【摘要】： 目的：對本實驗室建立的人鼻咽癌紫杉醇耐藥細胞株(CNE-1/Taxol、HNE-2/Taxol)的生物學特性進行觀察,檢測這兩株細胞系是否具有多藥耐藥性以及常見多藥耐藥基因MDR-1的表達情況。 方法：采用大劑量沖擊和逐步增加劑量相結合的方法,建立了兩株鼻咽癌紫杉醇耐藥細胞株(CNE-1/Taxol、HNE-2/Taxol),分別比較了這兩株耐藥細胞與其親本細胞的形態(tài)、生長曲線、倍增時間的差異；運用流式細胞儀檢測了其細胞周期變化；集落形成實驗檢測了它們對紫杉醇、順鉑、5-氟尿嘧啶、甲氨蝶呤和博來霉素五種化療藥的敏感性；使用real time RT-PCR比較了親本細胞與耐藥細胞MDR-1表達差異。 結果：兩株細胞系CNE-1/Taxol和HNE-2/Taxol的細胞大小及形態(tài)與親本細胞不一致,增殖速度減慢,倍增期延長,G2／M期細胞比例增多。集落形成實驗結果顯示CNE-1/Taxol、HNE-2/Taxol對紫杉醇的敏感性顯著低于其親本細胞CNE-1和HNE-2,前兩者的IC50值分別為9.59±0.26、7.62±0.21,而后兩者的IC50分別為1.14±0.08、0.92±0.08；同時,CNE-1/Taxol、HNE-2/Taxol對5-氟尿嘧啶和甲氨蝶呤的敏感性也顯著低于其親本細胞CNE-1和HNE-2,但耐藥指數(shù)顯著低于對紫杉醇的敏感性。然而,CNE-1/Taxol、HNE-2/Taxol對順鉑的敏感性顯著高于其親本細胞CNE-1和HNE-2,前兩者的IC50值分別為95.4±9.2、158.3±5.6,后兩者的IC50值分別為239.2±10.7、187.6±4.5；與親本細胞系比較,耐藥細胞系對博萊霉素的敏感性沒有變化(p0.05)；定量RT-PCR的結果顯示MDR-1在兩株親本細胞中表達均很低,且在耐藥細胞中表達無明顯增高,反而降低。 結論：CNE-1/Taxol、HNE-2/Taxol是兩株對紫杉醇耐藥的細胞株,是研究紫杉醇耐藥機制的重要實驗模型；兩株耐藥細胞對5-氟尿嘧啶、甲氨蝶呤產(chǎn)生了耐藥,具有多藥耐藥性的特點；紫杉醇耐藥細胞系對順鉑的敏感性更高；MDR-1在鼻咽癌細胞產(chǎn)生獲得性紫杉醇耐藥的過程中不起主要作用。
[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 鹵0.267.62 鹵0.21, respectively. The IC50 of the latter two was 1.14 鹵0.08 鹵0.92 鹵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 鹵9.158.3 鹵5.6) and IC50 (239.2 鹵10.7187.6 鹵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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R739.63

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