人肺腺癌紫杉醇耐药细胞株的建立及其生物学特性研究

Establishment and characteristics of a paclitaxel resistant human lung adenocarcinoma cell subline(SPC-A1/Taxol)

目的建立耐紫杉醇的人肺腺癌细胞株模型SPC-A1/Taxol,并初步研究其生物学特性。方法应用浓度递增和短时作用法,从人肺腺癌细胞株SPC-A1中诱导分离出对紫杉醇耐药的细胞亚株(SPC-A1/Taxol)。采用MTT法检测耐药细胞的耐药指数及对抗癌药物的敏感性;光镜和透射电镜观察细胞形态;以流式细胞术检测该耐药细胞的细胞周期分布;以免疫细胞化学染色检测多药耐药基因(mdr1)的表达产物P-糖蛋白(P-gp)。结果经MTT法检测,SPC-A1/Taxol细胞的紫杉醇半数抑制浓度(IC50)是亲代SPC-A1细胞的759.46倍,对Taxotere、NVB、ADM呈高度耐药状态,对VP-16和HCPT有轻度的耐药,而对DDP、GEM以及中药榄香烯乳无交叉耐药现象。耐药细胞的群体倍增时间是亲代细胞的1.32倍;细胞形态未见明显改变;G0+G1细胞比例减少,S期细胞增多。SPC-A1细胞无P-gp表达,SPC-A1/Taxol则高表达P-gp。结论SPC-A1/Taxol肺腺癌细胞株是一个明确的多药耐药模型,具有耐药细胞的基本生物学特性,推测其多药耐药性与P-gp的高表达相关。

Objective：To establish a new paclitaxel resistant human lung adenocarcinoma cell subline （SPC-A1/Taxol） and investigate its characteristics. Methods： A paclitaxel resistant human lung adenocarcinoma cell subline（SPC-A1/Taxol） was developed by intermittent exposure to gradually increasing concentration of Taxol from the parent cell line SPC-A1 in vitro. The multidrug resistance of SPC-A1/Taxol to anticancer agents was evaluated by MTT assay. The morphological features and the celluar ultrastructure characteristics were observed respectively by light microscopy and transmission electron microscopy. The distribution of its cell cycle was detected by flow cytometry. The expression of P-glycoprotein （P-gp） was detected by immunocytochemistry staining. Results：SPC-A1/ Taxol cells were of 759. 46-fold resistance to Taxol and displayed high resistance to Taxotere, NVB, ADM, but low resistance with VP-16and HCPT. No cross-resistance was observed to DDP,GEM and Elemene emusion. Comparing with the parent cells, the multiplication time of SPC-A1/Taxol cells was prolonged, their morphology was not changed obviously, and the proportion of cells in （G0 ＋ G1 ）-phase decreased while those in S-phase increased. The expression of P-gp in resistant cells was significantly higher than in their parent cells. Conclusions：SPC-A1/Taxol cell subline is a typical multidrug-resistant cell line which has basic characteristics of drugresistance cells. It was supposed that the multidrug resistance of SPC-A1/Taxol was related to the over expression of P-gp.