白血病细胞多药耐药细胞系的建立及白细胞介素4对其多药耐药的逆转研究

Establishment of multi drug resistant cell line HL-60/VCR and the reversal of multidrug resistance by interleukin4

目的 研究重组人白细胞介素 4(IL 4)对人白血病细胞 (HL 6 0 )多药耐药 (MDR)的逆转作用及可能机制。方法 采用长期、间隔、单一、小剂量用药的方法诱导HL 6 0细胞系成为MDR细胞长春新碱 (VCR)系HL 6 0 /VCR ,建立MDR细胞模型 ;以流式细胞仪间接免疫荧光染色检测HL 6 0、HL 6 0 /VCR及IL 4处理的HL 6 0 /VCR细胞中p 糖蛋白 (p gp)表达 ;四氮甲基唑蓝 (MTT)生物活性法及荧光分光光度法检测上述各种细胞对VCR、阿霉素 (14 羟柔红霉素 ,ADM)、柔红霉素 (DNR)、足叶乙甙 (VP 16 )等化疗药物的敏感性及ADM在肿瘤细胞中的聚集量。结果 HL 6 0 /VCR细胞中p gp呈高强度表达 ,阳性率为 (95 .4± 1.5 ) % ,较HL 6 0细胞 [p gp表达阳性率仅 (1.5± 0 .5 ) % ]差异有显著性(n =10 ,t=6 2 .5 ,P <0 .0 1) ;IL 4处理的HL 6 0 /VCR细胞株中的MDR瘤细胞p gp表达降低 [处理48hp gp表达降至 (11.3± 2 .9) % ],增加化疗药物的细胞内聚集量 ,恢复MDR细胞的药物敏感性 ,(耐药逆转倍数可达 3.0 ) ,此作用可被蛋白激酶C(PKC)激活剂———佛波酯 10 0ng/ml完全阻断。结论 长期、间断、单一、不足量的化疗可导致MDR ;IL 4可通过抑制肿瘤细胞的p gp表达 ,增加化疗药物在肿瘤细胞内的聚集而逆转白血病细胞的MDR ,此作用可能与?

Objective To investigate the reverse effect of interleukin 4 (IL 4) and its mechanism in multidrug resistance (MDR) of human leukemic cells. Methods A long term, intemittent, singular, uneffcient vincristine was added step by step into the culture medium of tumor cells to induce human promyeloid leukemia cell line HL 60 into multi drug resistant cell line HL 60/VCR; MTT assay was used to detect the sensitivity of these tumor cells to all sorts of chemotherapeutic agents such as VCR, Adriamycin (ADM), mitomycin (MMC) and etoposide (VP 16). Indirect immunofluorescence staining by flow cytometry was used to evalue the expression of p glycoprotein (p gp), which is one of the main multi drug resistant protein in tumor cells. Microquantitative analysis by fluorometer was employed to detect the intracellular accumulation of ADM. Results Over expression of p gp (the positive rate of p gp expression was 95.4%) was found in HL60/VCR cells while the p gp could only be detected in 1.5% of HL 60 cells, the difference was significant (n=10, t=62.5, P<0.01). HL 60/VCR treated with interleukin 4 could decrease the expression of p gp (the positive rate of p gp expression was down to 11.3% after 48 hours) and the quantity of intracellular accumulation of ADM was greatly increased. At the same time, the HL 60/VCR cells became again sensitive to all the above agents, the reversal index (RI) was 3.0 . These effects could be inhibited by 100 ng/ml 12 0 tetradecanoylphorbol 13 acetate (TPA), which is an activator of protein kinase C (PKC). Conclusion The treatment of leukemic cell with a longterm, intermittent,singular, unefficient chemotherapeutic agents could induce multi drug resistant cells. Interleukin 4 can reverse the multi drug resistance by inhibiting the expression of p gp and increasing the quantity of intracellular chemotherapeutic agents in tumor cells. The effects were probably related to the inhibition of the PKC activity.