【摘要】：宮頸癌是女性生殖系統(tǒng)最常見的惡性腫瘤之一。據(jù)統(tǒng)計(jì)數(shù)據(jù)顯示,宮頸癌是女性第四常見的腫瘤,并已成為15-44歲女性中僅次于乳腺癌的第二殺手；2012年全球有527,624例新發(fā)宮頸癌,85%以上發(fā)生在發(fā)展中國家,死亡病例為265,653例,死亡率為50.3%。高危型人乳頭瘤病毒(human papillomavirus, HPV)感染是宮頸癌的首要病因,但僅有少數(shù)HPV感染的女性發(fā)展為宮頸癌,提示其他因素也參與了宮頸癌的進(jìn)展。組織侵襲與轉(zhuǎn)移、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)的血管生成能力是癌細(xì)胞惡性進(jìn)展的主要特征。癌細(xì)胞在機(jī)體內(nèi)出現(xiàn)后,會(huì)遭受低氧、低糖、細(xì)胞與細(xì)胞之間或細(xì)胞與細(xì)胞外基質(zhì)(extracellular matrix, ECM)之間的支持不足、營養(yǎng)和生長因子缺乏等微環(huán)境壓力。只有成功適應(yīng)這些壓力,癌細(xì)胞才能在獲得抗凋亡、促增殖活性的同時(shí),維持血管生成、永生化、侵襲與轉(zhuǎn)移等特性。氧化還原平衡是維持細(xì)胞正常功能、確保細(xì)胞生存的根本機(jī)制。代謝缺陷、低氧、內(nèi)質(zhì)網(wǎng)應(yīng)激、癌基因均可引起活性氧(Reactive oxygen species, ROS)的產(chǎn)生。生長因子激活的PI3K/Akt途徑具有刺激葡萄糖轉(zhuǎn)運(yùn)和糖酵解的功能,當(dāng)生長因子缺乏時(shí)細(xì)胞PI3K/Akt信號(hào)途徑活性降低,細(xì)胞葡萄糖攝入能力下降,間接引起葡萄糖代謝缺陷,引起ROS的產(chǎn)生。過多的ROS會(huì)引起蛋白質(zhì)氧化、脂質(zhì)過氧化、線粒體與DNA的損傷。紅系衍生核因子相關(guān)因子2 (Nuclear factor (erythroid-derived 2)-related factor 2, Nrf2)是細(xì)胞抗氧化機(jī)制最重要的調(diào)控分子之一。在正常情況下Nrf2存在于胞漿中并與Keap1結(jié)合,經(jīng)泛素化降解；在某些誘導(dǎo)因素作用下Nrf2與Keap1解離進(jìn)入細(xì)胞核,結(jié)合多種抗氧化相關(guān)蛋白與Ⅱ相解毒酶基因調(diào)控區(qū)的抗氧化作用元件(antioxidant response element, ARE),驅(qū)動(dòng)這些基因的表達(dá),從而發(fā)揮其抗氧化作用。顆粒蛋白前體(progranulin, PGRN)是一種由71/2個(gè)富含半胱氨酸的GRN基序組成的生長因子,參與早期胚胎發(fā)育、骨發(fā)育、炎癥、損傷修復(fù)、神經(jīng)退行性病變、癌癥等生理和疾病進(jìn)程。本研究的前期工作證實(shí)PGRN在宮頸癌組織中高表達(dá),并可通過PI3K/Akt與Erk信號(hào)途徑促進(jìn)宮頸癌細(xì)胞增殖與惡性轉(zhuǎn)化。PGRN可以促進(jìn)細(xì)胞增殖、生存與遷移等,是正常細(xì)胞與癌細(xì)胞公認(rèn)的生存因子之一,能夠促進(jìn)細(xì)胞在化療藥物、低氧、酸性環(huán)境等壓力下的存活,但PGRN在腫瘤微環(huán)境壓力下癌細(xì)胞生存中的功能與調(diào)控機(jī)制尚不清楚。在明確PGRN在宮頸癌致癌機(jī)制中具有關(guān)鍵作用的基礎(chǔ)七,本論文采用血清饑餓模擬腫瘤微環(huán)境壓力,分析PGRN在宮頸癌細(xì)胞適應(yīng)微環(huán)境壓力中的功能,并探究PGRN促進(jìn)微環(huán)境壓力下宮頸癌細(xì)胞生存的抗氧化應(yīng)激分子機(jī)制。本論文的主要發(fā)現(xiàn)包括：(1) PGRN為血清應(yīng)答蛋白,血清饑餓導(dǎo)致宮頸癌細(xì)胞中PGRN蛋白的急劇下降；(2) PGRN能夠抵御血清饑餓誘導(dǎo)的宮頸癌細(xì)胞死亡及凋亡信號(hào),證明PGRN具有促進(jìn)癌細(xì)胞在腫瘤微環(huán)境壓力下生存的功能；(3) PGRN抑制血清饑餓處理的宮頸癌細(xì)胞內(nèi)ROS水平,并減輕蛋白質(zhì)氧化、脂質(zhì)過氧化、DNA氧化損傷以及線粒體功能失調(diào)等,證明PGRN可介導(dǎo)抗氧化應(yīng)激作用,促進(jìn)癌細(xì)胞適應(yīng)微環(huán)境壓力；(4)PGRN可通過增強(qiáng)ROS清除劑系統(tǒng)多種抗氧化蛋白與Ⅱ相脫毒酶的表達(dá)與活性、提高細(xì)胞內(nèi)關(guān)鍵還原性物質(zhì)水平實(shí)現(xiàn)其抗氧化作用；(5)PGRN能夠促進(jìn)血清饑餓處理的宮頸癌細(xì)胞中重要的抗氧化相關(guān)轉(zhuǎn)錄因子Nrf2的表達(dá),并促進(jìn)Nrf2與Keap1的解離,進(jìn)而轉(zhuǎn)移入核,發(fā)揮其轉(zhuǎn)錄活性；(6)血清饑餓壓力下PGRN的生存因子功能與抗氧化作用依賴于Nrf2/ARE信號(hào)途徑。本論文首次證實(shí)PGRN在血清饑餓壓力下胞內(nèi)蛋白水平急劇下降的表達(dá)特征；首次發(fā)現(xiàn)PGRN通過增強(qiáng)細(xì)胞內(nèi)ROS清除系統(tǒng)的活性,抵抗微環(huán)境壓力誘導(dǎo)的氧化應(yīng)激,促進(jìn)癌細(xì)胞的生存。這些發(fā)現(xiàn)提示PGRN除通過促進(jìn)細(xì)胞增殖與遷移外,還可介導(dǎo)癌細(xì)胞適應(yīng)微環(huán)境壓力驅(qū)動(dòng)癌癥的進(jìn)展,進(jìn)一步完善PGRN介導(dǎo)癌細(xì)胞惡性進(jìn)展的機(jī)理,為以PGRN及其介導(dǎo)的Nrf2/ARE信號(hào)途徑為靶點(diǎn)的包括宮頸癌的各類腫瘤的治療提供新的治療策略與研究證據(jù)；同時(shí)也為PGRN在炎癥應(yīng)答、神經(jīng)退行性疾病及其他生理和病理進(jìn)程中的功能提供抗氧化機(jī)制。
