无血清培养联合多柔比星诱导A549细胞耐药

目的： 探索快速建立肺癌耐药细胞模型的新方法，并探讨肺癌耐药和干细胞的相关性。方法： 应用无血清培养联合药物筛选法建立肺腺癌细胞耐药株A549/ADM；MTT法检测其对多种常用化疗药物的耐药程度；流式细胞仪检测细胞周期和凋亡、ABCB1和ABCG2的表达水平及细胞内多柔比星的浓度；细胞免疫荧光及流式细胞仪通过特异性表面抗原标记法（CD133）检测肿瘤干细胞含量。裸鼠皮下种植瘤试验评估A549/ADM的致瘤性。结果： A549细胞经无血清培养可见细胞球形成；A549/ADM细胞对多柔比星、长春新碱、紫杉醇均产生了耐药，其耐药指数分别为A549的13.167，12.86，3.4倍；与亲代A549相比， 耐药株A549/ADM S期细胞比例由(45.76±1.41)% 降低至(23.33±1.32)%,细胞凋亡百分率由(58.23±0.82)% 减少至(16.49±0.77)%；耐药株A549/ADM中ABCB1和ABCG2的表达水平明显增高(P<0.05)，且细胞内多柔比星浓度明显低于A549细胞； CD133细胞在A549/ADM和A549中的比例分别为(6.2±0.7)％和(1.9±0.2)％(P<0.05)，免疫荧光检测A549/ADM细胞CD133表达水平较A549细胞高；A549/ADM具有高致瘤性。结论： 无血清培养联合多柔比星诱导能高效富集肺癌干细胞，成功建立了人肺腺癌A549/ADM多药耐药细胞模型。

Establishment of multidrug resistant adenocarcinoma cell line A549/ADM model treated with serum free floating-culture system combined with doxorubicin

Objective: To establish a doxorubicin-resistant cell line A549/ADM and research the relationship between A549/ADM and human lung carcinoma stem cell.Methods: Doxorubicin-resistant lung adenocarcinoma cell line A549/ADM was obtained by treating lung adenocarcinoma A549 in vitro with increasing dosage of doxorubicin in serum free medium.The resistant to chemotherapeutic drugs was evaluated with MTT assay.Cell cycle,expressions of ABCB1 and ABCG2 and concentration of doxorubicin was determined by flow cytometry.CD133 positive cells explore were determined by the immunofluorescent staining and flow cytometry to examine lung carcinoma stem cell proportion.Tumorigenic potential was performed by nude mice xenograft transplant experiments.Results: A549/ADM cells were proved multidrug-resistant.The cells showed resistance to ADM,VCR,Taxol,and the resistant index of A549/ADM was 13.167,12.86,3.4.Cells in the period S was(45.76±1.41)% in A549 whereas(23.33±1.32)% in A549/ADM.The apoptosis of A549 cell was(58.23±0.82)% whereas(16.49±0.77)% in A549/ADM.Compared with the parental cell line,A549/ADM cell had a higher expression level of ABCB1 and ABCG2(P<0.05).The concentration of doxorubicin in the A549/ADM is obviously lower than that in the A549 cell.The immunofluorescent staining and flow cytometry analysis showed that the expression levels of CD133 were significantly higher in A549/ADM cell.A549/ADM cell were highly tumorigenic in nude mice.Conclusion: Doxorubicin-resistant lung adenocarcinoma cell line A549/ADM was obtained by treating lung adenocarcinoma A549 in vitro with increasing dosage of doxorubicin in serum free medium.cell line A549/ADM had a higher proportion of lung adenocarcinoma stem cells compared to its parental cell line A549.