非小細(xì)胞肺癌H3122克唑替尼耐藥細(xì)胞株的建立及耐藥機(jī)制的探究
發(fā)布時(shí)間：2019-06-06 06:24
【摘要】：非小細(xì)胞肺癌(non-small cell lung cancer,NSCLC)是危害人類生命最常見的惡性腫瘤之一,約有2/3左右的非小細(xì)胞肺癌患者就診時(shí)都已不能手術(shù)治療,單純的放療或化療的效果都不理想。以分子標(biāo)志物為靶點(diǎn)的靶向治療具有更強(qiáng)的針對(duì)性,療效更明顯,成為了NSCLC治療的研究熱點(diǎn)。不幸的是分子靶向藥物治療一段時(shí)間后會(huì)產(chǎn)生耐藥,給臨床造成了極大的困擾,所以研究肺癌靶向藥物的耐藥機(jī)制尤為重要。棘皮動(dòng)物微管蛋白樣4-間變性淋巴瘤激酶(echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase,EML4-ALK)融合基因是一個(gè)新的治療靶點(diǎn),在肺癌中的研究相對(duì)較少,有關(guān)耐藥機(jī)制的研究更少。鑒于以上問題,本課題旨在建立EML4-ALK靶向藥物克唑替尼的耐藥細(xì)胞模型,鑒定其耐藥性,并初步探討其耐藥機(jī)制。本實(shí)驗(yàn)采用克唑替尼濃度遞增的方法誘導(dǎo)非小細(xì)胞肺癌H3122細(xì)胞構(gòu)建非小細(xì)胞肺癌克唑替尼耐藥細(xì)胞H3122CR。倒置顯微鏡觀察親本細(xì)胞和耐藥細(xì)胞的形態(tài)學(xué)差異。采用CCK8法繪制細(xì)胞生長(zhǎng)曲線并測(cè)定親本細(xì)胞和耐藥細(xì)胞對(duì)克唑替尼的敏感度,計(jì)算耐藥指數(shù)。流式細(xì)胞術(shù)檢測(cè)肺癌H3122和H3122CR細(xì)胞的凋亡率以及周期分布的差異。實(shí)時(shí)熒光定量逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription-polymerase chain reaction,RT-PCR)檢測(cè)肺癌細(xì)胞H3122和H3122CR中耐藥相關(guān)基因EML4-ALK和乳腺癌耐藥蛋白(breast cancer resistance protein,BCRP)的mRNA表達(dá)水平�；驕y(cè)序技術(shù)測(cè)定肺癌H3122和H3122CR細(xì)胞中miRNA的表達(dá)變化,并進(jìn)一步篩選差異表達(dá)的miRNA。用熒光定量RT-PCR驗(yàn)證差異表達(dá)的mi RNA。采用脂質(zhì)體2000將miR-10a-5p mimics轉(zhuǎn)人H3122CR細(xì)胞,檢測(cè)轉(zhuǎn)染后細(xì)胞對(duì)克唑替尼敏感度的變化以及細(xì)胞周期的變化。歷時(shí)6個(gè)月成功建立了克唑替尼耐藥細(xì)胞H3122CR,耐藥后細(xì)胞體積增大,長(zhǎng)偽足,觸角伸長(zhǎng),耐藥指數(shù)為9.86。與H3122相比,H3122CR細(xì)胞凋亡率明顯降低(P0.001);細(xì)胞胞周期分布產(chǎn)生了顯著的變化,S期比例減少(P0.01),G1期和G2期比例增加(P0.05);耐藥相關(guān)基因EML4-ALK和BCRP的mRNA表達(dá)水平明顯上調(diào)。第二代測(cè)序和RT-PCR結(jié)果顯示,耐藥細(xì)胞H3122CR中hsa-mi R-30a-5p,hsa-miR-374c,hsa-miR-143-3p,has-miR-148a-5p,hsa-mi R-125b-5p表達(dá)上調(diào),hsa-miR-31-5p,hsa-miR-3182,hsa-mi R-148a-3p,hsa-miR-200c-3p,hsa-miR-7-5p和hsa-mi R-10a-5p表達(dá)下調(diào)。將miR-10a-5p mimics轉(zhuǎn)染H3122CR細(xì)胞結(jié)果發(fā)現(xiàn),高表達(dá)的mi R-10a-5p能一定程度改善耐藥細(xì)胞對(duì)克唑替尼的敏感度,同時(shí)使細(xì)胞在G1期比例下調(diào)。綜上所述,建立的H3122CR耐藥細(xì)胞對(duì)克唑替尼具有一定的耐藥性,為進(jìn)一步研究耐藥機(jī)制以及逆轉(zhuǎn)耐藥提供基礎(chǔ)。高表達(dá)的EML4-ALK和BCRP很可能參與了克唑替尼的耐藥,并且部分miRNAs可能也參與了克唑替尼的耐藥。
[Abstract]:Non-small cell lung cancer (non-small cell lung cancer,NSCLC) is one of the most common malignant tumors endangering human life. About 2 to 3 patients with non-small cell lung cancer can no longer be treated surgically. The effect of radiotherapy or chemotherapy alone is not ideal. Targeted therapy with molecular markers as the target has stronger pertinence and more obvious curative effect, which has become the research focus of NSCLC therapy. Unfortunately, drug resistance will occur after the treatment of molecular targeted drugs for a period of time, which has caused great clinical problems, so it is particularly important to study the mechanism of drug resistance of targeted drugs in lung cancer. Tubulin-like 4-anaplastic lymphocytic kinase (echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase,EML4-ALK) fusion gene is a new therapeutic target in echinoderm animals. There are relatively few studies in lung cancer and less research on the mechanism of drug resistance. In view of the above problems, the purpose of this study was to establish a drug-resistant cell model of EML4-ALK targeted drug cozodinib, to identify its drug resistance, and to explore the mechanism of drug resistance. In this study, the method of increasing the concentration of clozotinib was used to induce non-small cell lung cancer H _ 3122 cells to construct non-small cell lung cancer clozotinib resistant cell line H _ 3122CR. The morphological differences between parent cells and drug-resistant cells were observed by inverted microscope. The cell growth curve was drawn by CCK8 method and the sensitivity of parent cells and drug-resistant cells to clozotinib was measured, and the drug resistance index was calculated. The apoptosis rate and cycle distribution of lung cancer H _ 3122 and H3122CR cells were detected by flow cytometry. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction,RT-PCR) was used to detect the mRNA expression of drug resistance related gene EML4-ALK and breast cancer resistance protein (breast cancer resistance protein,BCRP in lung cancer cells H 3122 and H3122CR. The expression of miRNA in lung cancer H3122 and H3122CR cells was detected by gene sequencing, and the differentially expressed miRNA. was further screened. Verification of differentially expressed mi RNA. by fluorescence quantitative RT-PCR MiR-10a-5p mimics was transformed into human H3122CR cells by liposomes 2000. The sensitivity and cell cycle of the cells to clozotinil were detected. After 6 months, prazolinib resistant cells H 3122CR were successfully established. After drug resistance, the cell volume increased, pseudopodium, tentacles elongated, and the drug resistance index was 9.86. Compared with H _ 3122, the apoptosis rate of H3122CR cells was significantly decreased (P0.001), the distribution of cell cycle was significantly changed, the proportion of S phase was decreased (P 0.01), and the proportion of G _ 1 phase and G _ 2 phase was increased (P 0.05). The mRNA expression of drug resistance related genes EML4-ALK and BCRP was significantly up-regulated. The results of second generation sequencing and RT-PCR showed that the expression of HSA mi R-125b-5p was up-regulated and the expression of hsa mi R-125b-5p was up-regulated in drug-resistant H3122CR cells hsa-miR- 30a 5p, hsamiR 143c, hsamiR 143p, hasmiR 148a 5p. the expression of hsami R-125b-5p was up-regulated and the expression of hsami R-125b-5p was up-regulated in drug-resistant cells. The expressions of hsa-miR-3182,hsa-mi R 148a 3p, HSA miR 200c 3p, HSA miR 7 5p and hsa-miR- 10a-5p were down-regulated. The results of miR-10a-5p mimics transfection into H3122CR cells showed that the high expression of miR-10a-5p could improve the sensitivity of drug-resistant cells to clozotinib to a certain extent, and down-regulate the proportion of cells in G _ 1 phase. In conclusion, the established H3122CR resistant cells have certain resistance to clozotinib, which provides a basis for further study of drug resistance mechanism and reversal of drug resistance. High expression of EML4-ALK and BCRP may be involved in the drug resistance of clozotinib, and some miRNAs may also be involved in the drug resistance of clozotinib.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R734.2
本文編號(hào)：2494136
[Abstract]:Non-small cell lung cancer (non-small cell lung cancer,NSCLC) is one of the most common malignant tumors endangering human life. About 2 to 3 patients with non-small cell lung cancer can no longer be treated surgically. The effect of radiotherapy or chemotherapy alone is not ideal. Targeted therapy with molecular markers as the target has stronger pertinence and more obvious curative effect, which has become the research focus of NSCLC therapy. Unfortunately, drug resistance will occur after the treatment of molecular targeted drugs for a period of time, which has caused great clinical problems, so it is particularly important to study the mechanism of drug resistance of targeted drugs in lung cancer. Tubulin-like 4-anaplastic lymphocytic kinase (echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase,EML4-ALK) fusion gene is a new therapeutic target in echinoderm animals. There are relatively few studies in lung cancer and less research on the mechanism of drug resistance. In view of the above problems, the purpose of this study was to establish a drug-resistant cell model of EML4-ALK targeted drug cozodinib, to identify its drug