[Abstract]:Cervical cancer is one of the most common malignant tumors in female reproductive system. Cervical cancer is the fourth most common tumor in women and has become the second killer next to breast cancer among 15-44-year-olds, according to statistics. In 2012, 527,624 new cervical cancer, more than 85% occurred in developing countries, and death cases were 265, 653, with a mortality rate of 50.3%. High-risk human papillomavirus (HPV) infection is the primary cause of cervical cancer, but only a few HPV-infected women develop cervical cancer, suggesting that other factors are also involved in the progress of cervical cancer. Tissue invasion and metastasis, self-sufficiency of growth signals, tolerance of growth inhibition signals, escape of programmed death, unlimited replication potential, and sustained angiogenesis are the major features of malignant progression of cancer cells. In vivo, cancer cells suffer from hypoxia, low sugar, insufficient support between cells and cells or between cells and extracellular matrix (ECM), lack of nutritional and growth factors, and the like. Only by successfully adapting to these pressures, cancer cells can maintain the characteristics of angiogenesis, eternal life, invasion and metastasis while obtaining anti-apoptotic and pro-proliferative activity. The redox balance is the fundamental mechanism to maintain the normal function of cells and to ensure the survival of cells. Metabolic defects, hypoxia, ER stress and oncogene can cause reactive oxygen species (ROS) generation. The p38/ Akt pathway activated by the growth factor has the function of stimulating glucose transport and glycolysis, and when the growth factor is absent, the cell affinity/ Akt signal pathway activity is reduced, the cell glucose uptake capacity is reduced, the glucose metabolism defect is indirectly caused, and the generation of ROS is caused. Too much ROS can cause protein oxidation, lipid peroxidation, mitochondrial and DNA damage. Red-derived nuclear factor-related factor 2 (Nrf2) is one of the most important regulatory molecules in cell antioxidant mechanism. In normal cases, Nrf2 is present in cytoplasm and binds to Keap1, is degraded by ubiquitination, and Nrf2 dissociates into nucleus with Keap1 under the action of certain induction factors, and binds various anti-oxidation-related proteins and anti-oxidation active element (ARE) in the regulatory region of II-phase detoxication enzyme gene. The expression of these genes is driven so as to exert its anti-oxidation effect. Particle protein precursor (PGRN) is a growth factor composed of 71/ 2 cysteine-rich GRIN motifs, and is involved in the processes of early embryonic development, bone development, inflammation, injury repair, neurodegenerative diseases, and cancer. The preliminary work of this study confirms that PGRN is highly expressed in cervical cancer tissues, and can promote the proliferation and malignant transformation of cervical cancer cells by means of p38/ Akt and Erk signaling pathway. PGRN can promote cell proliferation, survival and migration and the like, is one of the well-known survival factors of normal cells and cancer cells, and can promote the survival of cells under pressure such as chemotherapy drugs, hypoxia, acidic