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目的 为研究脑胶质瘤的耐药、机理及逆转 ,建立了C6耐阿霉素细胞株 (C6/adr)MDR细胞系。方法 采用逐渐增加药物浓度联合反复短期暴露法建立了C6/adr细胞系 ,并对其形态学、超微结构及生长特点进行了研究 ,流式细胞术对细胞周期分布及DNA与RNA含量的变化进行了分析 ,观察了其克隆形成率 ,并对染色体进行了分析 ,用RT PCR法、免疫组织化学及流式细胞免疫学方法研究了多药耐药基因mdr 1及其表达的P gp定性、定量研究 ,用RT PCR法对端粒酶的活性进行了研究 ,建立了该耐药株的动物模型 ,并对其耐药性进行了分析 ,采用MTT法分析了耐药谱。结果 HE染色C6/adr与C6相似 ,透射电镜畸形核多见 ,胞浆内有较多的线粒体、核糖体及滑面内质网 ;群体倍增时间 5 5 2h ;传代 2、 3天 ,C6/adrDNA指数及RNA指数无变化 ;细胞周期分布以G0 +G1期为主 ,克隆形成率可达 5 5 % ;染色体众数多集中于 40～ 42条 ,以亚二倍体居多 ;mdr 1基因表达阳性 ,P gp含量为42 5 1%± 7 82 % ,P gp强阳性 ,主要位于细胞膜上 ,端粒酶呈强阳性 ;动物模型其形态学及P gp含量均无变化 ;该细胞株对ADM ,VM 2 6 ,VCR ,5 Fu ,VP 16 ,CDDP耐药 ,对MTX ,CCNU无耐药性。结论 该耐药细胞系C6/adr耐药表型主要为MDR 1,为研究脑胶质瘤的耐药、逆转提供
Objective To establish a C6 adriamycin-resistant cell line (C6/adr) MDR cell line for the study of drug resistance, mechanism, and reversal of glioma. Methods The C6/adr cell line was established by gradually increasing the drug concentration combined with repeated short-term exposure. The morphology, ultrastructure and growth characteristics of the cell line were studied. The cell cycle distribution and changes in DNA and RNA content were analyzed by flow cytometry. After analysis, the cloning rate was observed, and the chromosomes were analyzed. RT-PCR, immunohistochemistry, and flow cytometric immunoassay were used to study the multidrug resistance gene mdr 1 and its expressed P gp characterization. Quantitative studies were performed to investigate the activity of telomerase using RT PCR. An animal model of this drug resistant strain was established and its drug resistance was analyzed. The drug resistance spectrum was analyzed by MTT assay. Results The HE staining of C6/adr was similar to that of C6. The nucleus of transmission electron microscopy was more common. There were more mitochondria, ribosomes and smooth endoplasmic reticulum in the cytoplasm. The population doubling time was 552 hours; the passage was 2 or 3 days, C6/ There was no change in adrDNA index and RNA index; the cell cycle distribution was dominated by G0 + G1 phase, and the colony formation rate was up to 55%; the majority of chromosomes were concentrated in 40–42 bands, mostly subdiploids; mdr 1 gene expression Positive, P gp content of 42 5 1% 7 7 82%, P gp strong positive, mainly located on the cell membrane, telomerase was strongly positive; animal model its morphology and P gp content did not change; this cell line to ADM , VM 26, VCR, 5 Fu, VP 16, CDDP resistance, no resistance to MTX, CCNU. Conclusion The drug resistant cell line C6/adr resistant phenotype is mainly MDR1, which is provided for studying the resistance and reversal of glioma