resistance, and to explore the mechanism of drug resistance. In this study, the method of increasing the concentration of clozotinib was used to induce non-small cell lung cancer H _ 3122 cells to construct non-small cell lung cancer clozotinib resistant cell line H _ 3122CR. The morphological differences between parent cells and drug-resistant cells were observed by inverted microscope. The cell growth curve was drawn by CCK8 method and the sensitivity of parent cells and drug-resistant cells to clozotinib was measured, and the drug resistance index was calculated. The apoptosis rate and cycle distribution of lung cancer H _ 3122 and H3122CR cells were detected by flow cytometry. Real-time fluorescence quantitative reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction,RT-PCR) was used to detect the mRNA expression of drug resistance related gene EML4-ALK and breast cancer resistance protein (breast cancer resistance protein,BCRP in lung cancer cells H 3122 and H3122CR. The expression of miRNA in lung cancer H3122 and H3122CR cells was detected by gene sequencing, and the differentially expressed miRNA. was further screened. Verification of differentially expressed mi RNA. by fluorescence quantitative RT-PCR MiR-10a-5p mimics was transformed into human H3122CR cells by liposomes 2000. The sensitivity and cell cycle of the cells to clozotinil were detected. After 6 months, prazolinib resistant cells H 3122CR were successfully established. After drug resistance, the cell volume increased, pseudopodium, tentacles elongated, and the drug resistance index was 9.86. Compared with H _ 3122, the apoptosis rate of H3122CR cells was significantly decreased (P0.001), the distribution of cell cycle was significantly changed, the proportion of S phase was decreased (P 0.01), and the proportion of G _ 1 phase and G _ 2 phase was increased (P 0.05). The mRNA expression of drug resistance related genes EML4-ALK and BCRP was significantly up-regulated. The results of second generation sequencing and RT-PCR showed that the expression of HSA mi R-125b-5p was up-regulated and the expression of hsa mi R-125b-5p was up-regulated in drug-resistant H3122CR cells hsa-miR- 30a 5p, hsamiR 143c, hsamiR 143p, hasmiR 148a 5p. the expression of hsami R-125b-5p was up-regulated and the expression of hsami R-125b-5p was up-regulated in drug-resistant cells. The expressions of hsa-miR-3182,hsa-mi R 148a 3p, HSA miR 200c 3p, HSA miR 7 5p and hsa-miR- 10a-5p were down-regulated. The results of miR-10a-5p mimics transfection into H3122CR cells showed that the high expression of miR-10a-5p could improve the sensitivity of drug-resistant cells to clozotinib to a certain extent, and down-regulate the proportion of cells in G _ 1 phase. In conclusion, the established H3122CR resistant cells have certain resistance to clozotinib, which provides a basis for further study of drug resistance mechanism and reversal of drug resistance. High expression of EML4-ALK and BCRP may be involved in the drug resistance of clozotinib, and some miRNAs may also be involved in the drug resistance of clozotinib.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R734.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 蔣濤;周彩存;;ALK陽性非小細(xì)胞肺癌患者克唑替尼耐藥的機(jī)制和治療措施[J];中國(guó)肺癌雜志;2015年02期
2 郭蕾;劉秀云;邱田;凌云;山靈;謝永強(qiáng);;全自動(dòng)免疫組織化學(xué)法檢測(cè)肺腺癌組織中間變性淋巴瘤激酶融合基因表達(dá)情況[J];中華病理學(xué)雜志;2014年02期
3 賈守薇;劉韜;黃紅兵;;治療非小細(xì)胞肺癌的分子靶向藥物的研究進(jìn)展[J];中國(guó)藥房;2013年32期
4 王夢(mèng);楊繼元;李軍川;陳延煊;;EML4-ALK在非小細(xì)胞肺癌中的表達(dá)及臨床意義[J];臨床腫瘤學(xué)雜志;2013年08期
5 吳荻;于鴻;李佳美;;EML4-ALK抑制劑在非小細(xì)胞肺癌中發(fā)生耐藥的機(jī)制[J];中國(guó)肺癌雜志;2013年01期
6 錢健;秦超;殷長(zhǎng)軍;;腫瘤靶向藥物療效及毒性與基因多態(tài)性間相關(guān)性的研究進(jìn)展[J];中國(guó)腫瘤外科雜志;2012年03期
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