本文選題：宮頸癌　切入點(diǎn)：顆粒蛋白前體　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：宮頸癌作為最常見的女性生殖系統(tǒng)惡性腫瘤之一,已成為15-44歲女性的第二殺手,僅次于乳腺癌。2012年全球新發(fā)宮頸癌超過50萬例,其中85%以上發(fā)生在發(fā)展中國家,死亡病例超過26萬例。大量證據(jù)證明高危型HPV感染是宮頸癌致癌機(jī)制中的關(guān)鍵因素,但僅有很少部分的HPV女性感染者發(fā)展為宮頸癌,提示其他一些因素促進(jìn)了宮頸癌的進(jìn)展。癌細(xì)胞惡性進(jìn)展的主要特征包括:生長信號(hào)自給、生長抑制信號(hào)耐受、逃脫程序性死亡、無限的復(fù)制潛能、持續(xù)的血管生成、組織侵襲與轉(zhuǎn)移。轉(zhuǎn)移是腫瘤進(jìn)展的終極且最兇險(xiǎn)階段,超過90%的癌癥患者死于轉(zhuǎn)移而非原發(fā)性腫瘤。宮頸癌的進(jìn)展階段性明顯,包括正常宮頸、宮頸上皮內(nèi)瘤變、原位癌、局部侵襲性癌和遠(yuǎn)處轉(zhuǎn)移癌。早期宮頸癌(Ⅰa期-Ⅱa期)患者一般預(yù)后較好,但其中1/3死于轉(zhuǎn)移和復(fù)發(fā),晚期特別是有遠(yuǎn)處器官或淋巴結(jié)轉(zhuǎn)移的宮頸癌(Ⅱb期-Ⅳ期)患者則難以達(dá)到根治目的。盆腔淋巴結(jié)轉(zhuǎn)移是宮頸癌的重要擴(kuò)散途徑,有無淋巴結(jié)轉(zhuǎn)移直接影響患者的預(yù)后。探討宮頸癌的侵襲和轉(zhuǎn)移的機(jī)制對(duì)增強(qiáng)療效、防止復(fù)發(fā)和提高患者生存質(zhì)量具有重大意義。癌細(xì)胞轉(zhuǎn)移分為多個(gè)步驟,包括癌細(xì)胞從原位腫瘤脫離,侵入周圍組織,內(nèi)滲入血管或淋巴管,隨血流或淋巴系統(tǒng)散播,最終外滲并在遠(yuǎn)端器官生長。每一步驟均需要獨(dú)特的分子程序的參與,其中癌細(xì)胞黏附與遷移的調(diào)控和細(xì)胞骨架性質(zhì)發(fā)揮重要的作用。此外,腫瘤轉(zhuǎn)移的起始與癌細(xì)胞形態(tài)學(xué)改變,即上皮-間質(zhì)轉(zhuǎn)換(epithelial-mesenchymal transition,EMT)有關(guān)。顆粒蛋白前體(progranulin,PGRN)是一種新型多功能生長因子,具有促進(jìn)細(xì)胞增殖、生存、遷移、抗炎等功能特點(diǎn),參與多種重要的生理和疾病進(jìn)程。PGRN最初被鑒定為癌細(xì)胞的生長因子,在許多腫瘤中高水平表達(dá),與腫瘤的不良預(yù)后相關(guān),并介導(dǎo)乳腺癌、卵巢癌、前列腺癌、膀胱癌和肝癌等的發(fā)生與發(fā)展。PGRN與癌細(xì)胞侵襲轉(zhuǎn)移關(guān)系密切,可增強(qiáng)成纖維細(xì)胞的運(yùn)動(dòng)能力,驅(qū)動(dòng)SW13和MCF-7細(xì)胞穿過基質(zhì)膠濾膜,刺激乳腺癌細(xì)胞產(chǎn)生血管內(nèi)皮生長因子和血管生成素,并可促進(jìn)癌細(xì)胞基質(zhì)金屬蛋白酶的產(chǎn)生等,而近期的研究發(fā)現(xiàn)PGRN可通過增強(qiáng)EMT程序介導(dǎo)卵巢癌細(xì)胞的遷移與侵襲。我們的前期研究首次報(bào)道了 PGRN作為腫瘤微環(huán)境因素在宮頸癌中高表達(dá),并促進(jìn)宮頸癌細(xì)胞惡性增殖與轉(zhuǎn)化。然而,PGRN在宮頸癌細(xì)胞的遷移與侵襲中的功能,以及PGRN對(duì)癌細(xì)胞遷移、侵襲關(guān)鍵機(jī)制的調(diào)控作用仍未見報(bào)道。目的:本論文旨在明確PGRN在宮頸癌細(xì)胞運(yùn)動(dòng)、遷移與侵襲中的功能;分析PGRN對(duì)宮頸癌細(xì)胞EMT和細(xì)胞骨架重排的影響,探索PGRN促進(jìn)宮頸癌細(xì)胞遷移的分子機(jī)制。方法:利用重組人 PGRN(recombinant human PGRN,rhPGRN)處理宮頸癌細(xì)胞系(HeLa、SiHa)使用劃痕修復(fù)實(shí)驗(yàn)、transwell遷移和侵襲實(shí)驗(yàn),檢測PGRN對(duì)宮頸癌細(xì)胞運(yùn)動(dòng)、遷移和侵襲能力的影響;免疫印跡法分析EMT相關(guān)蛋白譜表達(dá)的變化;比較正常宮頸組織和宮頸癌組織中PGRN和p-mTOR的蛋白水平表達(dá)變化及其相關(guān)性,檢測mTOR下游蛋白的激活及PGRN調(diào)控mTOR的分子機(jī)制;采用F-actin免疫染色和激光共聚焦顯微鏡觀察,表征宮頸癌細(xì)胞遷移相關(guān)細(xì)胞結(jié)構(gòu)的改變;蛋白水平分析細(xì)胞骨架重排相關(guān)LIMK/cofilin信號(hào)途徑的活化水平及FAK等信號(hào)分子的磷酸化,并分析PGRN對(duì)Rho小GTP酶活性的影響;利用FAK、Rho小GTP酶等的抑制劑,分析PGRN調(diào)控宮頸癌細(xì)胞遷移與侵襲對(duì)FAK和Rho小GTP酶的依賴性。結(jié)果:rhPGRN能夠增強(qiáng)宮頸癌細(xì)胞運(yùn)動(dòng)、遷移和侵襲能力;PGRN對(duì)宮頸癌細(xì)胞EMT的影響較小,但可促進(jìn)細(xì)胞骨架重排,表現(xiàn)為壓力纖維增強(qiáng)、板狀偽足和絲狀偽足增多等;PGRN通過TNFR2激活mTOR信號(hào)通路,且在宮頸癌組織中與p-mTOR具有相關(guān)性,雷帕霉素抑制mTOR降低了宮頸癌細(xì)胞的遷移與侵襲;PGRN可激活細(xì)胞骨架重排相關(guān)的LIMK/cofilin信號(hào)途徑,同時(shí)促進(jìn)FAK的磷酸化水平增強(qiáng);PGRN通過mTOR信號(hào)途徑增強(qiáng)宮頸癌細(xì)胞中RhoA和Racl的活性,PGRN介導(dǎo)的宮頸癌細(xì)胞遷移和侵襲依賴于FAK、Racl和RhoA等的活化。結(jié)論:本論文證實(shí)了 PGRN具有促進(jìn)宮頸癌細(xì)胞運(yùn)動(dòng)、遷移與侵襲的能力,首次發(fā)現(xiàn)PGRN主要通過mTOR信號(hào)途徑促進(jìn)細(xì)胞骨架重排能力調(diào)控癌細(xì)胞遷移,并深入分析了 FAK和mTOR活化的Rho小GTP酶介導(dǎo)的信號(hào)途徑在其中的重要作用。這一研究發(fā)現(xiàn)了 PGRN促進(jìn)宮頸癌侵襲轉(zhuǎn)移,從而參與宮頸癌惡性進(jìn)展的新機(jī)制,為控制宮頸癌侵襲轉(zhuǎn)移提供了新的靶點(diǎn);本研究首次提出PGRN對(duì)細(xì)胞骨架重排的調(diào)控作用,并將PGRN介導(dǎo)的FAK與Rho/LIMK/cofilin信號(hào)途徑聯(lián)系起來,為PGRN信號(hào)通路及生物學(xué)功能研究提供了新的視角。
