摘要
目的采用依托泊苷诱导建立多药耐药细胞株U251／VP16。方法采用浓度递增法使U251细胞对依托泊苷耐药，细胞计数试剂盒（CCK-8）法检测其多药耐药性；瑞氏一姬姆萨染色观察亲本细胞U251和耐药细胞株U251／VP16的细胞形态；流式细胞仪检测细胞周期的变化；计算细胞倍增时间；逆转录-聚合酶链反应（RT—PCR）法检测多药耐药基因（MDRI）、B淋巴细胞／白血病-2（bcl-2）、多药耐药相关蛋白5（MRP5）、低密度脂蛋白受体相关蛋白1（LRP1）等耐药基因的表达变化。结果（1）成功建立多药耐药细胞株U251／VP16，其对VP-16、硫酸长春新碱、环磷酰胺、阿霉素、替莫唑胺的耐药倍数分别为：12．36、2．64、2．00、3．61、1．63。（2）光学显微镜下耐药细胞胞体增大，呈多形性，瑞氏一姬姆萨染色后可见增大的明显不规则的胞核，胞质内颗粒样物质增多。（3）细胞周期结果显示U251／VP16细胞株较亲本细胞G0／G1期和S期明显减少．G2／M期明显增多。（4）U251／VP16的倍增时间为（20．644-1．98）h，长于亲本细胞U251的（10．26±1．03）h。（5）RT—PCR检测结果显爪MDRl、bcl-2、MRP5、LRPl等耐药基因的表达上调。结论采用浓度递增法可成功建立多药耐药的人脑胶质瘤细胞株且耐药性稳定，其耐药性可能与bcl-2、MRP5、LRPI的表达上调有关。
Objective The multidrug resistant cell line U251/VP16 was established and induced by etoposide. Methods The U251 cells resistant to etoposide were make by increasing concentrations. The muhidrug resistance was detected by cell counting Kit-8 （CCK-8）. The morphology of U251 and U251/ VP16 cells was observed after Wright-Giemsa staining. Cell cycle was tested by flow cytometry. Cell dou- bling time was calculated. The expression of muhidrug resistance （ MDR1 ） , B lymphocytes/leukemia-2 （bcl-2）, multidrug resistance associated protein 5 （MRP5） and low density lipoprotein receptor related protein 1 （ LRP1 ） was assayed by using reverse transcripfion-polymerase chain reaction （RT-PCR）. Re- suits （ 1 ） Muhidrug resistant cell line was constructed and the cell line had resistance to cyclophospha- mide, etoposide, vincristine sulfate and doxorubicin hydrochloride; （2） The volume of multidrug resist- ante cell line was increased, showing pleomorphic under the optical microscope. Increased markedly itTeg- ular nucleus was observed and particles like substances were increased; （ 3 ） The results of cell cycle showed that the proportion of U251/VP16 cells in G0/G~ and S phases was decreased, while that in G2/M phase was increased significantly; （4） The doubling time of U251/VP16 cells was （20. 64 ＋ 1.98） h and that of U251 ceils was （ 10. 26 ＋ 1.03） h; （5） The expression of MDR1, bcl-2, MRP5 and LRP1 was positive. Conclusion The muhidrug resistance cell line was established and its drug resistance was stable. The multidrug resistance may be associated with bcl-2, MRP5, LRP1.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第9期1661-1663,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30973072）
