肝X受体激动剂GW3965对与EMT相关的非小细胞肺癌获得性吉非替尼耐药的逆转及机制研究

发布时间：2018-03-08 19:53

本文选题：EMT　切入点：GW3965　出处：《南京医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨上皮间质转化与获得性吉非替尼耐药的关系及相关标志物在获得性耐药前后的变化,并研究肝X受体的人工合成激动剂GW3965对获得性吉非替尼耐药的逆转与EMT过程相关性及具体分子机制,旨在为今后肝X受体及吉非替尼耐药研究提供新的思路。方法:取处于对数生长期的细胞,观察细胞形态并经倒置显微镜图像采集系统进行细胞拍照采集图像信息;Transwell实验检测不同细胞株侵袭能力差异,并用倒置显微镜采集图像并统计;调阅耐药株HCC827/GR-8-1与亲本HCC827细胞差异基因表达芯片数据EMT相关标志物编码基因表达差异;RT-PCR检测不同敏感性细胞株间VIM表达量差异;Western blot及免疫组化检测差异波形蛋白表达量差异;CCK-8实验检测LXR激动剂的单独应用对细胞毒力作用,及两药联合应用对吉非替尼敏感性的影响;克隆形成实验检测GW3965单独或联合应用吉非替尼对增殖的影响;Western blot进一步验证两药联用对细胞凋亡蛋白表达量的影响;Western blot检测两药联合过程对EMT相关标志物尤其是波形蛋白表达量的影响并分析两药联用后细胞增殖及蛋白表达量变化及其与细胞耐药性关系。结果:细胞形态观察显示耐药的细胞间质特征更明显。侵袭实验显示HCC827及H358侵袭能力弱,而H1299、H1975侵袭能力强,获得性耐药株侵袭能力也上升。对吉非替尼不敏感的细胞株VIM表达量高,Western blot及免疫组化同样显示H1975、H1299级获得性耐药株波形蛋白表达量显著增高。EMT及相关主要标志物波形蛋白与吉非替尼耐药性正相关。LXR激动剂单独或联合吉非替尼处理获得性耐药株HCC827/GR-8-1细胞后,CCK-8结果显示GW3965对细胞无明显毒力作用。但在多株耐药株中可显著增高细胞对吉非替尼敏感性,克隆形成实验同样显示,GW3965联合吉非替尼组细胞克隆形成率显著低于单用吉非替尼组。凋亡蛋白检测显示两药联用可增加凋亡蛋白如caspase-3等的表达,降低bcl-2蛋白的表达。检测两药联用中EMT相关标识物如波形蛋白的检测发现,应用吉非替尼后,胞内波形蛋白表达量异常增高,联用GW3965后,胞内异常增高的波形蛋白表达被遏制。结论:EMT与吉非替尼耐药正相关,细胞在获得吉非替尼耐药的同时也获得了EMT特征,以波形蛋白为代表的EMT标志物表达异常;LXR激动剂GW3965可逆转获得性吉非替尼耐药,并且这一过程与抑制EMT标志物如波形蛋白的表达有关。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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