本文關(guān)鍵詞： 卵巢高級(jí)別漿液性腺癌 卵泡刺激素 let-7b 高遷移率族蛋白A2 上皮間質(zhì)轉(zhuǎn)化　出處：《復(fù)旦大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：目前有學(xué)者認(rèn)為,部分卵巢高級(jí)別漿液性腺癌(high grade serous cancer,HGSC)可能起源于輸卵管傘端上皮細(xì)胞(主要是輸卵管上皮分泌細(xì)胞),在各種因子或環(huán)境等的刺激下出現(xiàn)“P53印跡”,隨后出現(xiàn)輸卵管上皮內(nèi)癌(serous tubal intraepithelial carcinoma, STIC),最后成為侵襲性的漿液性腺癌[1,2]。上皮細(xì)胞-間充質(zhì)細(xì)胞轉(zhuǎn)化(epithelial-mesenchymal transitions,EMT)指上皮細(xì)胞在形態(tài)學(xué)上發(fā)生成纖維細(xì)胞或間充質(zhì)細(xì)胞表型的轉(zhuǎn)變并獲得遷移的能力,與組織重建再生、腫瘤的發(fā)生發(fā)展、轉(zhuǎn)移密切相關(guān)。有研究表明,正常上皮細(xì)胞在各種因素作用下可發(fā)生EMT,參與致瘤[3]。高遷移率蛋白A2 (High mobility group proteinA2, HMGA2)是HMGA家族成員之一,其表達(dá)水平與腫瘤的惡性程度、轉(zhuǎn)移機(jī)率及預(yù)后密切相關(guān)[4]。目前國內(nèi)外很多研究都集中在"P53印跡”出現(xiàn)之后,尤其是STIC,但對(duì)“P53印跡”出現(xiàn)之前細(xì)胞的變化研究較少,而研究該階段出現(xiàn)的事件能為掌握HGSC的發(fā)生機(jī)制、研究早期診斷及早期靶向干預(yù)奠定基礎(chǔ)。籍于此,本研究探討了FSH作用于FTE后細(xì)胞是否發(fā)生EMT,且探究HMGA2在此過程中發(fā)揮的作用。第一部分HGSC和LGSC腫瘤未累及輸卵管傘端的鑒別目的：了解在P53突變之前,HGSC中未累及的輸卵管傘上皮細(xì)胞的某些指標(biāo)是否已經(jīng)發(fā)生了變化。方法：方法選擇輸卵管未累及的卵巢上皮高級(jí)別漿液性腺癌28例,低級(jí)別漿液性腺癌24例,另取因盆底功能障礙而切除全子宮雙附件5例做對(duì)照。選取輸卵管傘端組織行免疫組化檢測�；蛲蛔冎笜�(biāo)：P53；增殖指標(biāo)：pAKT;血管內(nèi)皮生長因子：VEGF;轉(zhuǎn)移指標(biāo)：E-cadherin;侵襲指標(biāo)：MMP-2;腫瘤微環(huán)境指標(biāo)：COX-2。結(jié)果：高級(jí)別漿液性腺癌傘端的pAKT和COX-2表達(dá)明顯高于低級(jí)別漿液性腺癌(分別為61% vs 8%和71 %vs 21%,P=0.005和0.007,).低級(jí)別漿液性腺癌傘端E-Cadherin的表達(dá)明顯高于高級(jí)別漿液性腺癌(83% vs 21%,P=0.003)。 MMP-2, VEGF和p53在高、低級(jí)別漿液性腺癌中的表達(dá)差異無統(tǒng)計(jì)學(xué)意義(分別為21% vs 13%,25% vs 21%和14% vs 8%,P=0.78,0.86和0.82)。結(jié)論：高級(jí)別漿液性卵巢腺癌中未受腫瘤累及的傘端細(xì)胞在p53突變發(fā)生前其增生、粘附力及炎癥微環(huán)境就可能已經(jīng)發(fā)生了變化。第二部分卵泡刺激素FSHHGSC傘端上皮細(xì)胞發(fā)生EMT目的：了解FSH能否促進(jìn)HGSC傘端上皮細(xì)胞發(fā)生EMT方法：取前期已成功原代培養(yǎng)的低級(jí)別漿液性腺癌、高級(jí)別漿液性腺癌腫瘤未累及、P53(-)的輸卵管傘端上皮細(xì)胞。將FSH以不同濃度(0、10、20、40、 80、160mIU/mL)和不同時(shí)間(分別作用于0、12、24、48、72h)作用于FTE,觀察細(xì)胞形態(tài)變化情況。Western-blot檢測EMT標(biāo)志物E-Cadherirn、 N-Cadheriin、波形蛋白(Vimentin)、MMP-2及P53蛋白表達(dá)情況。RT-PCR檢測mRNA表達(dá)情況。結(jié)果：FSH濃度為40mIU/ml作用于HGSC的FTE細(xì)胞48h后,上皮細(xì)胞外形演變?yōu)榧忓N形纖維細(xì)胞形態(tài),EMT標(biāo)志物E-Cadherin明顯下調(diào),而N-Cadherin、Vimentin、MMP-2明顯上調(diào),且呈FSH濃度-作用時(shí)間依賴性,初步證實(shí)了發(fā)生EMT。而在LGSC未見這些變化。FSH的作用不能促進(jìn)P53蛋白表達(dá)改變。結(jié)論：FSH促進(jìn)了HGSC傘端上皮細(xì)胞發(fā)生EMT。第三部分：HMGA2介導(dǎo)FSHHGSC輸卵管傘端上皮發(fā)生EMT目的：了解]HMGA2在FSH促進(jìn)HGSC輸卵管傘端上皮發(fā)生EMT中的作用。方法：取前期已成功原代培養(yǎng)的HGSC中瘤未累及、P53(-)的輸卵管傘端上皮細(xì)胞。將FSH以不同濃度(0、0、20、40、80mIU/mL)和不同時(shí)間(分別作用于0、12、24、48、72h)作用于FTE。 Western-blot檢測HMGA2蛋白表達(dá)情況。RT-PCR檢測let-7b mRNA表達(dá)情況。FTE細(xì)胞轉(zhuǎn)染]HMGA2 shRNA后,濃度為40mIU/mL的FSH作用,觀察細(xì)胞是否發(fā)生EMT及EMT相關(guān)分子E-cadherin, MMP-2, N-cadherin,vimentin蛋白表達(dá)變化情況。觀察分子生物學(xué)方法敲除let-7b后,HMGA2蛋白表達(dá)情況。結(jié)果：FSH作用HGSC的FTE細(xì)胞后,HMGA2蛋白表達(dá)逐漸增加,let-7b mRNA表達(dá)逐漸降低,并呈濃度-時(shí)間依賴性。FTE細(xì)胞轉(zhuǎn)染HMGA2 shRNA后,FSH不能促進(jìn)FTE細(xì)胞發(fā)生EMT,且EMT相關(guān)分子E-cadherin, MMP-2, N-cadherin,vimentin蛋白表達(dá)量均未出現(xiàn)明顯變化。Anti-let-7b轉(zhuǎn)染FTE細(xì)胞后,HMGA2蛋白表達(dá)升高。結(jié)論:HMGA2在介導(dǎo)FSH促進(jìn)FTE發(fā)生EMT中起了重要作用。let-7b能夠調(diào)控HMGA2的表達(dá)。綜上所述,FSH促進(jìn)了HGSC傘端上皮細(xì)胞發(fā)生EMT,且最佳條件為濃度40mIU/ml、作用時(shí)間48h。 FSH能調(diào)控FTE中l(wèi)et-7b的下調(diào)并通過其而上調(diào)HMGA2的表達(dá)。HMGA2介導(dǎo)FSH促進(jìn)HGSC的FTE細(xì)胞發(fā)生EMT,而在LGSC中未發(fā)現(xiàn)這種現(xiàn)象。本課題的研究結(jié)果提示了FSH通過HMGA2在人類高級(jí)別卵巢漿液性腺癌中具有促進(jìn)腫瘤進(jìn)展的作用,并初步探索了相關(guān)的分子調(diào)節(jié)機(jī)制,為進(jìn)一步闡明高級(jí)別漿液性卵巢腺癌發(fā)生、發(fā)展的機(jī)制提供了新的思路,并為其的診斷、治療提供了可能的新線索。
