本文選題：缺氧　切入點：KLF5　出處：《華中科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分 轉(zhuǎn)錄因子KLF5在缺氧性肺動脈高壓中的作用 目的：缺氧性肺動脈高壓是一種復(fù)雜的系統(tǒng)性疾病,肺小動脈重構(gòu)是其主要的病理特征。KLF5(Kruppel-like factor5)作為一種鋅指結(jié)構(gòu)的轉(zhuǎn)錄因子,參與了腫瘤細胞的增殖、凋亡、分化以及遷移過程。它在肺動脈高壓病人的肺動脈平滑肌細胞中高表達。研究發(fā)現(xiàn)KLF5參與了心血管重構(gòu)的形成過程。然而,目前KLF5在缺氧性肺動脈高壓中的作用及分子機制尚不清楚。 方法：實驗采用成年雄性SD大鼠(體重為200-250g),分別置于常氧或缺氧環(huán)境(10%氧濃度)中培養(yǎng)�！畽z測大鼠血流動力學(xué)指標,評估其肺動脈中膜厚度和右心室肥厚程度,行肺組織HE染色觀測大鼠血管重構(gòu)情況。在常氧或缺氧(5%氧濃度)培養(yǎng)箱中培養(yǎng)人肺動脈平滑肌細胞(human pulmonary artery smooth muscle cells, HPASMC)。在HPASMC中行CCK-8、BrdU標記、PI單染、Annexin-V/PI雙染、transwell遷移實驗檢測細胞活力、細胞增殖、細胞周期、細胞凋亡和細胞遷移能力。行免疫印跡、免疫組織化學(xué)、免疫熒光染色實驗檢測目的蛋白水平。行實時熒光定量聚合酶鏈反應(yīng)(polymerase chain reaction, PCR)檢測目的基因mRNA水平。提取暴露于常氧和缺氧環(huán)境的HPASMC裂解產(chǎn)物,通過免疫共沉淀實驗明確KLF5和缺氧誘導(dǎo)因子-1α(hypoxia inducible factor-la, HIF-1α)之間是否存在關(guān)聯(lián)。采用小干擾RNA (small interfering RNA, siRNA)下調(diào)HPASMC中KLF5和HIF-1α的表達。為上調(diào)KLF5水平,建立過表達KLF5基因的慢病毒載體并感染HPASMC。構(gòu)建KLF5基因短發(fā)卡結(jié)構(gòu)RNA (short hairpin RNA, shRNA)慢病毒載體,通過尾靜脈注射入大鼠體內(nèi)以下調(diào)KLF5表達。 結(jié)果：缺氧組大鼠的平均肺動脈壓力顯著升高,伴隨遠端肺小動脈中膜增厚及右心室肥厚。缺氧引起大鼠肺動脈中膜層PCNA、Ki67陽性細胞增多,TUNEL陽性細胞減少。缺氧HPASMC的增殖和遷移能力增強,細胞凋亡減少。沉默KLF5或HIF-1α可以逆轉(zhuǎn)這些生物學(xué)改變。在平滑肌細胞中,沉默KLF5后HIF-1α蛋白水平下調(diào),過表達KLF5可以上調(diào)HIF-1α水平,而沉默HIF-1α后KLF5表達無顯著改變。免疫共沉淀實驗提示KLF5和HIF-1α存在直接結(jié)合,缺氧刺激下沉淀的目的蛋白增多。細胞周期蛋白cyclin B1、cyclin D1和凋亡相關(guān)蛋白bax、bc-2、survivin、caspase-3和caspase-9參與了KLF5-HIF-1α軸對HPASMC生長的調(diào)控。KLF5沉默大鼠的肺動脈中HIF-1α表達顯著下調(diào),平均肺動脈壓和肺動脈重構(gòu)情況顯著改善,與細胞實驗的結(jié)果一致。 結(jié)論：缺氧促進肺動脈平滑肌細胞增殖、遷移增加,凋亡減少,血管重構(gòu)形成。本研究從細胞水平和在體水平驗證了KLF5-HIF-1α軸存在于缺氧大鼠的肺動脈平滑肌細胞,并且在缺氧性肺血管重構(gòu)中發(fā)揮了重要作用。KLF5是缺氧性肺動脈高壓治療的潛在靶點。 第二部分 轉(zhuǎn)錄因子KLF5在在缺氧對非小細胞肺癌細胞增殖和凋亡中的作用 目的：轉(zhuǎn)錄因子KLF5在腫瘤組織中廣泛表達,且是腫瘤的一個預(yù)后因子。然后,KLF5在非小細胞肺癌中的作用尚未闡明。缺氧通過HIF-1α在腫瘤的發(fā)展中起重要作用。本研究旨在探討KLF5在缺氧性肺癌細胞生長中的作用以及KLF5和HIF-1α的潛在關(guān)聯(lián)。 方法：我們通過對暴露于常氧(20%氧濃度)或缺氧(1%氧濃度)環(huán)境的A549細胞進行CCK-8實驗、克隆形成實驗、CFSE標記實驗、Annexin-V/PI實驗檢測細胞的活力、克隆形成能力、‘增殖和凋亡。通過合成小干擾RNA來下調(diào)KLF5和HIF-1α的表達。采用PCR和免疫印跡的方法檢測mRNA和蛋白的表達水平。 結(jié)果：研究顯示缺氧可以增強A549細胞活力、克隆形成能力,促進增殖,抑制細胞凋亡。缺氧上調(diào)HIF-1α和KLF5的表達,呈時間依賴性。沉默KLF5和HIF-1α可以通過下調(diào)cyclin B1、survivin,上調(diào)caspase-3逆轉(zhuǎn)缺氧引起的細胞活力增加和凋亡減少。免疫共沉淀實驗提示KLF5和HIF-1α存在直接關(guān)聯(lián),且缺氧環(huán)境中沉淀的目的蛋白更多。此外,沉默KLF5可以降低HIF-1α的水平,而KLF5表達不受HIF-1α的影響,確定了KLF5是HIF-1α的上游調(diào)控因子。 結(jié)論：本研究證實缺氧是一個促癌因素,在肺癌細胞中通過激活KLF5→HIF-1α→cyclin B1/survivin/caspase-3發(fā)揮作用。
