微乳丹参酮逆转肿瘤多药耐药的观察

背景:肿瘤多药耐药的出现,增加了肿瘤治疗的难度。肿瘤多药耐药的形成机制复杂,涉及多个环节,选择不同作用机制的药物联合应用,对肿瘤多药耐药的临床效果可能更有效。目的:观察中药新剂型微乳丹参酮对人白血病细胞株及其阿霉素耐药株的多药耐药性逆转效果。设计:观察对比实验。单位:吉林大学附属第一医院中心实验室。材料:人红白血病细胞株及其阿霉素耐药株,由中科院天津血研所提供。丹参酮、微乳丹参酮,由吉林大学药学院制剂室提供。阿霉素(辉瑞法玛西亚药业),P糖蛋白-PE荧光抗体、B细胞淋巴瘤/白血病-2基因-FITC荧光抗体(Immunotech公司),二甲基亚砜(北京化工厂),噻唑蓝(华美生物)。方法:实验于2003-03/2004-01在吉林大学附属一院中心实验室完成。①将人红白血病敏感细胞株与含100g/L小牛血清、100U/mL青霉素和链霉素的RMPI1640培养液中,在37℃,饱和湿度及体积分数为0.05的CO2细胞培养箱内传代;阿霉素耐药株连续培养于阿霉素上述培养液中,培养条件相同。②将中药制剂微乳丹参酮作为逆转剂,丹参酮和微乳体系作为对照,作用于人红白血病细胞株和阿霉素耐药株。③采用四甲基偶氮唑盐法检测微乳丹参酮,丹参酮及微乳的细胞毒性及抗药性逆转倍数。④分别在对数生长期的细胞株中加入10,20,40,60mg/L的阿霉素,采用荧光分光光度法检测微乳丹参酮,丹参酮及微乳对人红白血病细胞株及阿霉素耐药株中细胞内阿霉素浓度的影响。⑤流式细胞术检测给予10mg/L阿霉素的人红白血病细胞株及加入微乳丹参酮,丹参酮及微乳的阿霉素耐药株中P糖蛋白和B细胞淋巴瘤/白血病-2基因蛋白表达及肿瘤细胞凋亡碎片百分比。主要观察指标:①微乳丹参酮,丹参酮及微乳的细胞毒性及抗药性逆转倍数。②微乳丹参酮,丹参酮及微乳对人红白血病细胞株及阿霉素耐药株中细胞内阿霉素浓度的影响。③给予10mg/L阿霉素的人红白血病细胞株及加入微乳丹参酮,丹参酮及微乳的阿霉素耐药株中P糖蛋白和B细胞淋巴瘤/白血病-2基因蛋白表达及肿瘤细胞凋亡碎片百分比。结果:①微乳丹参酮,微乳体系,中药丹参酮均可显著降低阿霉素耐药株的耐药性,以浓度为0.2mg/L、0.5mg/L的微乳丹参酮的逆转倍数显著高于其他两组(P<0.05)。②微乳丹参酮,微乳体系,中药丹参酮均可使细胞内药物浓度升高,微乳丹参酮作用后的阿霉素耐药株细胞中阿霉素的浓度最高,差异有显著性(P<0.05)。③微乳丹参酮,微乳体系,丹参酮均可降低阿霉素耐药株的P糖蛋白和B细胞淋巴瘤/白血病-2基因表达水平,使肿瘤细胞凋亡比率上升,微乳丹参酮的作用最强,可显著降低P糖蛋白和B细胞淋巴瘤/白血病-2基因表达水平,使肿瘤细胞在ADM作用下的凋亡比率增加(P<0.05)。结论:微乳丹参酮0.5mg/L可引起阿霉素耐药株内药物浓度增加,与P糖蛋白、B细胞淋巴瘤/白血病-2基因表达下调的结论相印证;微乳丹参酮对多药耐药逆转作用较丹参酮和微乳逆转效果更显著。

Tanshinone microemulsion reverses multidrug resistance of tumor

BACKGROUND: Multidrug resistance (MDR) makes the treatment of tumor more difficult. Many factors contribute to the happening of MDR. The combination of two or more kinds of reverse reagent at the same time may be more effective to MDR.OBJECTIVE: To investigate the reversal effect of Tanshinone Microemulsion on MDR of human leukemia cell line (K562) and ADM resistant cell line (K562/ADM) cell line and the mechanism of it.DESIGN: An observational controlled experiment.SETTING: First Affiliated Hospital's Central Laboratory of Jilin University.MATERIALS: K562 and K562/ADM (Tianjin Blood Research Institute of CAS). Tanshinone and Tanshinone Microemulsion (Pharmaceutical College of Jilin University). ADM(Pfizer pharmaceutical corp). P-gp-PE fluorescent antibody and Bcl-2-FITC fluorescent antibody (Immunotech Biotech corp).DMSO(Beijing Chemical Plant).METHODS: The experiment was carried out in the Central Laboratory of First Hospital ffiliated to Jilin University from March 2003 to January 2004. ① K562 was cultured in liquid medium(RMPI1640) which contains 100 g/L calf serum, 100 U/mL penicillin and streptomycin. The culture tank's environment was kept at 37 ℃, with saturated humidity and 5%CO2. K562/ADM was cultured in the same environment as above, but ADM was added to the RMPI1640. ② The reverse effect of Tanshinone Microemulsion (Tanshinone and Microemulsion as control) on K562/ADM cells' MDR is observed. ③Tanshinone Microemulsion,Tanshinone and Microemulsion's MDR reverse fold to K562 cell line was detected by the method of MTT. ④ ADM of different concentrations (10, 20, 40, 60 mg/L)were added to grouped cells of exponential proliferation period. The intracellular drug concentration was observed by detecting fluorescent density.⑤ Expression rate of P-gp, Bcl-2 and apoptosis ratio of K562 and K562/ADM added with Tanshinone Microemulsion, Tanshinone and Microemulsion cell line were observed by the method of Flow Cytometry.MAIN OUTCOME MEASURES: ①Tanshinone Microemulsion, Tanshinone and Microemulsion's MDR reverse fold to K562 cell line. ②Tanshinone Microemulsion, Tanshinone and Microemulsion's effect to K562 cell's intracellular contration.③Tanshinone Microemulsion, Tanshinone and Microemulsion's influence to K562 cell's expression rate of P-gp, Bcl-2 and apoptosis ratio.RESULTS: ①Tanshinone Microemulsion, Tanshinone and Microemulsion could decrease the MDR of the K562/ADM. The effect of Tanshinone Microemulsion were significantly higher than Tanshinone and Microemulsion(P ＜ 0.05 ).②The intracellular concentration of ADM can be improved by Tanshinone Microemulsion, Microemulsion and Tanshinone. The ADM's concentration of K562/ADM treated by Tanshinone Microemulsion is highest and the difference is significant(P＜ 0.05). ③Tanshinone Microemulsion, Tanshinone and Microemulsion could reduce the expression level of P-gp and Bcl-2 in K562/ADM, and increase the apoptosis percentage of tumor cell. Tanshinone Microemulsion has the most significant effect on it,andincreases the apoptosis percentage of tumor cell with ADM (P ＜ 0.05 ).CONCLUSION: Tanshinone Microemulsion (0.5 mg/L) can increase the intracellular concentration of ADM, which is consist with the conclusion of down regulation of the expressions of P-gp and Bcl-2. Reversal effect of Tanshinone Microemulsion is more effective than that of Tanshinone and Microemulsion.