本文關(guān)鍵詞： ALX1 Snail 肺癌 EMT 侵襲 遷移 β-欖香烯　出處：《大連醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分ALX1在肺癌中的表達研究目的:比較ALX1在肺癌組織中表達差異情況,揭示ALX1表達水平與肺癌惡性程度以及預(yù)后的關(guān)系。方法:通過Real time-PCR實驗考察47例肺癌組織和癌旁組織樣本中ALX1m RNA的表達水平;利用免疫組織化學(xué)實驗考察發(fā)生淋巴結(jié)轉(zhuǎn)移的肺癌組織和未轉(zhuǎn)移的肺癌組織中ALX1蛋白的表達水平;利用Kaplan-Meier生存分析研究ALX1的表達水平與肺癌病人的預(yù)后情況。結(jié)果:1.47例肺癌組織均表達ALX1,且顯著高于癌旁組織樣本中ALX1 m RNA的表達水平。2.47例肺癌組織中,發(fā)生淋巴結(jié)轉(zhuǎn)移的肺癌組織的ALX1 m RNA的表達水平顯著高于未轉(zhuǎn)移的肺癌組織。3.免疫組化分析顯示,發(fā)生淋巴結(jié)轉(zhuǎn)移的肺癌組織中ALX1陽性細胞比例要顯著高于未轉(zhuǎn)移的肺癌組織中ALX1陽性細胞的比例。4.ALX1表達水平較低的肺癌患者的生存期顯著長于ALX1高表達組。結(jié)論:ALX1在肺癌組織中特異性高表達,發(fā)生淋巴結(jié)轉(zhuǎn)移的肺癌組織中表達水平更高。ALX1高表達縮短肺癌患者的生存期。第二部分構(gòu)建ALX1過表達和基因沉默細胞系目的:構(gòu)建ALX1穩(wěn)定轉(zhuǎn)染和基因沉默的細胞模型,為ALX1調(diào)控肺癌細胞侵襲和遷移的分子機制研究提供工具。方法:通過Real time-PCR實驗考察8種肺癌細胞系中ALX1 m RNA的表達水平。應(yīng)用逆轉(zhuǎn)錄病毒轉(zhuǎn)導(dǎo)構(gòu)建過表達人ALX1的H1975細胞模型,應(yīng)用sh RNA技術(shù)沉默H460細胞中ALX1的表達。通過Western blot實驗驗證模型構(gòu)建情況,采用MTT實驗觀察構(gòu)建細胞模型的增殖能力,并觀察模型細胞的形態(tài)變化。通過Western blot實驗和免疫熒光實驗考察ALX1誘導(dǎo)肺癌細胞EMT情況。結(jié)果:1.8種肺癌細胞系中ALX1表達水平各異,H1975細胞表達較低,而H460細胞表達較高,適用于細胞轉(zhuǎn)染研究。2.成功構(gòu)建了ALX1-p Babe-H1975細胞模型和ALX1-si RNA-H460細胞模型,Western blot實驗顯示過表達ALX1后,H1975細胞ALX1蛋白表達水平顯著高于mock-H1975和p Babe-H1975細胞;三組ALX1si RNA轉(zhuǎn)染細胞中,ALX1蛋白表達水平顯著低于對照細胞。3.MTT結(jié)果表明,過表達ALX1后,ALX1-p Babe-H1975細胞的增殖顯著快于對照細胞;沉默ALX1后,ALX1-si RNA-H460細胞的增殖能力下降。4.過表達ALX1后,ALX1-p Babe-H1975細胞呈現(xiàn)間質(zhì)細胞樣改變;沉默ALX1后,ALX1-si RNA-H460細胞呈現(xiàn)上皮樣細胞形態(tài)。5.過表達ALX1后,ALX1-p Babe-H1975細胞上皮細胞標(biāo)志蛋白E-cadherin和α-catenin表達降低,間質(zhì)細胞標(biāo)志蛋白Vimentin和N-cadherin表達上調(diào);沉默ALX1后,ALX1-si RNA-H460細胞E-cadherin和α-catenin表達上調(diào),而Vimentin和N-cadherin表達降低。免疫熒光實驗結(jié)果與Western blot實驗結(jié)果一致。結(jié)論:成功構(gòu)建了ALX1-p Babe-H1975和ALX1-si RNA-H460細胞模型,并成功運用于ALX1誘導(dǎo)肺癌細胞EMT的研究。第三部分ALX1誘導(dǎo)肺癌細胞EMT的機制研究目的:分析ALX1誘導(dǎo)肺癌細胞EMT的分子機制,揭示ALX1誘導(dǎo)EMT對肺癌細胞侵襲和遷移的影響。方法:利用構(gòu)建的ALX1高表達和基因沉默細胞模型,通過Transwell和基質(zhì)膠侵襲實驗、Real time-PCR實驗、Western blot實驗和熒光素酶實驗考察ALX1誘導(dǎo)肺癌細胞EMT的分子機制。結(jié)果:1.過表達ALX1增加ALX1-p Babe-H1975細胞通過Transwell膜和基質(zhì)膠的數(shù)量,侵襲和遷移能力增強。沉默ALX1后,ALX1-si RNA-H460細胞侵襲和遷移能力減弱。2.過表達ALX1不影響ALX1-p Babe-H1975細胞中ZEB1、Slug和Twist的m RNA和蛋白表達,顯著提高Snail的m RNA和蛋白表達水平。沉默ALX1后,ALX1-si RNA-H460細胞Snail的m RNA和蛋白表達水平顯著下調(diào)。3.過表達ALX1顯著提高H1975細胞中熒光素的強度。沉默ALX1后,ALX1-si RNA-H460細胞熒光素強度顯著下降。4.沉默Snail后,ALX1-p Babe-H1975細胞中低水平的E-cadherin和α-catenin表達恢復(fù),而高水平的Vimentin和N-cadherin表達下降;穿透基質(zhì)膠和Transwell膜的細胞個數(shù)減少。結(jié)論:ALX1通過Snail誘導(dǎo)肺癌細胞EMT,增強肺癌細胞的侵襲和遷移能力。這些發(fā)現(xiàn)為ALX1作為潛在治療靶點的進一步研究奠定了基礎(chǔ)。第四部分β-欖香烯逆轉(zhuǎn)ALX1誘導(dǎo)肺癌細胞EMT的機制研究目的:考察β-欖香烯對ALX1誘導(dǎo)的EMT的影響,揭示β-欖香烯逆轉(zhuǎn)肺癌EMT的分子機制。方法:利用構(gòu)建的ALX1高表達細胞模型,通過MTT、Transwell侵襲、Transwell遷移實驗和Western blot實驗、免疫熒光實驗考察β-欖香烯對ALX1誘導(dǎo)肺癌細胞EMT的影響和機制。結(jié)果:1.β-欖香烯處理后,ALX1過表達的ALX1-p Babe-H1975細胞形態(tài)呈現(xiàn)上皮樣改變,增殖速度減慢。2.β-欖香烯處理后,通過Transwell膜和基質(zhì)膠的ALX1-p Babe-H1975細胞數(shù)量減少,ALX1-p Babe-H1975細胞侵襲和遷移能力減弱。3.Western blot實驗表明β-欖香烯上調(diào)ALX1過表達的ALX1-p Babe-H1975細胞的E-cadherin和α-catenin,下調(diào)Vimentin和N-cadherin。免疫熒光分析發(fā)現(xiàn)E-cadherin熒光強度增強,而N-cadherin強度減弱。4.β-欖香烯降低ALX1-p Babe-H1975細胞Snail的蛋白水平。結(jié)論:β-欖香烯抑制Snail的表達,阻斷ALX1誘導(dǎo)的肺癌細胞EMT,減慢增殖速率,減弱細胞侵襲和遷移能力,β-欖香烯可以作為ALX1過表達的肺癌的潛在治療藥物加以開發(fā)利用。
