PDCD4在子宫内膜异位症中作用及其机制研究

发布时间：2018-05-29 01:26

本文选题：PDCD4 + 子宫内膜异位症　；参考：《山东大学》2017年硕士论文

【摘要】：实验目的:子宫内膜异位症是指子宫内膜上的腺上皮细胞和间质细胞出现在除子宫腔以外的其他部位,例如盆腔、肾上腺、肺,甚至是中枢神经系统。它是一种临床常见的依赖雌激素的妇科疾病,发病率约为10%-15%,常见于育龄期女性。子宫内膜异位症主要发生在卵巢(96.4%),其次是软组织(2.8%)、胃肠道(0.3%)还有泌尿道(0.2%)。子宫内膜异位症的临床症状主要表现为慢性盆腔痛,发生在大约50%病人,特别是在月经期。另外,还会导致不孕,发生率为30%-50%。尽管子宫内膜异位症是一种良性疾病,但它却有类似于恶性肿瘤的恶性生物学行为,如迁移、侵袭以及非限制性恶性增殖。现发现子宫内膜异位症的发病机制与上皮向间质转化过程有关,据报道大约有10%-15%的子宫内膜异位症患者的内膜组织具有更强的侵袭力,极易侵入到相关的组织及器官上,从而产生一系列严重的临床症状,而且深部浸润型的子宫内膜异位症患者对激素的抵抗治疗效果不佳。现在被大家广为接受的子宫内膜异位症的发病机制是"经血逆流学说",但几乎所有的育龄期女性在月经时期都会有经血逆流的现象,而发现子宫内膜异位症的发病率只有10%-15%左右。随着越来越多的研究,我国郎景和教授又提出了"在位内膜决定论"的假说,即由于子宫内膜上某些基因的变化引起了子宫内膜异位症的发生。但是子宫内膜异位症的在位内膜的细胞和分子方面的特性尚未完全阐明,需要进一步的研究,为子宫内膜异位症寻找有效的治疗方法。PDCD4最初是作为一个促凋亡因子被发现的,随着后来的研究发现PDCD4还可以通过抑制某些蛋白翻译和基因转录来发挥抗肿瘤抑制作用。据报道在多种恶性肿瘤中PDCD4的mRNA和蛋白水平都是明显下调的,例如肝癌、胶质瘤、肺癌、胃癌和卵巢癌。PDCD4的过表达可以明显诱导肿瘤细胞的凋亡、周期阻滞、增加肿瘤细胞对药物的敏感性、抑制肿瘤细胞的迁移、侵袭及血管形成作用。另外,PDCD4还可作为炎性因子参与肥胖、脂肪组织炎症、动脉粥样硬化、LPS/D-GalN诱导肝损伤的发生。鉴于PDCD4在肿瘤及炎症中的作用,我们推测PDCD4在子宫内膜异位症中发挥着一定的作用。在本研究中,我们检测子宫内膜异位症患者的在位内膜和异位内膜以及对照子宫内膜中PDCD4的表达,利用体外实验研究PDCD4过表达或干扰后对子宫内膜细胞HEC-1-A和Ishikawa增殖、迁移及侵袭的影响及其机制,同时在原代子宫内膜腺上皮细胞和间质细胞中进行验证。本实验的研究目的是探究PDCD4对子宫内膜异位症的作用,并对其机制进一步探讨。通过本研究有利于解释子宫内膜异位症的发病机制,寻找新的治疗靶点。实验方法:1.PDCD4在对照组子宫内膜和子宫内膜异位症患者的在位内膜及异位内膜中的表达收集35例对照组子宫内膜(control)及26例卵巢型子宫内膜异位症(EM)患者的在位及异位子宫内膜,利用qRT-PCR、western blot及免疫组化检测对照组子宫内膜和子宫内膜异位症患者的在位及异位子宫内膜中PDCD4 mRNA及蛋白表达水平。2.雌二醇、孕酮对子宫内膜细胞中PDCD4表达水平的影响利用不同浓度的雌二醇(0.1nM、1nM、10nM、100nM和1000nM)刺激子宫内膜细胞HEC-1-A和Ishikawa,然后检测PDCD4表达水平。利用不同浓度的孕酮(0.01uM、0.1uM、1uM、10uM 和 20uM)刺激 HEC-1-A 和 Ishikawa 细胞,48h后收集蛋白,然后检测PDCD4表达变化。然后,用10uM的孕酮刺激HEC-1-A和Ishikawa细胞不同的时间点(6h,12h,24h,36h,48h),检测PDCD4表达水平的变化。