中药复方君子汤联合阿霉素逆转白血病K562/VCR细胞多药耐药及诱导凋亡的机制研究  

Mechanism of Chinese Medicine Compound Junzi Recipe Combined with Doxorubicin for Reversing Multidrug Resistance and Apoptosis of Leukemia K562/VCR Cells

作　　者：林婷[1] 廖斌 徐成波 齐彦 陈毅宁 陈佳薇 

机构地区：[1]福建中医药大学,福建福州350108 [2]福建中医院大学附属人民医院,福建福州350004

出　　处：《广州中医药大学学报》2017年第1期95-100,共6页Journal of Guangzhou University of Traditional Chinese Medicine

基　　金：福建省自然科学基金资助项目(编号:2014J01348;201501327)

摘　　要：【目的】探讨中药复方君子汤(FFJZ)联合阿霉素(DOX)逆转白血病K562/VCR细胞多药耐药及诱导凋亡的机制。【方法】采用CCK8法检测细胞耐药性及耐药性逆转作用;采用流式细胞术检测细胞总凋亡率和早期凋亡率;采用实时荧光定量逆转录—聚合酶链反应(RT-PCR)检测多药耐药基因1(MDR1)、P糖蛋白(P-gp)、乳腺癌耐药相关蛋白(BCRP)、B淋巴细胞瘤-2相关X蛋白(Bax)和B淋巴细胞瘤-2(Bcl-2)的基因表达水平;Western blot法检测Bax、Bcl-2蛋白表达。【结果】CCK-8检测结果显示:无细胞毒性的FFJZ(6 mg/m L)、DOX(5 mg/m L)单独用药可显著降低K562/VCR的半数抑制浓度指数(IC_(50))(P<0.05),两药联合应用对K562/VCR逆转指数显著高于单独应用(P<0.05)。流式细胞术结果显示:作用浓度为4、6 mg/m L的FFJZ联合DOX(5 mg/m L)能够提高K562/VCR细胞总凋亡率和早期凋亡率,与DOX对照组比较差异有统计学意义(P<0.05)。RT-PCR结果显示:FFJZ联合DOX应用可下调MDR1、BCRP、P-gp m RNA表达,与DOX对照组比较,差异无统计学意义(P>0.05);FFJZ联合DOX应用可上调Bax m RNA、下调Bcl-2 m RNA表达,与DOX对照组比较,差异有统计学意义(P<0.01)。Western blot结果显示:FFJZ联合DOX与对照组比较,Bax蛋白表达显著上调(P<0.05),Bcl-2蛋白表达显著下调(P<0.05),与Bax、Bcl-2 m RNA表达一致。【结论】无细胞毒性的FFJZ、DOX对K562/VCR细胞耐药性均有逆转作用,两药联合应用具有明显的协同效应,其机制可能与其上调促凋亡基因Bax和下调抗凋亡基因Bcl-2的表达有关。Objective To explore the mechanism of Chinese medicine compound recipe, Fufang Junzi Recipe（FFJZ）,combined with doxorubicin（DOX）for reversing multidrug resistance（MDR）and apoptosis of leukemia K562/VCR cells. Methods CCK8 assay was used to detect the drug resistance of the cell lines and the reversal of drug resistance. Overall apoptotic rate and early apoptotic rate were detected by flow cytometry. The expression levels of multidrug resistance 1（MDR1）,permeability glycoprotein（P-gp）,breast cancer resistance（BCRP）,B-cell lymphoma-2（Bcl-2）,Bcl-2 associated X protein（Bax）mRNA were determined by real-time fluorescent quantitative reverse transcription ploymerase chain reaction（RT-PCR）. The expression levels of Bax and Bcl-2proteins were assessed by Western blotting method. Results Cell counting Kit-8 assay results showed that FFJZ at non-toxic concentration of 6 mg/m L or DOX at non-toxic concentration of 5mg/m L could decreased the half maximal inhibitory concentration（IC_（50））of K562/VCR cell lines（P 0.05）,and the effect of combined use of FFJZ and DOX was superior to that of the single application（P 0.05）. The flow cytometry results indicated that FFJZ（4,6 mg/m L） combined with DOX（5 mg/m L） increased the overall apoptotic rate and early apoptotic rate of K562/VCR cells, and the difference was significant compared with DOX group（P 0.05）.RT-PCR results showed that the expression levels of MDR1, BCRP, P-gp mRNA were down-regulated by FFJZ combined with DOX（P 0.05 compared with the DOX control group）,and FFJZ combined with DOX up-regulated Bax mRNA expression and down-regulated Bcl-2 mRNA expression（P 0.01 compared with the DOX control group）. Western blotting results showed that the expression level of Bcl-2 protein was down-regulated and that of Bax protein was up-regulated by FFJZ combined with DOX, the difference being significant compared with those of the DOX control group（P 0.05）,and the results were consistent with those of mRNA expr

关 键 词：中药复方君子汤/药理学 阿霉素/药理学 多药耐药 白血病/中西医结合疗法 K562/VCR 基因表达调控 细胞培养 

分 类 号：R285.5[医药卫生—中药学]