鼻咽癌紫杉醇耐药细胞以及其亲本细胞的细胞周期分布

发布时间：2018-03-31 00:01

本文选题：鼻咽肿瘤　切入点：紫杉醇　出处：《中南大学》2010年硕士论文

【摘要】： 肿瘤化疗药物抵抗的分子机制十分复杂,目前归纳为基因突变-药物抵抗假说和染色体不平衡-药物抵抗假说。目前,鼻咽癌化疗药物抵抗过程中,其细胞周期改变及细胞周期调控因子机制尚不清楚,深入研究十分重要,对指导鼻咽癌的治疗有重要价值。目的：探讨鼻咽癌亲本细胞系及耐药细胞系细胞周期改变及细胞周期调控因子变化情况,了解细胞周期改变及细胞周期调控因子与鼻咽癌化疗耐药发生发展中的关系；试图发现细胞周期改变及细胞周期调控因子在鼻咽癌化疗耐药过程中的作用。方法：1.以鼻咽癌细胞CNE-1, HNE2,5-8F及被诱导产生的紫杉醇耐药细胞CNE-1/Taxol, HNE-2/Taxol,5-8F/Taxol为研究对象,用集落形成实验检测所有细胞系的生长抑制率,并计算其紫杉醇和顺铂半数抑制剂量(IC50)。 2.流式细胞仪检测不同浓度的紫杉醇(0 ng/ml、5 ng/ml、10 ng/ml、15ng/ml、20ng/ml、25ng/ml)和顺铂(0ng/ml、100ng/ml、200ng/ml、300 ng/ml、400 ng/ml、500 ng/ml)处理鼻咽癌亲本细胞系及紫杉醇耐药细胞系后细胞周期分布情况。 3.荧光定量PCR检测鼻咽癌亲本细胞系和紫杉醇耐药系细胞周期蛋白依赖性激酶CDK1和有丝分裂纺锤体检控点基因BubR1的表达。结果：1.CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol紫杉醇的IC50值分别为9.61±0.43、11.48±0.52、9.9±0.48 ng/ml,相应亲本细胞的IC50值分别为1.14±0.05、1.78±0.08、1.63±0.06ng／ml,其耐药指数分别为8.43±0.42、6.45±0.31、6.11±0.35；顺铂作用于鼻咽癌亲本细胞CNE-1、HNE-2、5-8F的IC50值分别为：380.83±16.42ng/ml、362.12±15.31ng/ml、316.82±14.65ng/ml,作用于耐药细胞CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol的IC50值分别为177.43±7.65ng/ml、69.94±3.53ng/ml、147.91±6.53ng/ml。 2.10ng/ml紫杉醇处理鼻咽癌亲本细胞系(CNE-1, HNE-2)出现G2／M期阻滞,并随浓度增加G2／M期阻滞逐渐增加；15ng／ml紫杉醇处理鼻咽癌耐药细胞系(CNE-1/Taxol, HNE-2/Taxol)才引起G2/M期阻滞,并随浓度增加G2／M期逐渐增加；相同浓度紫杉醇引起鼻咽癌亲本细胞G2/M阻滞明显高于鼻咽癌紫杉醇耐药细胞系(p0.05)；顺铂可以导致鼻咽癌亲本细胞系及耐药细胞系细胞周期S期明显增加,相同浓度顺铂处理时,鼻咽癌紫杉醇耐药细胞系S期分布明显高于亲本细胞系(p0.05)。 3.鼻咽癌紫杉醇耐药细胞(HNE-2/Taxol)中细胞周期蛋白依赖性激酶CDK1表达比在亲本细胞(HNE-2)中上调约3倍。结论：1.鼻咽癌细胞诱导成耐药细胞后,其细胞周期时相分布没有显著变化,但引起其发生G2/M期和S期阻滞的紫杉醇和顺铂的最低浓度发生了显著变化。 2.鼻咽癌耐药细胞G2/M期和S期阻滞的药物浓度变化可能与细胞周期G2/M期检控点关键分子、细胞周期蛋白依赖性激酶CDK1表达上调有关,而与纺锤体检控点蛋白BubR1无关。
[Abstract]:The molecular mechanism of chemotherapeutic drug resistance in nasopharyngeal carcinoma is very complicated, which is summarized as gene mutation drug resistance hypothesis and chromosome imbalance drug resistance hypothesis. The changes of cell cycle and the mechanism of cell cycle regulatory factors are not clear. It is very important to further study the mechanism of cell cycle regulation, which is of great value in guiding the treatment of nasopharyngeal carcinoma. Objective: to investigate the changes of cell cycle and cell cycle regulatory factors in nasopharyngeal carcinoma (NPC) parent cell line and drug resistant cell line, and to understand the relationship between cell cycle change, cell cycle regulatory factor and chemotherapeutic resistance in nasopharyngeal carcinoma (NPC). The purpose of this study was to explore the role of cell cycle changes and cell cycle regulators in chemotherapeutic resistance of nasopharyngeal carcinoma (NPC). Methods: cell lines CNE-1, HNE2O5-8F and CNE-1 / Taxoll, HNE-2 / Taxol5-8F / Taxol were used as subjects. Colony formation assay was used to detect the growth inhibition rate of all cell lines, and the amount of paclitaxel and cisplatin 50% inhibitor was calculated. 