摘要
目的:探讨紫杉醇诱导筛选的喉癌Hep-2耐药细胞的多药耐药特性,为逆转喉癌的多药耐药提供理论依据。方法:以浓度逐渐递增法诱导筛选喉癌Hep-2细胞的耐药细胞株Hep-2T,比较2种细胞的形态和倍增时间。以ATP酶检测法确定细胞的IC50和细胞的耐药倍数。流式细胞仪检测2种细胞的周期分布情况、细胞凋亡以及2种细胞内的罗丹明聚集情况。以实时定量RT-PCR法检测MDR1和MRP1基因,Western-botting法检测相应的蛋白表达情况。结果:耐药细胞株的耐药倍数是非耐药株的104倍。耐药株对多柔吡星、吉西它宾、5-Fu及顺铂的耐药倍数分别是非耐药株的46.78,1.95,2.50和1.05倍。与非耐药株比,耐药细胞G0/G1期的细胞较后者升高(P<0.05),而G2/M和S期细胞则较后者明显降低(P<0.05)。当作用于Hep-2的IC50浓度时,Hep-2细胞的凋亡细胞数较Hep-2T明显高。罗丹明染色显示,Hep-2细胞内的罗丹明较Hep-2T明显增高。Hep-2T细胞MDR1/GAPDH表达在基因和蛋白水平明显高于Hep-2细胞。MRP1/GAPDH在基因和蛋白水平的表达前者亦高于后者,但增高程度不如MDR1/GAPDH明显。结论:在寻找逆转喉癌多药耐药的方法时,应着重于MDR1/P-gp,当应用化疗药时,应考虑非P-gp作用底物的药物。并选择应用周期特异性作用的药物。
Objective：To build the multiple drug resistant human laryngeal cancer cell line and investigate its characteristics. Method.. Human laryngeal cancer cells were exposed in stepwise escalating concentration of Taxol until the resistant cell line was developed. The IC50 and the resistance folds of muhidrug resistance were detected by an ATP assay. The differences of cell cycle distribution, apoptosis, and Rhodamine accumulation between Hep-2 and Hep-2T cells were studied through flow cytometry. The MDR1 and MRP1 gene were detected through realtime quantitative RT-PCR, and the corresponding protein was detected through western-blotting. Result.. A multidrug resistance cell line-Hep-2T induced by Taxol was effectively developed, whose drug resistance was 104 times that of Hep-2 cells. Doxorubicin, Gemcitabine,5-Fu,Cisplatin all increased the drug resistance by 46.78, 1.95, 2.50, 1.05 folds. The cell cycle distribution altered. The apoptosis of Hep-2 cells was quite greater than that of Hep-2T cells （45.32% vs 4.26%, P 〈0.01, flow cytometry）, （54.47±8.95 vs 9.84±2.53 P 〈0.01, hoechest staining） after Hep-2 and Hep-2T exposed to Taxol at ICs0 to Hep-2. The copy ratio of MDR1/GAPDH mRNA of Hep-2T was 64.2±36.7 times that of Hep-2 （ P 〈0.05）, while MRP1/GAPDH of Hep-2T was only 1.20± 0.09 folds more than that of Hep-2 （P〈0.05）. The proteins of MDR1/P-gp were greatly over expressed in Hep- 2T cells compared with Hep-2 cells （ P 〈0.01） whose was in the same trend （ P 〈0.05）, while the elevated expression of MRP1 was lower than that of MDR1/P-gp. Conclusion：When considering the possible methods to reverse MDR of SCCHN, more emphasis should be laid on MDR1/P-gp, and when combining this with chemotherapy the non-P-gp substrate chemotherapeutic agents should be considered. At the same time, more attention should be paid to the changes of cell cycle distribution during the drug selection.
出处
《临床耳鼻咽喉头颈外科杂志》
CAS
CSCD
北大核心
2007年第18期843-847,共5页
Journal of Clinical Otorhinolaryngology Head And Neck Surgery
基金
上海市卫生局科技发展计划基金资助(No:044024)
关键词
喉肿瘤
紫杉醇
多药耐药
Laryngeal neoplasms
Paclitaxel Muhidrug resistance
