly located in the Subendometrium. The expression of PDCD4 in the eutopic and ectopic endometrium of endometriosis patients was significantly lower than that in the control group. At the same time, the expression of PDCD4 in the endometrium endometrium in the control group was significantly higher than that in the secretory phase. The expression of PDCD4 in the eutopic endometrium and ectopic endometrium of endometriosis patients was significantly lower than that in the endometrium of the control group. The expression of.2. progesterone in endometrium cells could decrease the expression of PDCD4 in endometrium cells. The expression of estradiol on PDCD4 was not significantly affected by the difference of progesterone and estradiol exciton. After collecting protein in endometrium epithelial cells, it is found that progesterone can obviously reduce the expression of PDCD4 protein in endometrium cells, but estradiol has no significant influence on the expression of PDCD4, and the expression of PDCD4 protein can obviously reduce the proliferation of.3.PDCD4 cells by stimulating the expression of PDCD4 protein with the concentration of progesterone with 10uM concentration. HEC-1-A and Ishikawa cells were transfected with PDCD4 overexpression vector, and the effect of PDCD4 on cell proliferation was detected after transfection of 24h. The results showed that the transfection of PDCD4 overexpression vector could obviously inhibit cell proliferation. The result of two siRNA transfected cells in Ishikawa cells showed that PDCD4 siRNA transfection group was fine after the transfection of PDCD4. The cell viability was significantly higher than that of the control siRNA transfection group. The cloning formation test was used to detect the effect of PDCD4 on the cell clone formation ability. The results showed that the cloning and formation ability of HEC-1-A and Ishikawa cells could be inhibited obviously after the overexpression of PDCD4. The cloning ability of the cells after transfection of two kinds of siRNA in Ishikawa cells was obviously superior to that of the cells. The effect of.4.PDCD4 on cell cycle and apoptosis was not obvious in endometrial cells. Flow cytometry was used to detect the effect of PDCD4 on cell cycle. The results showed that no significant effect of PDCD4 on cell cycle was detected or interfered. The effect of PDCD4 on cell apoptosis was also detected by flow cytometry. The expression or interference of PDCD4 had no obvious effect on the apoptosis of cells..5.PDCD4 could inhibit the autophagy of endometrium cells by using PDCD4 overexpression vector or siRNA transfected cells. After transfection of 48h to the protein, the result showed that the expression level of LC3B was significantly reduced after PDCD4 expression was expressed, and the level of LC3B expression increased obviously after the reduction of PDCD4, the level of LC3B expression increased obviously.6.P. DCD4 could inhibit the migration and invasion of endometrium cells and transfect cells with PDCD4 overexpression vector and transfected 24h to do Transwell experiments. The results showed that the number of cells migrated in Ishikawa cells transfected with PDCD4 overexpression vector was significantly lower than that of the empty cell transfected group, and the same phenomenon was found in HEC-1-A fine cells. Two siRNA transfected Ishikawa cells for PDCD4 showed that the number of cells migrated in the PDCD4 siRNA transfected group was more than that of the control siRNA transfection group. Meanwhile, the effect of PDCD4 on the invasion of the cells was detected by the Transwell test (paving matrix glue). The results showed that the PDCD4 overexpression could obviously inhibit HEC-1-A and Ishikawa fine. The invasiveness of the cell, after interfering with PDCD4, obviously promotes the invasion ability of Ishikawa cells,.7.PDCD4 inhibits the migration and invasion of endometrial cells by inhibiting the NF- kappa B/MMP2/MMP9 pathway and uses western blot to explore the mechanism of PDCD4 inhibition of migration and invasion. The results show that p-P65, MMP2, and inhibits after HEC-1-A cells overexpress PDCD4. The expression of p-P65, MMP2 and MMP9 obviously increased the expression of p-P65, MMP2 and MMP9 to inhibit the primary endometrium epithelial cells, the migration and proliferation of interstitial cells in order to further verify the role of PDCD4 in endometriosis and the isolation and culture of the normal endometrium in the secretory phase. The effects of PDCD4 on the proliferation of primary glandular epithelial cells and interstitial cells were detected by CCK8 kit. The results showed that the proliferation of cells was obviously inhibited by overexpression of PDCD4 in the primary cells, and the proliferation of cells was obviously promoted after interfering with PDCD4. Then, the Transwell test was used to detect PDCD4 on the primary gland. The effects of the migration of skin cells and interstitial cells showed that over expression of PDCD4 in the primary cells significantly inhibited the migration of cells. After interfering with PDCD4, the migration of.9.PDCD4 significantly inhibited the primary endometrial glandular epithelial cells by inhibiting the NF- kappa B/MMP2/MMP9 pathway, and the migration of interstitial cells further utilized Western blot. The mechanism of the inhibition of migration of PDCD4 in the primary epithelial cells and interstitial cells was examined. The results showed that the expression of p-P65, MMP2 and MMP9 decreased obviously after the overexpression of PDCD4 in the primary cells. The expression of p-P65, MMP2 and MMP9 increased obviously after the interference of PDCD4 in the primary cells. The experimental conclusion: 1.PDCD4 in the comparison of the endometrium table .2. was significantly higher than that in endometrium and ectopic endometrium of patients with endometriosis.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R711.71

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