摘要
目的探讨转多药耐药基因mdr1的K562/MDR细胞株作为单机制耐药模型的可行性,为进一步研究肿瘤耐药及其逆转奠定基础。方法实验分为3部分:(1)在电子显微镜下观察慢性髓细胞白血病急性红白变敏感细胞系K562,阿霉素(adriamycin,ADM)诱导耐药细胞株K562/ADM和K562/MDR耐药细胞株的生物学行为;同时测定3种细胞系的群体倍增时间;以观察药物诱导和基因转移是否对细胞的生物学行为造成影响。(2)以K562细胞为对照,用MTT法分别测定阿霉素、柔红霉素(daunorubicin,DNR)、长春新碱(vincristine,VCR)对3种细胞的半数致死量(IC50)。(3)多药耐药相关基因与蛋白的检测。免疫细胞化学法观察mdr1基因编码的P-糖蛋白(P-gp)的表达;流式细胞术检测P-gp、bcl-2的表达百分率;生化法测定细胞内谷胱甘肽S-转移酶(GSTs)活性;RT-PCR法检测拓扑异构酶(to-poisomeraseⅡ,topoⅡ)mRNA的表达变化。结果(1)在超微结构上,K562/ADM的细胞器—线粒体出现水肿,K562和K562/MDR未见明显异常;K562的群体倍增时间为19.67±3.10d;K562/MDR为20.40±1.80d;K562/ADM为28.47±1.75d;(2)K562/ADM和K562/MDR细胞对ADM的耐药倍数分别为23.1和1.2倍;对DNR为84.9和14.4倍;对VCR为298.3和10.1倍。(3)与K562比较,K562/ADM细胞的P-gp和Bcl-2蛋白表达率高且topoⅡcDNA片段大小发生变化;K562/MDR仅P-gp表达率高。结论K562/MDR的生物学行为与亲本细胞K562相似,耐药机制单一,可作为单机制耐药模型,对某一耐药基因进行更为深入精确的研究,也可针对该耐药基因准确地筛选相应的逆转剂。
Objective To explore the feasibility of K562/MDR as a model possessing single drug resistant mechanism in order to lay a foundation for further studies of tumors＇ multidrug resistance and its reversal. Methods Our work was composed of three parts： （1） Observing biological characters of K562, K562/ADM and K562/MDR cell lines with electron microscopy and counting multiplication time. （2） Using K562 as the control, applying MTT method to examine the IC50 value of the three cell lines to ADM, DNR and VCR. （3） Detection of multidrug resistance-related genes and proteins. Immunocytochemistry was used to observe the expr,ession of P-glycoprotein （P-gp）, flow cytometry to quatitatively determine the expression rate of P-gp and Bcl-2, biochemistry to detect the activity of GSTs, and RT-PCR to determine topoisomerase Ⅱ mRNA expression. Results （1） Under electron microscope, KS62/ADM - showed swelling mitochondria. （2） The resistance multiple of K562/ADM and K562/MDR to ADM, DNR and VCR was 23. 1 and 1.2, 84.9 and 14.4,298. 3 and 10. 1. （3） Compared with K562, K562/ADM showed high expression level of multidrug resistance-related genes and proteins and changed topoisomerase Ⅱ cDNA. K562/MDR only had high expression rate of P-gp. Conclusion The biological charaters of KS62/MDR are similar to those of its drug sensitive parent cell K562. K562/MDR has single drug resistance mechanism, which makes it possible to further study a certain drug-resistant gene and exactly screen the relative reversal agents.
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2006年第1期66-71,共6页
Chinese Journal of Histochemistry and Cytochemistry
基金
辽宁省教委重点项目基金资助(9707111010)
关键词
多药耐药
基因转移
生物学行为
耐药机制
Multidrug resistance
Gene transfer
Biological characterization
Mechanism of drug resistance