[Abstract]:At present, some scholars believe that some high grade ovarian serousadenocarcinoma (high grade serous cancer, HGSC) may originate from fallopian tube epithelial cells (mainly secretory cells of the oviduct), in a variety of factors or environmental stimuli "P53 blot, followed by tubal intraepithelial carcinoma (serous tubal intraepithelial carcinoma, STIC), and finally become invasive serous carcinoma [1,2]. epithelial mesenchymal transformation (epithelial-mesenchymal transitions EMT) refers to the transformation of epithelial cells of fiber cells or mesenchymal cell phenotype in morphology and migration ability of regeneration and tissue reconstruction, the occurrence and development of tumor. Metastasis. Studies have shown that normal epithelial cells occurred in EMT under the influence of various factors, involved in the tumorigenic [3]. high mobility protein A2 (High mobility group proteinA2, HMGA2) Is a member of the HMGA family, its expression and tumor malignant degree, metastasis and prognosis of [4]. probability at home and abroad, many studies have focused on the "P53 mark", especially the STIC, but the P53 imprinting changes of cells before, and on the stage of the event to to grasp the mechanism of HGSC on the early diagnosis and targeted intervention to lay the foundation. Base on this, this study investigated the effect of FSH on whether FTE cells occurred after the EMT, and explore the play the role of HMGA2 in this process. The first part of the HGSC and LGSC to identify tumor uninvolved fallopian tube: understanding in P53 the mutation before, some indicators did not involve HGSC in fallopian tube epithelial cells have changed. Methods: select 28 cases of tubal ovarian high grade serous adenocarcinoma without involvement of the lower level 24 cases of serous adenocarcinoma, another for pelvic floor dysfunction and resection of the uterus double Appendix 5 cases in the control. Select the fallopian tube tissue by immunohistochemistry. Gene mutation index: P53; proliferation index: pAKT; vascular endothelial growth factor VEGF; transfer index: E-cadherin; MMP-2; tumor invasion index micro environmental indicators: COX-2. results: high grade serous adenocarcinoma of the fimbria and pAKT expression of COX-2 was significantly higher than that of low-grade serous carcinoma (61% vs 8% and 71%vs 21%, P=0.005 and 0.007). The expression of low grade serous adenocarcinoma fimbria E-Cadherin were significantly higher than those of high grade serous adenocarcinoma (83% vs 21% P=0.003). MMP-2, VEGF, and p53 in high, no statistically significant differences in the expression of low grade serous adenocarcinoma (21% vs 13%, 25% vs 21% and 14% vs 8%, P=0.78,0.86 and 0.82). Conclusion: swollen without high-grade serous ovarian adenocarcinoma Umbrella end cell tumor involved in p53 mutation occurred before the proliferation, adhesion and inflammatory microenvironment may have changed. The second part EMT to FSHHGSC follicle stimulating epithelial cells: understanding FSH fimbria can promote HGSC fimbria epithelial cells. EMT