3.PDCD4对子宫内膜细胞的增殖、迁移及侵袭的影响我们首先在RNA水平和蛋白水平检测子宫内膜细胞HEC-1-A和Ishikawa细胞中PDCD4的表达水平,发现PDCD4在Ishikawa细胞中相对高表达,在HEC-1-A细胞中低表达。利用PDCD4过表达载体(pEGFP-C1-PDCD4)转染HEC-1-A和Ishikawa细胞,转染24h后利用CCK8试剂盒、克隆形成及Transwell实验检测PDCD4对细胞增殖、迁移及侵袭的影响,在Ishikawa细胞中用两种siRNA转染干扰PDCD4以后对细胞增殖、迁移及侵袭的的影响。4.PDCD4在子宫内膜细胞中对细胞周期及凋亡的影响利用流式细胞术检测转染HEC-1-A和Ishikawa细胞(过表达或干扰PDCD4)48h后对细胞周期的影响及转染24h对细胞凋亡的作用。5.PDCD4对子宫内膜细胞自噬水平的影响利用western blot方法分别检测过表达HEC-1-A细胞24h后的蛋白和干扰Ishikawa细胞48h后的蛋白中自噬标志物LC3B的表达变化。6.PDCD4抑制子宫内膜细胞的迁移和侵袭相关机制利用PDCD4过表达载体转染HEC-1-A和Ishikawa细胞,或利用特异性针对PDCD4的两种siRNA转染Ishikawa细胞,转染24h后分别收蛋白,利用western blot实验检测p-P65、MMP2和MMP9的表达情况,探讨PDCD4对迁移及侵袭作用的机制。7.PDCD4对原代子宫内膜腺上皮细胞及间质细胞增殖和迁移的作用及其机制分离培养处于分泌期子宫内膜的腺上皮细胞及间质细胞。利用免疫细胞化学检测腺上皮细胞标志物(角蛋白)及间质细胞标志物(波形蛋白)的表达鉴别细胞类别,用CCK8试剂盒检测PDCD4对原代腺上皮细胞及间质细胞增殖能力的影响。然后又利用Transwell实验检测PDCD4对原代腺上皮细胞及间质细胞迁移的影响。进一步利用western blot实验检测在原代腺上皮细胞和间质细胞中过表达PDCD4或干扰PDCD4后p-P65、MMP2和MMP9的表达情况,探讨PDCD4抑制细胞迁移的机制。实验结果:1.在子宫内膜异位症患者在位内膜及异位内膜中PDCD4表达明显降低利用qRT-PCR和western blot检测结果显示子宫内膜异位症患者在位子宫内膜中PDCD4的表达低于对照子宫内膜。免疫组化的结果显示PDCD4表达主要位于子宫内膜腺上皮细胞的胞浆中,而在间质细胞中几乎不表达。与对照组子宫内膜相比,子宫内膜异位症患者在位内膜及异位内膜中PDCD4的表达明显降低。同时发现对照组子宫内膜中位于增生期的内膜PDCD4表达明显高于分泌期的内膜,另外,与对照组处于增生期的子宫内膜相比,子宫内膜异位症患者增生期的在位内膜及异位内膜中PDCD4的表达明显降低。2.孕酮在子宫内膜细胞中可以下调PDCD4表达,雌二醇对PDCD4的表达没有明显的影响分别用不同浓度的孕酮、雌二醇刺激子宫内膜上皮细胞后收集蛋白,发现孕酮能够明显下调子宫内膜细胞PDCD4蛋白的表达,但雌二醇对PDCD4的表达没有明显的影响;利用浓度 10uM的孕酮分别刺激细胞不同的时间也可以明显下调PDCD4蛋白的表达。3.PDCD4抑制子宫内膜细胞的增殖及克隆形成能力利用PDCD4过表达载体转染HEC-1-A和Ishikawa细胞,转染24h后检测PDCD4对细胞增殖的影响,结果显示转染PDCD4过表达载体后可明显抑制细胞增殖能力,在Ishikawa细胞中用两种siRNA转染细胞干扰PDCD4以后结果显示PDCD4 siRNA转染组细胞活力明显高于对照siRNA转染组。利用克隆形成实验检测PDCD4对细胞克隆形成能力的影响,结果显示过表达PDCD4后可明显抑制HEC-1-A和Ishikawa细胞的克隆形成能力,在Ishikawa细胞中用两种siRNA转染干扰PDCD4以后细胞的克隆形成能力明显上调。4.PDCD4在子宫内膜细胞中对细胞周期及凋亡没有明显的作用利用流式细胞术检测PDCD4对细胞周期的影响,结果显示无论过表达或干扰PDCD4对细胞周期都没有明显的影响。