[Abstract]:Objective to study the expression of ALX1 in lung cancer in the first part: the difference of ALX1 expression in lung cancer tissues, revealing the expression level of ALX1 and the degree of malignancy of lung cancer and the prognosis. Methods: To investigate the expression level of 47 cases of lung cancer tissues and paracancerous tissue samples in ALX1m RNA by Real time-PCR by immunohistochemical experiment; experimental studying lymph node metastasis of lung cancer and the expression level of ALX1 protein in non metastatic lung carcinoma; prognosis analysis to study the expression of ALX1 and Kaplan-Meier in lung cancer patients with survival. Results: ALX1 was expressed in 1.47 cases of lung cancer, and was significantly higher than that in adjacent tissue samples in ALX1 m RNA.2.47 expression level in lung cancer patients the expression level of ALX1, m lymph node metastasis of RNA lung cancer was significantly higher than that in lung cancer without metastasis of.3. immunohistochemical analysis showed that the occurrence of lymph node Metastasis of lung cancer tissues was significantly higher than the proportion of ALX1 positive cells and ALX1 positive cells without metastasis in lung cancer survival ratio of.4.ALX1 expressed low levels of lung cancer patients was significantly longer than that of ALX1 high expression group. Conclusion: high expression of ALX1 in lung cancer tissue specific, higher expression level occurred in lung cancer lymph node metastasis in the high expression of.ALX1 reduced survival of patients with lung cancer. The second part is the construction of ALX1 overexpression and gene silencing cell line Objective: cell model was constructed and stably transfected with ALX1 gene silencing, provide a tool for the study of molecular mechanism of invasion and migration of ALX1 regulation of lung cancer cell. Methods: the effects of 8 kinds of lung cancer cell lines ALX1 m RNA expression the level of Real by time-PCR experiment. Application of retroviral transduction to construct H1975 cell model expressing human ALX1, the expression of ALX1 using SH RNA technology to silence H460 cells by Wes. The construction of tern blot experiment model, MTT to observe the construction of cell proliferation model, and to observe the morphological changes of model cells. By Western blot assay and immunofluorescence experiments to investigate ALX1 induced lung cancer cell