非小细胞肺癌H3122克唑替尼耐药细胞株的建立及耐药机制的探究

发布时间：2019-06-06 06:24

【摘要】：非小细胞肺癌(non-small cell lung cancer,NSCLC)是危害人类生命最常见的恶性肿瘤之一,约有2/3左右的非小细胞肺癌患者就诊时都已不能手术治疗,单纯的放疗或化疗的效果都不理想。以分子标志物为靶点的靶向治疗具有更强的针对性,疗效更明显,成为了NSCLC治疗的研究热点。不幸的是分子靶向药物治疗一段时间后会产生耐药,给临床造成了极大的困扰,所以研究肺癌靶向药物的耐药机制尤为重要。棘皮动物微管蛋白样4-间变性淋巴瘤激酶(echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase,EML4-ALK)融合基因是一个新的治疗靶点,在肺癌中的研究相对较少,有关耐药机制的研究更少。鉴于以上问题,本课题旨在建立EML4-ALK靶向药物克唑替尼的耐药细胞模型,鉴定其耐药性,并初步探讨其耐药机制。本实验采用克唑替尼浓度递增的方法诱导非小细胞肺癌H3122细胞构建非小细胞肺癌克唑替尼耐药细胞H3122CR。倒置显微镜观察亲本细胞和耐药细胞的形态学差异。采用CCK8法绘制细胞生长曲线并测定亲本细胞和耐药细胞对克唑替尼的敏感度,计算耐药指数。流式细胞术检测肺癌H3122和H3122CR细胞的凋亡率以及周期分布的差异。实时荧光定量逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)检测肺癌细胞H3122和H3122CR中耐药相关基因EML4-ALK和乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)的mRNA表达水平。基因测序技术测定肺癌H3122和H3122CR细胞中miRNA的表达变化,并进一步筛选差异表达的miRNA。用荧光定量RT-PCR验证差异表达的mi RNA。采用脂质体2000将miR-10a-5p mimics转人H3122CR细胞,检测转染后细胞对克唑替尼敏感度的变化以及细胞周期的变化。历时6个月成功建立了克唑替尼耐药细胞H3122CR,耐药后细胞体积增大,长伪足,触角伸长,耐药指数为9.86。与H3122相比,H3122CR细胞凋亡率明显降低(P0.001);细胞胞周期分布产生了显著的变化,S期比例减少(P0.01),G1期和G2期比例增加(P0.05);耐药相关基因EML4-ALK和BCRP的mRNA表达水平明显上调。第二代测序和RT-PCR结果显示,耐药细胞H3122CR中hsa-mi R-30a-5p,hsa-miR-374c,hsa-miR-143-3p,has-miR-148a-5p,hsa-mi R-125b-5p表达上调,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表达下调。将miR-10a-5p mimics转染H3122CR细胞结果发现,高表达的mi R-10a-5p能一定程度改善耐药细胞对克唑替尼的敏感度,同时使细胞在G1期比例下调。综上所述,建立的H3122CR耐药细胞对克唑替尼具有一定的耐药性,为进一步研究耐药机制以及逆转耐药提供基础。高表达的EML4-ALK和BCRP很可能参与了克唑替尼的耐药,并且部分miRNAs可能也参与了克唑替尼的耐药。
[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.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R734.2

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