[Abstract]:Cervical cancer is one of the most common malignant tumor of the female reproductive system, has become a 15-44 year old female second killer after breast cancer.2012 global new cervical cancer more than 500 thousand cases, of which more than 85% of deaths occur in developing countries, more than 260 thousand cases. A lot of evidence of high-risk HPV infection is the key factor for cervical cancer carcinogenesis the only small part of HPV infected women develop cervical cancer, suggesting that some other factors to promote the progression of cervical cancer. Key features include: the malignant progression of cancer cell growth inhibitory signal signal self-sufficiency, tolerance, evasion of programmed death, unlimited replicative potential, sustained angiogenesis, tissue invasion and transfer. Transfer is the ultimate and most dangerous tumor progression stage, more than 90% of cancer patients died of metastasis and primary tumor. Advanced stage cervical cancer significantly, including Normal cervix, cervical intraepithelial neoplasia and carcinoma in situ, local invasive carcinoma and metastasis carcinoma. Early cervical cancer (stage I a - II a period) the general prognosis is better, but the 1/3 died of recurrence and metastasis, especially in advanced distant organs or lymph node metastasis of cervical cancer (stage II B - IV) patients is difficult to achieve the purpose of cure. Pelvic lymph node metastasis is an important way to spread of cervical cancer, lymph node metastasis directly affect the prognosis of patients of cervical cancer. The invasion and metastasis mechanism to enhance the curative effect, prevent recurrence and is of great significance to improve the quality of life of patients. The metastasis of cancer cells is divided into multiple steps, including cancer cells from the tumor in situ, invade surrounding tissue, infiltrated the blood or lymphatic vessels, with blood or lymphatic system spread, eventually extravasation and growth in the distal organs. Each step requires the participation of unique molecular programs, including cancer The regulation of cytoskeleton and cell adhesion and migration properties play an important role. In addition, the initial change of tumor metastasis and tumor cell morphology, namely epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT). Progranulin (progranulin, PGRN) is a multifunctional growth factor, can promote cell proliferation survival, migration, anti-inflammatory and other features, is involved in many important physiological and disease progression of.PGRN was originally identified as a growth factor in cancer cells, the expression level is high in many cancers, poor prognosis associated with tumor, and mediated breast cancer, ovarian cancer, prostate cancer, metastasis and invasion of bladder cancer and hepatocellular carcinoma the occurrence and development of.PGRN and cancer cells, can enhance the ability of motion of fiber cells, drive SW13 and MCF-7 cells through Matrigel membrane, stimulated breast cancer cells and vascular endothelial growth factor production Ang, and can promote cancer cell matrix metalloproteinase production, but recent studies have found that PGRN can be enhanced by the invasion and migration of ovarian cancer cells mediated by EMT program. Our previous study reports for the first time PGRN as the tumor microenvironment factors in cervical cancer and high expression, and promote malignant cervical cancer cells cell proliferation and transformation. However, PGRN on migration and invasion of cervical cancer