鼻咽癌紫杉醇耐药细胞系的生物学特性及多药耐药性检测
发布时间：2019-02-14 19:16
【摘要】： 目的：对本实验室建立的人鼻咽癌紫杉醇耐药细胞株(CNE-1/Taxol、HNE-2/Taxol)的生物学特性进行观察,检测这两株细胞系是否具有多药耐药性以及常见多药耐药基因MDR-1的表达情况。 方法：采用大剂量冲击和逐步增加剂量相结合的方法,建立了两株鼻咽癌紫杉醇耐药细胞株(CNE-1/Taxol、HNE-2/Taxol),分别比较了这两株耐药细胞与其亲本细胞的形态、生长曲线、倍增时间的差异；运用流式细胞仪检测了其细胞周期变化；集落形成实验检测了它们对紫杉醇、顺铂、5-氟尿嘧啶、甲氨蝶呤和博来霉素五种化疗药的敏感性；使用real time RT-PCR比较了亲本细胞与耐药细胞MDR-1表达差异。 结果：两株细胞系CNE-1/Taxol和HNE-2/Taxol的细胞大小及形态与亲本细胞不一致,增殖速度减慢,倍增期延长,G2／M期细胞比例增多。集落形成实验结果显示CNE-1/Taxol、HNE-2/Taxol对紫杉醇的敏感性显著低于其亲本细胞CNE-1和HNE-2,前两者的IC50值分别为9.59±0.26、7.62±0.21,而后两者的IC50分别为1.14±0.08、0.92±0.08；同时,CNE-1/Taxol、HNE-2/Taxol对5-氟尿嘧啶和甲氨蝶呤的敏感性也显著低于其亲本细胞CNE-1和HNE-2,但耐药指数显著低于对紫杉醇的敏感性。然而,CNE-1/Taxol、HNE-2/Taxol对顺铂的敏感性显著高于其亲本细胞CNE-1和HNE-2,前两者的IC50值分别为95.4±9.2、158.3±5.6,后两者的IC50值分别为239.2±10.7、187.6±4.5；与亲本细胞系比较,耐药细胞系对博莱霉素的敏感性没有变化(p0.05)；定量RT-PCR的结果显示MDR-1在两株亲本细胞中表达均很低,且在耐药细胞中表达无明显增高,反而降低。 结论：CNE-1/Taxol、HNE-2/Taxol是两株对紫杉醇耐药的细胞株,是研究紫杉醇耐药机制的重要实验模型；两株耐药细胞对5-氟尿嘧啶、甲氨蝶呤产生了耐药,具有多药耐药性的特点；紫杉醇耐药细胞系对顺铂的敏感性更高；MDR-1在鼻咽癌细胞产生获得性紫杉醇耐药的过程中不起主要作用。
[Abstract]:Objective: to observe the biological characteristics of paclitaxel resistant human nasopharyngeal carcinoma cell line (CNE-1/Taxol,HNE-2/Taxol) established in our laboratory. To detect whether the two cell lines have multidrug resistance and the expression of common multidrug resistance gene MDR-1. Methods: two taxol resistant nasopharyngeal carcinoma cell lines (CNE-1/Taxol,HNE-2/Taxol) were established by the combination of high dose shock and stepwise increasing dose, and the morphology of these two cell lines were compared with those of their parents. Growth curve, difference of doubling time; The cell cycle changes were detected by flow cytometry, and the sensitivity to five chemotherapeutic agents, paclitaxel, cisplatin, 5-fluorouracil, methotrexate and bleomycin, were detected by colony forming assay. Real time RT-PCR was used to compare the difference of MDR-1 expression between parent cells and drug resistant cells. Results: the cell size and morphology of the two cell lines CNE-1/Taxol and HNE-2/Taxol were different from those of their parents, the proliferation rate was slow, the multiplication period was prolonged, and the proportion of G2 / M phase cells was increased. The results of colony formation test showed that the sensitivity of CNE-1/Taxol,HNE-2/Taxol to paclitaxel was significantly lower than that of CNE-1 and HNE-2, which were 9.59 卤0.267.62 卤0.21, respectively. The IC50 of the latter two was 1.14 卤0.08 卤0.92 卤0.08; Meanwhile, the sensitivity of CNE-1/Taxol,HNE-2/Taxol to 5-fluorouracil and methotrexate was significantly lower than that of CNE-1 and HNE-2, but the drug resistance index was significantly lower than that of paclitaxel. However, the sensitivity of CNE-1/Taxol,HNE-2/Taxol to cisplatin was significantly higher than that of CNE-1 and HNE-2, (95.4 卤9.158.3 卤5.6) and IC50 (239.2 卤10.7187.6 卤4.5) respectively. Compared with parent cell line, the sensitivity of drug resistant cell line to bleomycin did not change (p0.05). The results of quantitative RT-PCR showed that the expression of MDR-1 in both parent cells was very low, and the expression of MDR-1 in resistant cells was not significantly increased, but decreased. Conclusion: CNE-1/Taxol,HNE-2/Taxol is two taxol resistant cell lines and is an important experimental model for studying the mechanism of paclitaxel resistance. Two drug-resistant cell lines developed resistance to 5-fluorouracil and methotrexate with multidrug resistance, and paclitaxel resistant cell lines were more sensitive to cisplatin. MDR-1 does not play a major role in the production of acquired paclitaxel resistance in nasopharyngeal carcinoma cells.
