本文選題：EMT　切入點(diǎn)：GW3965　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討上皮間質(zhì)轉(zhuǎn)化與獲得性吉非替尼耐藥的關(guān)系及相關(guān)標(biāo)志物在獲得性耐藥前后的變化,并研究肝X受體的人工合成激動(dòng)劑GW3965對(duì)獲得性吉非替尼耐藥的逆轉(zhuǎn)與EMT過(guò)程相關(guān)性及具體分子機(jī)制,旨在為今后肝X受體及吉非替尼耐藥研究提供新的思路。方法:取處于對(duì)數(shù)生長(zhǎng)期的細(xì)胞,觀察細(xì)胞形態(tài)并經(jīng)倒置顯微鏡圖像采集系統(tǒng)進(jìn)行細(xì)胞拍照采集圖像信息;Transwell實(shí)驗(yàn)檢測(cè)不同細(xì)胞株侵襲能力差異,并用倒置顯微鏡采集圖像并統(tǒng)計(jì);調(diào)閱耐藥株HCC827/GR-8-1與親本HCC827細(xì)胞差異基因表達(dá)芯片數(shù)據(jù)EMT相關(guān)標(biāo)志物編碼基因表達(dá)差異;RT-PCR檢測(cè)不同敏感性細(xì)胞株間VIM表達(dá)量差異;Western blot及免疫組化檢測(cè)差異波形蛋白表達(dá)量差異;CCK-8實(shí)驗(yàn)檢測(cè)LXR激動(dòng)劑的單獨(dú)應(yīng)用對(duì)細(xì)胞毒力作用,及兩藥聯(lián)合應(yīng)用對(duì)吉非替尼敏感性的影響;克隆形成實(shí)驗(yàn)檢測(cè)GW3965單獨(dú)或聯(lián)合應(yīng)用吉非替尼對(duì)增殖的影響;Western blot進(jìn)一步驗(yàn)證兩藥聯(lián)用對(duì)細(xì)胞凋亡蛋白表達(dá)量的影響;Western blot檢測(cè)兩藥聯(lián)合過(guò)程對(duì)EMT相關(guān)標(biāo)志物尤其是波形蛋白表達(dá)量的影響并分析兩藥聯(lián)用后細(xì)胞增殖及蛋白表達(dá)量變化及其與細(xì)胞耐藥性關(guān)系。結(jié)果:細(xì)胞形態(tài)觀察顯示耐藥的細(xì)胞間質(zhì)特征更明顯。侵襲實(shí)驗(yàn)顯示HCC827及H358侵襲能力弱,而H1299、H1975侵襲能力強(qiáng),獲得性耐藥株侵襲能力也上升。對(duì)吉非替尼不敏感的細(xì)胞株VIM表達(dá)量高,Western blot及免疫組化同樣顯示H1975、H1299級(jí)獲得性耐藥株波形蛋白表達(dá)量顯著增高。EMT及相關(guān)主要標(biāo)志物波形蛋白與吉非替尼耐藥性正相關(guān)。LXR激動(dòng)劑單獨(dú)或聯(lián)合吉非替尼處理獲得性耐藥株HCC827/GR-8-1細(xì)胞后,CCK-8結(jié)果顯示GW3965對(duì)細(xì)胞無(wú)明顯毒力作用。但在多株耐藥株中可顯著增高細(xì)胞對(duì)吉非替尼敏感性,克隆形成實(shí)驗(yàn)同樣顯示,GW3965聯(lián)合吉非替尼組細(xì)胞克隆形成率顯著低于單用吉非替尼組。凋亡蛋白檢測(cè)顯示兩藥聯(lián)用可增加凋亡蛋白如caspase-3等的表達(dá),降低bcl-2蛋白的表達(dá)。檢測(cè)兩藥聯(lián)用中EMT相關(guān)標(biāo)識(shí)物如波形蛋白的檢測(cè)發(fā)現(xiàn),應(yīng)用吉非替尼后,胞內(nèi)波形蛋白表達(dá)量異常增高,聯(lián)用GW3965后,胞內(nèi)異常增高的波形蛋白表達(dá)被遏制。結(jié)論:EMT與吉非替尼耐藥正相關(guān),細(xì)胞在獲得吉非替尼耐藥的同時(shí)也獲得了EMT特征,以波形蛋白為代表的EMT標(biāo)志物表達(dá)異常;LXR激動(dòng)劑GW3965可逆轉(zhuǎn)獲得性吉非替尼耐藥,并且這一過(guò)程與抑制EMT標(biāo)志物如波形蛋白的表達(dá)有關(guān)。
[Abstract]:Objective: to investigate the relationship between epithelial mesenchymal transformation and acquired gefitinib resistance and the changes of relative markers before and after acquired drug resistance. To study the relationship between the reversal of acquired gefitinib resistance and the process of EMT and the specific molecular mechanism of the synthetic agonist GW3965 of liver X receptor. The aim of this study was to provide new ideas for the study of liver X receptor and gefitinib resistance in the future. Methods: cells in logarithmic growth phase were taken. The cell morphology was observed and the image information was collected by the inverted microscope image acquisition system. Transwell experiment was used to detect the difference of invasion ability of different cell lines, and the images were collected by inverted microscope and counted. Analysis of differentially expressed genes in HCC827/GR-8-1 and parent HCC827 cells by reverse transcriptase reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining of different vimentin tables (vimentin tables) among different sensitive cell lines, we detected the difference in VIM expression levels between different cell lines and in different sensitive cell lines by reverse transcription-polymerase chain reaction (RT-PCR). CCK-8 assay was used to detect the cytotoxicity of LXR agonists alone. The effect of the two drugs and their combination on the sensitivity of gefitinib; Detection of the effect of GW3965 alone or in combination with Gifitinib on Proliferation by Clone formation Assay further validation of the effect of combination of two drugs on the expression of apoptotic protein by Western blot; Western blot detection of the combined process of the two drugs on the expression of EMT markers, especially. The effect of vimentin on the expression of vimentin and the changes of cell proliferation, protein expression and their relationship with cell drug resistance were analyzed. Results: cell morphological observation showed that the intercellular interstitial characteristics of drug resistance were more obvious. The results showed that HCC827 and H358 were weak in invasiveness. However, H1299 and H1975 have strong invasiveness. The invasive ability of acquired drug resistant strain was also increased. The expression of VIM in the cell line which was insensitive to gefitinib was also increased. Western blot and immunohistochemistry also showed that the expression of vimentin in H1975 / H1299 class acquired drug resistance strain was significantly increased. Vimentin is positively correlated with drug resistance of gefitinib. LXR agonist alone or in combination with gefitinib treatment of acquired drug resistant HCC827/GR-8-1 cell line CCK-8 results show that GW3965 has no obvious virulence to cells, but can be significantly increased in multiple drug resistant strains. Cell sensitivity to gefitinib, Clonogenic assay also showed that the colony formation rate of GW3965 combined with gefitinib group was significantly lower than that of single gifitinib group, and the expression of apoptotic protein, such as caspase-3, was increased by the combination of two drugs. Detection of EMT related markers such as vimentin in combination of two drugs showed that the expression of vimentin in cells was increased after gifitinib, and the expression of vimentin was increased in combination with GW3965. Conclusion the expression of vimentin was positively correlated with gefitinib resistance, and the cell line obtained the EMT characteristics as well as gefitinib resistance. The abnormal expression of EMT markers represented by vimentin GW3965 can reverse the acquired gefitinib resistance and this process is related to the inhibition of the expression of EMT markers such as vimentin.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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本文編號(hào)：1585324