methods: Previous studies have successfully cultured lower level serous adenocarcinoma and high-grade serous ovarian cancer without involvement, P53 (-) of the fallopian tube epithelial cells. FSH at different concentrations (0,10,20,40, 80160mIU/mL) and different time (respectively for 0,12,24,48,72h) to FTE, observe the cell morphology changes of.Western-blot detection of EMT markers E-Cadherirn, N-Cadheriin, vimentin (Vimentin), the expression of MMP-2 and P53 protein expression in.RT-PCR mRNA assay. Results: the concentration of FSH is FTE 48h 40mIU/ml in HGSC cells, epithelial cells were spindle shape evolution Form of fiber cells, EMT marker E-Cadherin was down regulated, while N-Cadherin, Vimentin, MMP-2 and FSH were significantly increased, the concentration time dependence, preliminary confirmed the occurrence of EMT. in LGSC was.FSH these changes cannot promote the expression of P53 protein. Conclusion: FSH promotes HGSC fimbria epithelial cells EMT. the third part: HMGA2 FSHHGSC mediated by fallopian tube epithelial EMT Objective: to understand the]HMGA2 FSH HGSC in promoting fallopian tube epithelial EMT in vitro. Methods: Previous studies have successfully cultured HGSC tumor uninvolved, P53 (-) of the fallopian tube epithelial cells with FSH. Different concentration (0,0,20,40,80mIU/mL) and different time (respectively for 0,12,24,48,72h) to detect HMGA2 protein expression of.RT-PCR FTE. Western-blot mRNA to detect let-7b expression of.FTE cells transfected with]HMGA2 after shRNA, the concentration of FSH 40mIU/mL, EMT and EMT were observed whether related molecules E-cadherin, MMP-2, N-cadherin, vimentin protein expression changes. To observe the molecular biological methods let-7b knockdown the expression of HMGA2 protein. Results: the FSH function of HGSC FTE cells, HMGA2 protein expression increased, let-7b expression of mRNA decreased gradually, and a concentration time dependent.FTE cells transfected with HMGA2 shRNA, FSH can promote FTE cell EMT, EMT and MMP-2, related molecular E-cadherin, N-cadherin, vimentin protein expression showed no obvious change in.Anti-let-7b after transfection into FTE cells, the expression of HMGA2 protein increased. Conclusion: HMGA2 mediated FSH promote EMT play the important role of.Let-7b can regulate the expression of HMGA2 FTE. In summary, FSH promoted HGSC fimbria epithelial cells EMT, and the best conditions for the concentration of 40mIU/ml, reaction time 48h. FSH The down-regulation of let-7b in regulation of FTE and the expression of.HMGA2 mediated upregulation of HMGA2 by FSH HGSC FTE promote EMT cells, and this phenomenon was not found in LGSC. The results of this study suggest that FSH can promote tumor progression in human ovarian serous adenocarcinoma in high level by HMGA2, and preliminary exploration of the regulatory mechanism of related molecules, in order to further clarify the high-grade serous ovarian adenocarcinoma, provides new ideas for the development of the mechanism, and its diagnosis, treatment may provide a new clue.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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相關(guān)期刊論文 前1條

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本文編號(hào)：1464500