CAJ | 학술논문

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-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(MDR1)、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-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(20.64±1.98)h,长于亲本细胞U251的(10.26±1.03)h.(5)RT-PCR检测结果显示MDR1、bcl-2、MRP5、LRP1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、LRP1的表达上调有关.
목적 채용의탁박감유도건립다약내약세포주U251/VP16.방법 채용농도체증법사U251세포대의탁박감내약,세포계수시제합(CCK-8)법검측기다약내약성；서씨-희모살염색관찰친본세포U251화내약세포주U251/VP16적세포형태；류식세포의검측세포주기적변화；계산세포배증시간；역전록-취합매련반응(RT-PCR)법검측다약내약기인(MDR1)、B림파세포/백혈병-2(bcl-2)、다약내약상관단백5(MRP5)、저밀도지단백수체상관단백1(LRP1)등내약기인적표체변화.결과 (1)성공건립다약내약세포주U251/VP16,기대VP-16、류산장춘신감、배린선알、아매소、체막서알적내약배수분별위:12.36、2.64、2.00、3.61、1.63.(2)광학현미경하내약세포포체증대,정다형성,서씨-희모살염색후가견증대적명현불규칙적포핵,포질내과립양물질증다.(3)세포주기결과현시U251/VP16세포주교친본세포G0/G1기화S기명현감소,G2/M기명현증다.(4) U251/VP16적배증시간위(20.64±1.98)h,장우친본세포U251적(10.26±1.03)h.(5)RT-PCR검측결과현시MDR1、bcl-2、MRP5、LRP1등내약기인적표체상조.결론 채용농도체증법가성공건립다약내약적인뇌효질류세포주차내약성은정,기내약성가능여bcl-2、MRP5、LRP1적표체상조유관.
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 multidrug 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 doubling time was calculated.The expression of multidrug 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 transcription-polymerase chain reaction (RT-PCR).Results (1) Multidrug resistant cell line was constructed and the cell line had resistance to cyclophosphamide,etoposide,vincristine sulfate and doxorubicin hydrochloride; (2) The volume of multidrug resistance cell line was increased,showing pleomorphic under the optical microscope.Increased markedly irregular 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/G1 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 cells was (10.26 ± 1.03) h; (5) The expression of MDR1,bcl-2,MRP5 and LRP(1) was positive.Conclusion The multidrug resistance cell line was established and its drug resistance was stable.The multidrug resistance may be associated with bcl-2,MRP5,LRP1.