2. Flow cytometry was used to detect the cell cycle distribution of nasopharyngeal carcinoma parent cell line and paclitaxel resistant cell line treated with different concentrations of paclitaxel 0 ng / ml 5 ng / ml 10 ng / ml 10 ng / ml 10 ng / ml 10 ng / ml 20 ng / ml 25 ng / ml) and cisplatin 0 ng / ml / ml 100 ng / ml / ml 100 ng / ml / ml 200 ng / ml / ml 300 ng / ml 400 ng / ml 400 ng / ml 500 ng / ml of nasopharyngeal carcinoma parent cell line and paclitaxel resistant cell line. 3. Fluorescence quantitative PCR was used to detect the expression of cyclin dependent kinase (CDK1) and mitotic spindle point gene (BubR1) in nasopharyngeal carcinoma cell line and paclitaxel resistant cell line. Results the IC50 value of HNE-2 / CNE-1 / Taxolan5-8Ftaxol / taxol was 9.61 卤0.43 卤0.53 卤0.48 卤0.48 卤0.48 ng / ml, respectively, and the IC50 value of the corresponding parent cells was 1.14 卤0.051.78 卤0.081.63 卤0.06 ng / ml, respectively. Its drug resistance index was 8.43 卤0.42n 6.45 卤0.31 6.11 卤0.35 mg / ml; the IC50 value of cisplatin on CNE-1H- 25-8F cells was 3.380.83 卤16.42ngr / ml 362.12 卤315.1ngml / ml respectively, and the IC50 value of CNE-1H1-H-25-8F on CNE-1ne-25-8F cells was 177.43 卤6.65nml 卤6.65nml 卤177.43 卤6.65nml / ml of CNE-1nne-25-8F, respectively, and the IC50 value of the corresponding parent cells was 1.14 卤0.051.78 卤0.081.63 卤0.06ngml.Results the index of drug resistance was 8.43 卤0.426.45 卤0.316.11 卤0.35ngTaxol; the IC50 value of CNE-1H- 25-8F treated with cisplatin was 3.380.83 卤16.42ngml-362.12 卤315.1ngml / ml, respectively. 2.10ng/ml paclitaxel-treated nasopharyngeal carcinoma cell line CNE-1 (HNE-2) showed G _ 2 / M phase arrest, and the G _ 2 / M phase arrest increased gradually with the concentration increasing, and then increased gradually with the increase of concentration in CNE-1 / Taxol, HNE-2Taxol-1 / ml paclitaxel treated nasopharyngeal carcinoma resistant cell line (CNE-1 / Taxol, HNE-2 / Taxol-1), and increased gradually with the increase of concentration. The G2 / M block of nasopharyngeal carcinoma parental cells induced by the same concentration of paclitaxel was significantly higher than that of nasopharyngeal carcinoma paclitaxel resistant cell line p0.05, and cisplatin could significantly increase the S-phase of cell cycle of both parent and resistant nasopharyngeal carcinoma cell lines, and when the same concentration of cisplatin was treated with the same concentration of cisplatin, The distribution of taxol resistant nasopharyngeal carcinoma cell line S phase was significantly higher than that of parent cell line p0.05. 3.The expression of cyclin dependent kinase CDK1 in paclitaxel-resistant nasopharyngeal carcinoma cell line (HNE-2 / Taxolol) was increased by about 3 times than that in parent cell line (HNE-2). Conclusion: 1. The cell cycle phase distribution of nasopharyngeal carcinoma cells induced into drug resistant cells did not change significantly, but the lowest concentrations of paclitaxel and cisplatin, which resulted in G 2 / M phase and S phase arrest, changed significantly. 2. The changes of drug concentration in G _ 2 / M phase and S phase arrest of nasopharyngeal carcinoma cells may be related to the up-regulation of cyclin dependent kinase (CDK1) expression in G _ 2 / M phase of cell cycle, but not to spindle point protein (BubR1).
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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