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[摘要]
目的通过观察人参皂苷Rh2对人白血病多药耐药(MDR)细胞K562/VCR生长、凋亡的作用和逆转耐药的情况,探索该药在抗人白血病MDR方面的应用价值。方法将人参皂苷Rh2与K562、K562/VCR细胞共培养48 h后采用MTT法分析其对细胞生长的抑制率将其与K562/VCR细胞于37℃孵育30 min后,采用An-nexin V和PI双染法在流式细胞仪上检测细胞凋亡情况并观察其对K562/VCR细胞摄取柔红霉素(DNR)能力及细胞表面P-糖蛋白(P-gp)表达的影响在DNR与K562/VCR培养体系中加入不同质量浓度人参皂苷Rh2,孵育48 h后观察人参皂苷Rh2对K562/VCR细胞耐药的逆转情况。结果人参皂苷Rh2可明显抑制K562和K562/VCR细胞的生长,并呈量效关系,人参皂苷Rh2对K562和K562/VCR的半数抑制浓度(IC50)分别为44.5、59.4μg/mL。K562/VCR经人参皂苷Rh2在37℃作用30 min后,细胞的凋亡明显增加,随人参皂苷Rh2质量浓度的增加,凋亡细胞的比例明显增加[人参皂苷Rh2300μg/mL,Annexin V+细胞(51.5±6.9)%]。K562细胞经长春新碱(VCR)诱导耐药后,P-gp表达率明显提高(从4.28%到93.80%),DNR摄取率减少,25μg/mL以上质量浓度的人参皂苷Rh2即可明显提高K562/VCR对DNR的摄取,但P-gp的表达无明显改变。同时,它可明显提高DNR对K562/VCR的杀伤率,50μg/mL人参皂苷Rh2可使K562/VCR对DNR的敏感性提高到原来的6.30倍。结论人参皂苷Rh2能抑制K562/VCR细胞生长,诱导其凋亡,还可以逆转K562/VCR的耐药,是一种具有广阔开发前景的抗白血病药物。
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[Abstract]
Objective To study the therapeutic effects and their mechanisms of ginsenoside Rh2 on multi-drug resistance of(MDR) leukemic cells by observing the effects of ginsenoside Rh2 on proliferation,apoptosis,and resistance to Vincristine(VCR) of human erythroleukemia cell line K562/VCR.MethodsFirst,ginsenoside Rh2 with different concentration was co-cultured with K562 and K562/VCR cells in 96 wells cell culture plates.The inhibitory rates and 50% inhibitory concentration(IC50) were determined and calculated 48 h later by MTT assay.Second,ginsenoside Rh2 with different concentration was co-cultured with K562/VCR cells in water bath at 37 ℃ for 30 min,then the apoptosis rates were examined by Annexin V/PI apoptosis kit on flow cytometry.Third,ginsenoside Rh2 with different concentration was co-cultured with K562/VCR cells in water bath at 37 ℃ for 30 min followed by adding Daunorubicin(DNR) after washing with PBS.The intake of DNR and the expression of P-glycoprotein(P-gp) were analyzed 30 min later on flow cytometry.Finally,ginsenoside Rh2 with different concentration was co-cultured with K562/VCR cells in 96 well cell culture plates,which were then treated by DNR.The inhibitory rates and reverse effects were evaluated 48 h later by MTT assay.Results K562 and K562/VCR cells' growth were obviously inhibited by ginsenoside Rh2 in a dose-dependent manner.The IC50 values of K562 and K562/VCR were 44.5 and 59.4 μg/mL,respectively.Ginsenoside Rh2 could induce apoptosis of K562/VCR [Rh2 300 μg/mL,Annexin V+ cell(51.5±6.9)%].The apoptosis rate of K562/VCR increased in accordance with the rise of ginsenoside Rh2 concentration.The expression of P-gp increased(4.28% to 93.80%) and the intake of DNR decreased when K562 was resistant to VCR.However,ginsenoside Rh2 with a concentration of 25 μg/mL or higher could greatly enhance the intake of DNR.The inhibitory effects of DNR on K562/VCR could be greatly increased by Rh2.The reverse index was 6.30 when Rh2 concentration was 50 μg/mL.Conclusion Ginsenoside Rh2 could inhibit the growth,induce the apoptosis,and reverse the MDR of K562/VCR.It could be an excellent anti-leukemic agent.
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[基金项目]
浙江省中医药科技计划项目(2005C168);浙江省教育厅科研项目(20061263)