同时也利用流式细胞术检测PDCD4对细胞凋亡的影响,结果显示无论过表达或干扰PDCD4对细胞凋亡都没有明显的影响。5.PDCD4能够抑制子宫内膜细胞的自噬水平利用PDCD4过表达载体或siRNA转染细胞,转染48h后收蛋白,结果显示过表达PDCD4后,LC3B表达水平明显降低;下调细胞PDCD4后,LC3B表达水平明显升高。6.PDCD4可以抑制子宫内膜细胞的迁移和侵袭作用利用PDCD4过表达载体转染细胞,转染24h后做Transwell实验,结果显示在转染PDCD4过表达载体的Ishikawa细胞迁移的细胞数明显低于空载体转染组,在HEC-1-A细胞中也发现了同样的现象。利用特异性针对PDCD4的两种siRNA转染Ishikawa细胞,结果显示PDCD4 siRNA转染组细胞中迁移的细胞数目明显的多于对照siRNA转染组。同时利用Transwell实验(铺基质胶)检测过表达PDCD4后对细胞侵袭的影响,结果显示PDCD4过表达后可明显抑制HEC-1-A和Ishikawa细胞的侵袭能力,干扰PDCD4后则明显的促进Ishikawa细胞的侵袭能力。7.PDCD4通过抑制NF-κB/MMP2/MMP9通路抑制子宫内膜细胞的迁移和侵袭利用western blot实验探讨PDCD4抑制迁移及侵袭的机制,结果显示在HEC-1-A细胞过表达PDCD4以后,p-P65、MMP2和MMP9的表达明显的降低。对Ishikawa细胞干扰PDCD4以后发现p-P65、MMP2和MMP9的表达明显的升高。8.PDCD4抑制原代子宫内膜腺上皮细胞、间质细胞的迁移和增殖为了进一步的验证PDCD4在子宫内膜异位症中的作用,分离培养正常的位于分泌期的子宫内膜的腺上皮细胞及间质细胞。用CCK8试剂盒检测PDCD4对原代腺上皮细胞及间质细胞增殖能力的影响,结果显示在原代细胞中过表达PDCD4后明显抑制细胞的增殖,干扰PDCD4后明显的促进细胞的增殖。然后又利用Transwell实验检测PDCD4对原代腺上皮细胞及间质细胞迁移作用的影响,结果显示在原代细胞中过表达PDCD4后明显抑制细胞的迁移,干扰PDCD4后明显的促进细胞的迁移。9.PDCD4通过抑制NF-κB/MMP2/MMP9通路抑制原代子宫内膜腺上皮细胞、间质细胞的迁移进一步利用western blot实验探讨在原代腺上皮细胞及间质细胞中PDCD4抑制迁移的机制,结果显示在原代细胞过表达PDCD4以后,p-P65、MMP2和MMP9的表达明显的降低。在原代细胞中干扰PDCD4以后发现p-P65、MMP2和MMP9的表达明显的升高。实验结论:1.PDCD4在对照子宫内膜的表达明显高于子宫内膜异位症患者的在位内膜和异位内膜中的表达。2.孕酮可以下调子宫内膜细胞中PDCD4表达水平,雌二醇对子宫内膜细胞中PDCD4的表达没有明显的作用。3.PDCD4能够明显抑制子宫内膜细胞的增殖及克隆形成能力,但对细胞周期及凋亡没有明显的作用。4.PDCD4可以抑制子宫内膜细胞的自噬水平。5.PDCD4可以通过NF-κB/MMP2/MMP9通路抑制子宫内膜细胞的迁移和侵袭作用。6.PDCD4可以抑制原代子宫内膜腺上皮细胞及间质细胞的迁移和增殖,抑制迁移的机制与NF-κB/MMP2/MMP9通路有关。创新性及意义:1.首次探讨了PDCD4与子宫内膜异位症之间的关系,发现PDCD4在子宫内膜异位症患者在位内膜及异位内膜中的表达明显降低。2.在体外,PDCD4可以明显抑制子宫内膜细胞的增殖、克隆形成能力、迁移及侵袭作用。3.我们的研究发现可能为子宫内膜异位症的治疗提供一个新的治疗靶点。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.71

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