cells and the function of PGRN on cancer cell migration, invasion and regulation of key mechanism has not been reported. Objective: the aim of this paper is to clear the PGRN movement in the cervical cancer cell migration and invasion in function; analysis on the effect of PGRN on cervical cancer cell line EMT and cytoskeletal rearrangement, to explore the molecular mechanism of PGRN promoting cervical cancer cell migration. Methods: using recombinant human PGRN (recombinant human PGRN, rhPGRN) treatment of cervical cancer cell lines (HeLa, SiHa) using scratch repair After experiment, the invasion and migration of experimental Transwell, detection of PGRN on cervical cancer cells, effects of migration and invasion; analysis of the changes of the expression of EMT related protein immunoblotting; protein levels of PGRN and p-mTOR in normal cervical tissue and cervical cancer tissue expression changes and correlation detection, molecular mechanism of activation and regulation of PGRN mTOR mTOR downstream proteins; using the microscope F-actin staining and confocal laser, related to the change of cell structure characterization of cervical cancer cell migration; protein level analysis and activation level of FAK signaling molecules cytoskeleton related LIMK/ cofilin signaling pathway, phosphorylation, and analysis the influence of PGRN on Rho GTP activity; the use of FAK, GTP enzyme inhibitor Rho small, analysis of PGRN expression of cervical cancer cell migration and invasion depends on FAK and Rho GTP enzyme. Results: rhPGRN can enhance the cervical Cancer cell motility, migration and invasion; PGRN has little effect on cervical cancer EMT cells, but could promote the cytoskeleton, as pressure reinforced, lamellipodia and filopodia increased; PGRN through TNFR2 and mTOR signal pathway in cervical cancer tissues and the correlation with p-mTOR, mTOR reduced the inhibitory effect of rapamycin the migration and invasion of cervical cancer cells; PGRN activation of LIMK/cofilin signaling pathway related to cytoskeleton rearrangement, while promoting the phosphorylation level of FAK increased; PGRN enhanced RhoA and Racl activity in cervical cancer cells through the mTOR signaling pathway, PGRN mediated migration and invasion of cervical cancer cells depends on the activation of Racl and RhoA FAK. Etc.. Conclusion: This study confirmed that PGRN can promote the movement of cervical cancer cell migration and invasion ability, for the first time that PGRN mainly through the mTOR signaling pathway promotes cell skeleton rearrangement regulating ability Control of cancer cell migration, and in-depth analysis of the FAK signaling pathway and activation of mTOR Rho GTP enzyme mediated in the important role. This study found that PGRN promotes the invasion and metastasis of cervical cancer, a new mechanism to participate in the malignant progression of cervical cancer, for the control of cervical cancer invasion and metastasis will provide a new target; this is the first study of the role of PGRN in regulation of cytoskeletal rearrangement, and the link between FAK and Rho/LIMK/cofilin signaling pathway mediated by PGRN, provides a new perspective for the study of PGRN signaling pathway and biological function.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.33

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