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(dá)研究目的:比較ALX1在肺癌組織中表達(dá)差異情況,揭示ALX1表達(dá)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(shí)驗(yàn)考察47例肺癌組織和癌旁組織樣本中ALX1m RNA的表達(dá)水平;利用免疫組織化學(xué)實(shí)驗(yàn)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(dá)水平;利用Kaplan-Meier生存分析研究ALX1的表達(dá)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(dá)ALX1,且顯著高于癌旁組織樣本中ALX1 m RNA的表達(dá)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(dá)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(xì)胞比例要顯著高于未轉(zhuǎn)移的肺癌組織中ALX1陽性細(xì)胞的比例。4.ALX1表達(dá)水平較低的肺癌患者的生存期顯著長于ALX1高表達(dá)組。結(jié)論:ALX1在肺癌組織中特異性高表達(dá),發(fā)生淋巴結(jié)轉(zhuǎn)移的肺癌組織中表達(dá)水平更高。ALX1高表達(dá)縮短肺癌患者的生存期。第二部分構(gòu)建ALX1過表達(dá)和基因沉默細(xì)胞系目的:構(gòu)建ALX1穩(wěn)定轉(zhuǎn)染和基因沉默的細(xì)胞模型,為ALX1調(diào)控肺癌細(xì)胞侵襲和遷移的分子機(jī)制研究提供工具。方法:通過Real time-PCR實(shí)驗(yàn)考察8種肺癌細(xì)胞系中ALX1 m RNA的表達(dá)水平。應(yīng)用逆轉(zhuǎn)錄病毒轉(zhuǎn)導(dǎo)構(gòu)建過表達(dá)人ALX1的H1975細(xì)胞模型,應(yīng)用sh RNA技術(shù)沉默H460細(xì)胞中ALX1的表達(dá)。通過Western blot實(shí)驗(yàn)驗(yàn)證模型構(gòu)建情況,采用MTT實(shí)驗(yàn)觀察構(gòu)建細(xì)胞模型的增殖能力,并觀察模型細(xì)胞的形態(tài)變化。通過Western blot實(shí)驗(yàn)和免疫熒光實(shí)驗(yàn)考察ALX1誘導(dǎo)肺癌細(xì)胞EMT情況。結(jié)果:1.8種肺癌細(xì)胞系中ALX1表達(dá)水平各異,H1975細(xì)胞表達(dá)較低,而H460細(xì)胞表達(dá)較高,適用于細(xì)胞轉(zhuǎn)染研究。2.成功構(gòu)建了ALX1-p Babe-H1975細(xì)胞模型和ALX1-si RNA-H460細(xì)胞模型,Western blot實(shí)驗(yàn)顯示過表達(dá)ALX1后,H1975細(xì)胞ALX1蛋白表達(dá)水平顯著高于mock-H1975和p Babe-H1975細(xì)胞;三組ALX1si RNA轉(zhuǎn)染細(xì)胞中,ALX1蛋白表達(dá)水平顯著低于對照細(xì)胞。3.MTT結(jié)果表明,過表達(dá)ALX1后,ALX1-p Babe-H1975細(xì)胞的增殖顯著快于對照細(xì)胞;沉默ALX1后,ALX1-si RNA-H460細(xì)胞的增殖能力下降。4.過表達(dá)ALX1后,ALX1-p Babe-H1975細(xì)胞呈現(xiàn)間質(zhì)細(xì)胞樣改變;沉默ALX1后,ALX1-si RNA-H460細(xì)胞呈現(xiàn)上皮樣細(xì)胞形態(tài)。5.過表達(dá)ALX1后,ALX1-p Babe-H1975細(xì)胞上皮細(xì)胞標(biāo)志蛋白E-cadherin和α-catenin表達(dá)降低,間質(zhì)細(xì)胞標(biāo)志蛋白Vimentin和N-cadherin表達(dá)上調(diào);沉默ALX1后,ALX1-si RNA-H460細(xì)胞E-cadherin和α-catenin表達(dá)上調(diào),而Vimentin和N-cadherin表達(dá)降低。