【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
本文编号：2422515
[Abstract]:Objective: to observe the biological characteristics of paclitaxel resistant human nasopharyngeal carcinoma cell line (CNE-1/Taxol,HNE-2/Taxol) established in our laboratory. To detect whether the two cell lines have multidrug resistance and the expression of common multidrug resistance gene MDR-1. Methods: two taxol resistant nasopharyngeal carcinoma cell lines (CNE-1/Taxol,HNE-2/Taxol) were established by the combination of high dose shock and stepwise increasing dose, and the morphology of these two cell lines were compared with those of their parents. Growth curve, difference of doubling time; The cell cycle changes were detected by flow cytometry, and the sensitivity to five chemotherapeutic agents, paclitaxel, cisplatin, 5-fluorouracil, methotrexate and bleomycin, were detected by colony forming assay. Real time RT-PCR was used to compare the difference of MDR-1 expression between parent cells and drug resistant cells. Results: the cell size and morphology of the two cell lines CNE-1/Taxol and HNE-2/Taxol were different from those of their parents, the proliferation rate was slow, the multiplication period was prolonged, and the proportion of G2 / M phase cells was increased. The results of colony formation test showed that the sensitivity of CNE-1/Taxol,HNE-2/Taxol to paclitaxel was significantly lower than that of CNE-1 and HNE-2, which were 9.59 卤0.267.62 卤0.21, respectively. The IC50 of the latter two was 1.14 卤0.08 卤0.92 卤0.08; Meanwhile, the sensitivity of CNE-1/Taxol,HNE-2/Taxol to 5-fluorouracil and methotrexate was significantly lower than that of CNE-1 and HNE-2, but the drug resistance index was significantly lower than that of paclitaxel. However, the sensitivity of CNE-1/Taxol,HNE-2/Taxol to cisplatin was significantly higher than that of CNE-1 and HNE-2, (95.4 卤9.158.3 卤5.6) and IC50 (239.2 卤10.7187.6 卤4.5) respectively. Compared with parent cell line, the sensitivity of drug resistant cell line to bleomycin did not change (p0.05). The results of quantitative RT-PCR showed that the expression of MDR-1 in both parent cells was very low, and the expression of MDR-1 in resistant cells was not significantly increased, but decreased. Conclusion: CNE-1/Taxol,HNE-2/Taxol is two taxol resistant cell lines and is an important experimental model for studying the mechanism of paclitaxel resistance. Two drug-resistant cell lines developed resistance to 5-fluorouracil and methotrexate with multidrug resistance, and paclitaxel resistant cell lines were more sensitive to cisplatin. MDR-1 does not play a major role in the production of acquired paclitaxel resistance in nasopharyngeal carcinoma cells.
【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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