EMT. Results: 1.8 different levels of expression of ALX1 in lung cancer cell lines, H1975 cells and H460 expression is low. Expression is high, suitable for cell transfection studies of.2. to construct ALX1-p model of Babe-H1975 cells and ALX1-si RNA-H460 cells Western model, blot assay showed that the overexpression of ALX1, H1975 cells ALX1 protein expression level was significantly higher than that of mock-H1975 and P in Babe-H1975 cells; three groups of ALX1si RNA in the transfected cells, the expression level of ALX1 protein was significantly lower than the control cells.3.MTT the results show that the overexpression of ALX1 and ALX1-p on the proliferation of Babe-H1975 cells significantly faster than control cells; after ALX1 was silenced, ALX1-si RNA-H460 Cell proliferation decreased expression of.4. ALX1, ALX1-p Babe-H1975 cells showed interstitial cells changed; after ALX1 was silenced, ALX1-si RNA-H460 cells showed epithelioid cell morphology after overexpression of ALX1.5., ALX1-p Babe-H1975 cell marker of epithelial cells and decrease the expression of protein E-cadherin and -catenin alpha, interstitial cell marker expression of protein Vimentin and N-cadherin; silence ALX1, ALX1-si RNA-H460 E-cadherin cells and alpha -catenin expression, while Vimentin and N-cadherin expression decreased. Immunofluorescence experiment results are consistent with the experimental results of Western blot. Conclusion: the successful construction of ALX1-p Babe-H1975 and ALX1-si RNA-H460 cell model, and successfully applied to ALX1 induced lung cancer cell EMT. The third part is the research objective mechanism of ALX1 induced lung cancer EMT cells: analysis of molecular mechanism of ALX1 induced lung cancer cell line EMT, ALX1 induced EMT of lung cancer revealed Effect of cell migration and invasion. Methods: the high expression of ALX1 and gene silencing cell model was constructed by Transwell, and Matrigel invasion assay Real time-PCR experiment, Western blot experiment and experimental study of molecular mechanism of ALX1 induced luciferase EMT lung cancer cells. Results: 1. overexpression of ALX1 increased the number of ALX1-p Babe-H1975 cells by Transwell and membrane Matrigel, enhance the invasion and migration ability. After ALX1 was silenced, ALX1-si RNA-H460 cell invasion and migration ability of.2. decreased expression of ALX1 ZEB1 ALX1-p does not affect Babe-H1975 cells, the expression of M protein and RNA Slug and Twist, significantly increased m RNA and protein expression level of Snail. After ALX1 was silenced, the expression level of M and RNA ALX1-si RNA-H460 protein Snail cells significantly reduced.3. expression of ALX1 increased significantly in H1975 cells. Fluorescence intensity of ALX1 silencing, ALX1-si RNA-H460 cells Guang Suqiang. 搴︽樉钁椾笅闄，

本文編號：1464990