本文選題：鼻咽腫瘤　切入點(diǎn)：紫杉醇　出處：《中南大學(xué)》2010年碩士論文

【摘要】： 腫瘤化療藥物抵抗的分子機(jī)制十分復(fù)雜,目前歸納為基因突變-藥物抵抗假說和染色體不平衡-藥物抵抗假說。目前,鼻咽癌化療藥物抵抗過程中,其細(xì)胞周期改變及細(xì)胞周期調(diào)控因子機(jī)制尚不清楚,深入研究十分重要,對(duì)指導(dǎo)鼻咽癌的治療有重要價(jià)值。 目的：探討鼻咽癌親本細(xì)胞系及耐藥細(xì)胞系細(xì)胞周期改變及細(xì)胞周期調(diào)控因子變化情況,了解細(xì)胞周期改變及細(xì)胞周期調(diào)控因子與鼻咽癌化療耐藥發(fā)生發(fā)展中的關(guān)系；試圖發(fā)現(xiàn)細(xì)胞周期改變及細(xì)胞周期調(diào)控因子在鼻咽癌化療耐藥過程中的作用。 方法：1.以鼻咽癌細(xì)胞CNE-1, HNE2,5-8F及被誘導(dǎo)產(chǎn)生的紫杉醇耐藥細(xì)胞CNE-1/Taxol, HNE-2/Taxol,5-8F/Taxol為研究對(duì)象,用集落形成實(shí)驗(yàn)檢測(cè)所有細(xì)胞系的生長抑制率,并計(jì)算其紫杉醇和順鉑半數(shù)抑制劑量(IC50)。 2.流式細(xì)胞儀檢測(cè)不同濃度的紫杉醇(0 ng/ml、5 ng/ml、10 ng/ml、15ng/ml、20ng/ml、25ng/ml)和順鉑(0ng/ml、100ng/ml、200ng/ml、300 ng/ml、400 ng/ml、500 ng/ml)處理鼻咽癌親本細(xì)胞系及紫杉醇耐藥細(xì)胞系后細(xì)胞周期分布情況。 3.熒光定量PCR檢測(cè)鼻咽癌親本細(xì)胞系和紫杉醇耐藥系細(xì)胞周期蛋白依賴性激酶CDK1和有絲分裂紡錘體檢控點(diǎn)基因BubR1的表達(dá)。 結(jié)果：1.CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol紫杉醇的IC50值分別為9.61±0.43、11.48±0.52、9.9±0.48 ng/ml,相應(yīng)親本細(xì)胞的IC50值分別為1.14±0.05、1.78±0.08、1.63±0.06ng／ml,其耐藥指數(shù)分別為8.43±0.42、6.45±0.31、6.11±0.35；順鉑作用于鼻咽癌親本細(xì)胞CNE-1、HNE-2、5-8F的IC50值分別為：380.83±16.42ng/ml、362.12±15.31ng/ml、316.82±14.65ng/ml,作用于耐藥細(xì)胞CNE-1/Taxol、HNE-2/Taxol、5-8F/Taxol的IC50值分別為177.43±7.65ng/ml、69.94±3.53ng/ml、147.91±6.53ng/ml。 2.10ng/ml紫杉醇處理鼻咽癌親本細(xì)胞系(CNE-1, HNE-2)出現(xiàn)G2／M期阻滯,并隨濃度增加G2／M期阻滯逐漸增加；15ng／ml紫杉醇處理鼻咽癌耐藥細(xì)胞系(CNE-1/Taxol, HNE-2/Taxol)才引起G2/M期阻滯,并隨濃度增加G2／M期逐漸增加；相同濃度紫杉醇引起鼻咽癌親本細(xì)胞G2/M阻滯明顯高于鼻咽癌紫杉醇耐藥細(xì)胞系(p0.05)；順鉑可以導(dǎo)致鼻咽癌親本細(xì)胞系及耐藥細(xì)胞系細(xì)胞周期S期明顯增加,相同濃度順鉑處理時(shí),鼻咽癌紫杉醇耐藥細(xì)胞系S期分布明顯高于親本細(xì)胞系(p0.05)。 3.鼻咽癌紫杉醇耐藥細(xì)胞(HNE-2/Taxol)中細(xì)胞周期蛋白依賴性激酶CDK1表達(dá)比在親本細(xì)胞(HNE-2)中上調(diào)約3倍。 結(jié)論：1.鼻咽癌細(xì)胞誘導(dǎo)成耐藥細(xì)胞后,其細(xì)胞周期時(shí)相分布沒有顯著變化,但引起其發(fā)生G2/M期和S期阻滯的紫杉醇和順鉑的最低濃度發(fā)生了顯著變化。 2.鼻咽癌耐藥細(xì)胞G2/M期和S期阻滯的藥物濃度變化可能與細(xì)胞周期G2/M期檢控點(diǎn)關(guān)鍵分子、細(xì)胞周期蛋白依賴性激酶CDK1表達(dá)上調(diào)有關(guān),而與紡錘體檢控點(diǎn)蛋白BubR1無關(guān)。
[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 鹵0.43 鹵0.53 鹵0.48 鹵0.48 鹵0.48 ng / ml, respectively, and the IC50 value of the corresponding parent cells was 1.14 鹵0.051.78 鹵0.081.63 鹵0.06 ng / ml, respectively. Its drug resistance index was 8.43 鹵0.42n 6.45 鹵0.31 6.11 鹵0.35 mg / ml; the IC50 value of cisplatin on CNE-1H- 25-8F cells was 3.380.83 鹵16.42ngr / ml 362.12 鹵315.1ngml / ml respectively, and the IC50 value of CNE-1H1-H-25-8F on CNE-1ne-25-8F cells was 177.43 鹵6.65nml 鹵6.65nml 鹵177.43 鹵6.65nml / ml of CNE-1nne-25-8F, respectively, and the IC50 value of the corresponding parent cells was 1.14 鹵0.051.78 鹵0.081.63 鹵0.06ngml.Results the index of drug resistance was 8.43 鹵0.426.45 鹵0.316.11 鹵0.35ngTaxol; the IC50 value of CNE-1H- 25-8F treated with cisplatin was 3.380.83 鹵16.42ngml-362.12 鹵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).
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.63

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