[Abstract]:Part one
The role of transcription factor KLF5 in hypoxic pulmonary hypertension
Objective: hypoxic pulmonary hypertension is a complex systemic disease, pulmonary vascular remodeling is the main pathological features of.KLF5 (Kruppel-like factor5) is a zinc finger transcription factor structure, involved in tumor cell proliferation, apoptosis, differentiation and migration process. Its high expression in patients with pulmonary hypertension lung arterial smooth muscle cells. The study found that KLF5 is involved in the formation process of cardiovascular remodeling. However, the role of KLF5 in hypoxic pulmonary hypertension and its molecular mechanism is unclear.
Methods: adult male SD rats (weight 200-250g), respectively in normoxia or hypoxia environment (10% oxygen) in vitro. "Dynamic detection indexes of rats to evaluate the pulmonary artery blood flow, film thickness and right ventricular hypertrophy, lung tissue HE staining observation of rat vascular remodeling. Normoxia or hypoxia (5% oxygen) in cultured human pulmonary artery smooth muscle cells in the box (human pulmonary artery smooth muscle cells, HPASMC). Mark BrdU in HPASMC CCK-8, Bank of China, PI staining, Annexin-V/PI staining, Transwell migration assay cell viability, cell proliferation, cell cycle, apoptosis and cell migration the ability for. Western blotting, immunohistochemistry and immunofluorescence staining experiments to detect protein level. For real-time fluorescence quantitative polymerase chain reaction (polymerase chain reaction, PCR) to detect mRNA gene level. The extraction of exposure to The pyrolysis products of HPASMC in normoxic and hypoxic environment, through co immunoprecipitation experiments clearly KLF5 and hypoxia inducible factor -1 alpha (hypoxia inducible Factor-la, HIF-1 a) between whether there is correlation. Using small interfering RNA (siRNA small interfering RNA) expression of KLF5 alpha and HIF-1 HPASMC. In order to increase the level of KLF5, established the lentiviral expression vector of KLF5 gene and HPASMC. KLF5 gene infection construct short hairpin structure RNA (short hairpin RNA, shRNA) lentiviral vector, by intravenous injection of rats following KLF5 expression.
