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目的研究孕激素拮抗剂米非司酮对白血病多药耐药细胞 K562/A02的逆转作用及其机制。方法 MTT 法检测米非司酮作用72 h 后 K562/A02细胞增殖及其对阿霉素杀伤敏感性的变化;流式细胞术检测米非司酮作用前后 K562/A02细胞表面 P 糖蛋白的表达和细胞内柔红霉素的浓度;免疫组化法观察米非司酮作用前后 K562/A02细胞凋亡相关蛋白 bcl-2、Bax、caspase-3的表达;RT-PCR 检测米非司酮作用后对 K562/A02细胞内葡萄糖神经酰胺合成酶(GcS)mRNA 表达的影响。结果2.5、5.0和10.0μmol/L 米非司酮不抑制 K562/A02细胞的增殖,但上述浓度的米非司酮作用后K562/A02细胞对阿霉素的敏感性较前分别增强了1.68、4.17和10.71倍。K562/A02细胞表面 P 糖蛋白的表达为(49.03±5.32)%,10 μmol/L 米非司酮作用72 h 后降低到(28.60±2.13)%(P<0.01);K562/A02细胞内柔红霉素的浓度为(61.07±8.61)%,而10 μmol/L米非司酮作用后升高到(92.72±3.48)%(P<0.01)。经10 μmol/L 米非司酮作用后,bcl-2蛋白表达由(56±9)%降低到(37±6)%(P<0.05);Bax 蛋白由(40±5)%升高到(87±10)%(P<0.01);caspase-3蛋白则由(36±7)%升高到(89±6)%(P<0.01)。RT-PCR 结果显示 K562/A02细胞 GcS mRNA 的表达较K562细胞明显升高,10 μmol/L 米非司酮能明显降低 K562/A02细胞内 GcS mRNA 的表达。结论米非司酮可逆转白血病 K562/A02细胞的多药耐药,且具有剂量依赖性。10 μmol/L 米非司酮能明显逆转白血病 K562/A02细胞的多药耐药,其机制与降低 P 糖蛋白的水平,调节凋亡相关蛋白 bcl-2、Bax、caspase-3的表达,降低 GcS mRNA 有关。
Objective To study the reversal effect and mechanism of progestin antagonist mifepristone on multidrug resistant K562 / A02 leukemia cells. Methods The proliferation of K562 / A02 cells induced by mifepristone and the sensitivity to adriamycin were detected by MTT assay. The expression of P-glycoprotein on K562 / A02 cells was detected by flow cytometry And the concentration of intracellular daunorubicin. The expression of apoptosis-related proteins bcl-2, Bax and caspase-3 in K562 / A02 cells before and after mifepristone treatment were observed by immunohistochemistry. The effects of mifepristone On K562 / A02 intracellular glucose ceramide synthase (GcS) mRNA expression. Results Mifepristone 2.5, 5.0 and 10.0 μmol / L did not inhibit the proliferation of K562 / A02 cells. However, the sensitivity to doxorubicin of K562 / A02 cells increased by 1.68 after the above concentration of mifepristone, 4.17 and 10.71 times. The expression of P-glycoprotein on K562 / A02 cells was (49.03 ± 5.32)% and decreased to 28.60 ± 2.13% (P <0.01) after treated with 10 μmol / The concentration of erythromycin was (61.07 ± 8.61)%, while it increased to 92.72 ± 3.48% (P <0.01) after 10 μmol / L mifepristone treatment. The expression of bcl-2 decreased from (56 ± 9)% to (37 ± 6)% (P <0.05) after 10 μmol / L mifepristone treatment. The Bax protein increased from (40 ± 5)% to (87 ± 10)% (P <0.01). The protein level of caspase-3 increased from (36 ± 7)% to (89 ± 6)% (P <0.01). The results of RT-PCR showed that the expression of GcS mRNA in K562 / A02 cells was significantly higher than that in K562 cells. Mifepristone (10 μmol / L) significantly decreased the expression of GcS mRNA in K562 / A02 cells. Conclusion Mifepristone can reverse the multidrug resistance of K562 / A02 cells in a dose-dependent manner. Mifepristone at a concentration of 10 μmol / L reversed the multidrug resistance of leukemic K562 / A02 cells, and its mechanism was related to the decrease of P-glycoprotein level and the expression of bcl-2, Bax and caspase-3 GcS mRNA related.