environment and the like, However, the function and regulation mechanism of PGRN in cancer cell survival at micro-ambient pressure is unclear. In order to clarify the key role of PGRN in carcinogenesis of cervical cancer, this paper uses serum starvation to simulate the micro-environmental stress of tumor, and analyses the function of PGRN in cervical cancer cells to adapt to micro-environmental pressure. and explore the anti-oxidative stress molecular mechanism of PGRN for promoting the survival of cervical cancer cells under micro-ambient pressure. The main findings of this paper include: (1) PGRN is a serum-responsive protein, serum starvation leads to a sharp decrease in PGRN protein in cervical cancer cells; (2) PGRN is capable of resisting the death and apoptosis signals of cervical cancer cells induced by serum starvation, It is proved that PGRN has the function of promoting the survival of cancer cells in tumor microenvironment; (3) PGRN inhibits the level of ROS in cervical cancer cells treated by serum starvation, and reduces protein oxidation, lipid peroxidation, DNA oxidative damage and mitochondrial dysfunction, etc. It is proved that PGRN can mediate anti-oxidative stress and promote the adaptation of cancer cells to microambient pressure; (4) PGRN can increase the level of key reducing substances in cells by enhancing the expression and activity of various anti-oxidation proteins of ROS scavenger system and II-phase detoxification enzymes, and improve the level of key reducing substances in cells. (5) PGRN can promote the expression of important anti-oxidation-related transcription factor Nrf2 in cervical cancer cells treated by serum starvation, and promote the dissociation of Nrf2 and Keap1, and then transfer into the nucleus to exert its transcriptional activity; (6) The survival factor function and anti-oxidation effect of PGRN under serum starvation pressure depend on the Nrf2/ ARE signal pathway. For the first time, the expression of PGRN in the intracellular protein level in serum starvation was confirmed; the first time we found that PGRN enhanced the activity of ROS scavenging system in the cell, resist oxidative stress induced by microenvironmental stress, and promote the survival of cancer cells. These findings suggest that, in addition to promoting cell proliferation and migration, PGRN can mediate cancer cell adaptation to micro-ambient pressure to drive cancer progression, and further improve the mechanism of PGRN-mediated malignant progression of cancer cells. To provide new therapeutic strategies and research evidence for the treatment of various tumors including cervical cancer with PGRN and its mediated Nrf2/ ARE signaling pathway; meanwhile, it also provides antioxidant mechanism for the function of PGRN in inflammatory response, neurodegenerative diseases and other physiological and pathological processes.
【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】：2016
【分類號(hào)】：R737.33

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