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【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 investigate the mechanism of FFJZ combined with doxorubicin (DOX) in reversing multidrug resistance and inducing apoptosis in leukemia K562 / VCR cells. 【Methods】 CCK8 assay was used to detect the drug resistance and drug resistance reversal effect. Flow cytometry was used to detect the total apoptosis rate and early apoptosis rate. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) (MDR1), P-glycoprotein (P-gp), BCRP, Bax and B-lymphoblastoma-2 (Bcl-2) gene expression; Western blot detection of Bax, Bcl-2 protein expression. 【Results】 The results of CCK-8 showed that the cytotoxicity of FFJZ (6 mg / m L) and DOX (5 mg / m L) alone could significantly reduce the half inhibitory concentration index (IC 50) of K562 / VCR (P <0.05). The reversal index of K562 / VCR was significantly higher than that of K562 alone (P <0.05). Flow cytometry showed that FFJZ combined with DOX (5 mg / mL) at a concentration of 4,6 mg / mL could increase the total apoptosis rate and early apoptosis rate of K562 / VCR cells compared with DOX control group There was statistical significance (P <0.05). The results of RT-PCR showed that FFJZ combined with DOX could down-regulate the expression of MDR1, BCRP and P-gp m RNA, but there was no significant difference compared with DOX control group (P> 0.05) Compared with DOX control group, the difference was statistically significant (P <0.01). Western blot results showed that Bax protein expression was significantly upregulated (P <0.05), Bcl-2 protein expression was significantly down-regulated (P <0.05), and was consistent with the expression of Bax and Bcl-2 mRNA in FFJZ combined with DOX. 【Conclusion】 The cytotoxicity of FFJZ and DOX reverses the drug resistance of K562 / VCR cells. The combination of the two drugs has obvious synergistic effect. The mechanism may be related to its up-regulation of Bax and down-regulation of anti-apoptotic gene Bcl -2 expression.