汉防己甲素干预K562细胞mdr1基因表达的研究

目的 观察汉防己甲素(TTD)对阿霉素诱导的K562细胞mdr1基因表达的影响,并初步探讨其机制.方法 采用MTT法观察TTD对K562细胞的毒性作用,以0.6μg/ml阿霉素单独作用或0.6μg/ml阿霉素联合不同浓度的TTD(0.5、1.0、2.0μg/ml)作用于K562细胞,用RT-PCR法检测mdr1 mRNA、NF-kB mRNA水平,用流式细胞术检测P-gP的表达情况,用胞内罗丹明123(Rho123)积聚试验检测P-gP的功能.结果 空白对照K562细胞未见明显mdr1 mRNA及P-gP表达(水平分别为0.171±0.012、7.85±0.15),细胞内Rho123平均荧光强度(反映P-gP功能)为711.9±63.6,NF-kBmRNA水平为0.783±0.090;0.6μg/ml阿霉素作用24 h后mdr1 mRNA、P-gP、NF-kB mRNA表达上调为0.428±0.012、73.68±1.84、1.075±0.047,细胞内Rho123平均荧光强度下降为347.8±60.6(P值均＜0.05);2.0μg/ml TTD预作用24 h再与0.6 μg/ml阿霉素联合作用24 h能显著抑制阿霉素诱导的mdr1 mRNA、P-gp表达及功能、NF-kB mRNA的上调(分别为0.148±0.006、7.18 ±0.38、799.7±45.8、0.627±0.098)(P＜0.05),而0.5、1.0μg/TTD无明显影响.结论 TTD以浓度依赖性的方式干预阿霉素诱导的mdr1 mRNA、P-gp表达和P-gp功能的上调,其机制可能与TTD抑制了阿霉素诱导的NF-kB的表达有关.

Effect of tetrandrine on the doxorubicin-induced expression of mdr1 gene in K562 cells

Objective To investigate the effect of tetrandrine(TTD)on doxorubicin-induced mdr1 gene expression and its mechanism.Methods MTF assay was used to detect the cytotoxicity of TTD to K562 cells.K562 cells were treated with doxorubicin alone or 0.6μg/ml doxorubicin combined with various concentrations of TTD.RT-PCR was used to detect the mRNA expression of mdr1 and NF-kB.Flow cytometry was used to assay the expression of P-glycoprotein(P-gP).Intracellular rhedamine 123(Rho123)retention assay was applied to test the P-gp function.Results After treatment with 0.6μg/ml doxorubicin for 24 hours,the expressions of mdr1 mRNA,NF-kB mRNA and P-gP in K562 cells were increased from 0.171±0.012,0.783±0.090,7.85±0.15 to 0.428±0.012,1.075±0.047 and 73.68±1.84,respectively.The intracellular Rho123 retention was decreased from 711.9±63.6 to 347.8 ±60.6.indicating up-regulation of P-go function(P＜0.05).Pretreatment of K562 ceUs with 2.0μg/ml TTD for 24 hours and then incubated for another 24 h with doxorubicin,the expressions of mdr1 mRNA,NF-kB mRNA,P-gp and up-regulation of P-gP function induced by doxorubicin were prevented in K562 cells(0.148±0.006.0.627±0.098,7.18±0.38and 799.7±45.8,respectively P＜0.05).But 0.5μg/ml and 1.0μg/ml TTD had little effect.Conclusions TTD inhibits the expression of mdr1 mRNA,P-gP and up-regulated P-gP function induced by doxorubicin in a dose dependent manner.The mechanism of this effect may be down-regulation of NF-kB by TTD.