Results: the mean pulmonary artery pressure in hypoxia group rats increased significantly with distal pulmonary arterioles in membrane thickening and hypertrophy of right ventricle. Hypoxia induced pulmonary arteries of rats in the film PCNA, Ki67 positive cells, TUNEL positive cells decreased. To enhance the ability of proliferation and migration of HPASMC cells reduced apoptosis of hypoxia, silencing of KLF5 or HIF-1 alpha. These biological changes can be reversed. In smooth muscle cells, down-regulation of HIF-1 protein level of KLF5 silencing, overexpression of KLF5 can increase the levels of HIF-1, HIF-1 and alpha silence the expression of KLF5 was not significantly changed. The experiment showed that the KLF5 and HIF-1 alpha are direct binding immunoprecipitation, protein precipitation increased under hypoxia stimulation of cell cycle. Protein cyclin B1, D1 cyclin and apoptosis related proteins Bax, BC-2, survivin, caspase-3 and caspase-9 are involved in the regulation of.KLF5 KLF5-HIF-1 alpha axis on the growth of HPASMC silencing rat pulmonary artery in HIF- The expression of 1 alpha was significantly down, and the average pulmonary arterial pressure and pulmonary artery remodeling were significantly improved, and the results were in agreement with the results of cell experiments.
Conclusion: hypoxia induced proliferation of pulmonary artery smooth muscle cell migration increased, apoptosis, vascular remodeling formation. This study from the cellular level and in vivo validation of pulmonary artery smooth muscle cells KLF5-HIF-1 alpha axis exists in hypoxic rats, and hypoxic pulmonary vascular remodeling plays an important role in the.KLF5 is a potential target for the treatment of hypoxia pulmonary arterial hypertension.
The second part
Role of transcription factor KLF5 in the proliferation and apoptosis of non small cell lung cancer cells in anoxia
Purpose: the transcription factor KLF5 is widely expressed in tumor tissue, and is a prognostic factor in cancer. Then, the role of KLF5 in non small cell lung cancer has not been elucidated. Hypoxia plays an important role in the development of cancer by HIF-1 alpha. This study aimed to investigate the role of KLF5 in hypoxia induced lung cancer cell growth and the the potential association between KLF5 and HIF-1 alpha.
Methods: we exposed to normoxia (20% oxygen) or hypoxia (1% oxygen) environment A549 cell clone formation assay, CCK-8 test, CFSE labeling experiment, Annexin-V/PI assay, cell viability, colony forming ability, cell proliferation and apoptosis. The expression of "synthetic small interfering RNA to downregulation of KLF5 and HIF-1 alpha. MRNA and protein expression detected by PCR and Western blot.
Results: the study shows that hypoxia can enhance A549 cell viability, colony formation ability, promote the proliferation and inhibition of apoptosis. The expression of HIF-1 is upregulated by hypoxia and KLF5 alpha, is time dependent. The silencing of KLF5 and HIF-1 alpha through the down-regulation of cyclin B1, survivin, upregulation of Caspase-3 caused by lack of oxygen to reverse the increase in cell viability and apoptosis of experimental reduced. KLF5 and HIF-1 a direct link between the co immunoprecipitation and protein precipitation in the anoxic environment more. In addition, silencing of KLF5 can reduce the level of HIF-1 alpha, KLF5 expression was not affected by HIF-1 alpha, the KLF5 is the upstream regulatory factor HIF-1 alpha.
Conclusion: This study confirmed that hypoxia is a cancer promoting factor, and it plays a role in lung cancer cells by activating KLF5 to HIF-1 alpha to cyclin B1/survivin/caspase-3.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R544.1

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