中文摘要：

目的建立耐高三尖杉酯碱人SKM-1细胞系，初步探讨该细胞系的生物学特性及多药耐药机制。方法通过较大剂量间断冲击逐渐增加药物浓度的方法培养人骨髓增生异常综合征转急性髓系白血病细胞系SKM-1细胞，建立耐高三尖杉酯碱的SKM-1细胞系，命名为SKM-1／HHT。光学显微镜下观察耐药细胞系和亲本细胞系的一般形态学变化，MTT法测定倍增时间和耐药指数，绘制生长曲线；利用流式细胞术检测细胞周期分布及细胞内柔红霉素含量；应用常规遗传学方法进行核型分析；采用荧光相对实时定量PCR方法检测多药耐药相关基因（mdrl及MRP）及topo-IIa的表达。结果历经7个月建立了耐药细胞系SKM一1／HHT。耐药细胞与亲本细胞的形态及免疫表型接近；耐药细胞核型与SKM-1细胞基本相同，但更为复杂，存在20、x、4、5、9、11号染色体差异；耐药细胞G，期细胞增多（64．04％对41．91％），S期细胞减少（34．92％对53．53％），G：期细胞减少（1．04％对4．56％）；耐药细胞对高三尖杉酯碱、长春新碱、柔红霉素、依托泊苷耐药性明显增加，耐药指数分别为17．94、8．75、5．99和13．76；耐药细胞内DNR荧光量明显低于亲本细胞（698±36对858±54）；耐药细胞mdr1表达水平明显增高，其2^△Ct值为亲本细胞的20．1倍[（3．42±0．46）×10^12对（0．17±0．01）×10^-2，P〈0．05]；MRP表达亦明显升高，2^△Ct值为亲本细胞的3．56倍[（4．77±0．87）×10^-3对（1．34±0．56）×10^-3，P〈0．05]；topo—IIa基因表达在耐药细胞有所下降，耐药细胞与亲本细胞的2^△Ct值之比为0．619：1[（1．91±0．30）×10^-4对（3．08±0．21）×10^-4，P〈0．05]。结论建立了对高三尖杉酯碱耐药的SKM-1／HHT细胞系，其耐药机制主要涉及mdr1的过度表达而导致的细胞外排增强，MRP及topo-IIa表达水平的改变可能也部分参与了这?

英文摘要：

Objective To estabilish a homoharringtonine （HHT）-resistant SKM-1 cell line and ex- plore its biologic characteristics and mechanisms for drug resistance. Methods The HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short- time duration, and gradually elevating the drug concentration to an endurable level. The morphology of the resistant and parental cell lines was observed through optical microscope. The MTF assay was used to determine the doubling time and the resistance index to draw growth curve. The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry, and the karyo- types by R-banding. Semi-quantitative real-time PCR was performed to evaluate the expression levels of mdrl, MRP and topo- II a. Results The HHT-resistant cell line SKM-1/HHT was eventually established following 7-month drug induction. Both the resistant and the parental cell lines were similar with regard to morphology and immunophenotpye. The karyotypes of the former was more complicated with differences located in chromosome 20, X, 4,5,9 and 11. The resistant cell line had more Gt phase cells （64.04% vs 41.91% ）, less S phase cells（34.92% vs 53.53% ）, and less G2 phase cells（ 1.04% vs 4.56% ） compared with the parental cell line. The SKM-1/HHT cell line showed significant drug resistance to HHT,VCR, DNR and etoposide, the resistance indices of HHT, VCR, DNR and etoposide were 17.94, 8.75, 5.99 and 13.76 respectively. DNR accumulation was impaired in SKM-1/HHT cell line as less fluorescence of DNR（698 ± 36 vs 858 ± 54）. The expression of mdrl increased dramatically in the resistant cell line, its 2^△Ct value was 20. 1 higher than that of the parental cell line [ （ 3.42 ± 0.46）×10^-2 vs （0. 17 ± 0.01 ）×10^-2, p 〈 0.05 ）, while MRP also increased in the resistant by 3.56 folds [ （4.77 ± 0.87）×10^-3 vs （1.34 ± 0.56） ×10^-3, P 〈 0.05 ] ; However there was