免疫熒光實(shí)驗(yàn)結(jié)果與Western blot實(shí)驗(yàn)結(jié)果一致。結(jié)論:成功構(gòu)建了ALX1-p Babe-H1975和ALX1-si RNA-H460細(xì)胞模型,并成功運(yùn)用于ALX1誘導(dǎo)肺癌細(xì)胞EMT的研究。第三部分ALX1誘導(dǎo)肺癌細(xì)胞EMT的機(jī)制研究目的:分析ALX1誘導(dǎo)肺癌細(xì)胞EMT的分子機(jī)制,揭示ALX1誘導(dǎo)EMT對肺癌細(xì)胞侵襲和遷移的影響。方法:利用構(gòu)建的ALX1高表達(dá)和基因沉默細(xì)胞模型,通過Transwell和基質(zhì)膠侵襲實(shí)驗(yàn)、Real time-PCR實(shí)驗(yàn)、Western blot實(shí)驗(yàn)和熒光素酶實(shí)驗(yàn)考察ALX1誘導(dǎo)肺癌細(xì)胞EMT的分子機(jī)制。結(jié)果:1.過表達(dá)ALX1增加ALX1-p Babe-H1975細(xì)胞通過Transwell膜和基質(zhì)膠的數(shù)量,侵襲和遷移能力增強(qiáng)。沉默ALX1后,ALX1-si RNA-H460細(xì)胞侵襲和遷移能力減弱。2.過表達(dá)ALX1不影響ALX1-p Babe-H1975細(xì)胞中ZEB1、Slug和Twist的m RNA和蛋白表達(dá),顯著提高Snail的m RNA和蛋白表達(dá)水平。沉默ALX1后,ALX1-si RNA-H460細(xì)胞Snail的m RNA和蛋白表達(dá)水平顯著下調(diào)。3.過表達(dá)ALX1顯著提高H1975細(xì)胞中熒光素的強(qiáng)度。沉默ALX1后,ALX1-si RNA-H460細(xì)胞熒光素強(qiáng)度顯著下降。4.沉默Snail后,ALX1-p Babe-H1975細(xì)胞中低水平的E-cadherin和α-catenin表達(dá)恢復(fù),而高水平的Vimentin和N-cadherin表達(dá)下降;穿透基質(zhì)膠和Transwell膜的細(xì)胞個數(shù)減少。結(jié)論:ALX1通過Snail誘導(dǎo)肺癌細(xì)胞EMT,增強(qiáng)肺癌細(xì)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(diǎn)的進(jìn)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(xì)胞EMT的機(jī)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(jī)制。方法:利用構(gòu)建的ALX1高表達(dá)細(xì)胞模型,通過MTT、Transwell侵襲、Transwell遷移實(shí)驗(yàn)和Western blot實(shí)驗(yàn)、免疫熒光實(shí)驗(yàn)考察β-欖香烯對ALX1誘導(dǎo)肺癌細(xì)胞EMT的影響和機(jī)制。結(jié)果:1.β-欖香烯處理后,ALX1過表達(dá)的ALX1-p Babe-H1975細(xì)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(xì)胞數(shù)量減少,ALX1-p Babe-H1975細(xì)胞侵襲和遷移能力減弱。3.Western blot實(shí)驗(yàn)表明β-欖香烯上調(diào)ALX1過表達(dá)的ALX1-p Babe-H1975細(xì)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(qiáng)度增強(qiáng),而N-cadherin強(qiáng)度減弱。4.β-欖香烯降低ALX1-p Babe-H1975細(xì)胞Snail的蛋白水平。結(jié)論:β-欖香烯抑制Snail的表達(dá),阻斷ALX1誘導(dǎo)的肺癌細(xì)胞EMT,減慢增殖速率,減弱細(xì)胞侵襲和遷移能力,β-欖香烯可以作為ALX1過表達(dá)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