姜黄素抗胆囊癌GBC-SD细胞增殖效应的研究

Anti-proliferation effect of curcumin on human gallbladder carcinoma cell line

目的观察姜黄素对人胆囊癌细胞株GBC—SD的抗增殖效应，并探讨其作用机制。方法采用噻唑蓝（M1Tr）法检测0、10、30、50、70mg／L浓度的姜黄素对胆囊癌GBC—SD细胞24h的生长抑制作用；检测30mg／L浓度的姜黄素在0、12、24、48、72h对GBC．SD细胞的抑制作用。依照MTF结果设立对照组、10mg／L姜黄素组、30mg／L姜黄素组及50mg／L姜黄素组分别作用GBC—SD细胞24h。运用流式细胞术检测细胞的凋亡率，使用Westernblot法和逆转录-聚合酶链反应（RT—PCR）法分别检测姜黄素对核因子（NF）-κB与细胞周期素（Cyclin）D1表达的影响，通过实时定量聚合酶链反应（Real-timePCR）法检测姜黄素对bax、B淋巴细胞／白血病-2（bel-2）mRNA表达的影响。结果姜黄素以时间．剂量的方式抑制胆囊癌GBC—SD细胞的活力。10、30、50mg／L浓度的姜黄素作用24h后，细胞存活率分别为（95．34±9．21）％、（70．63±11．09）％、（50．58±6．31）％，与对照组比较，30、50mg／L姜黄素组的差异有统计学意义（P〈0．05）；上述各组细胞的凋亡率分别为（9．83±1．98）％、（37．25±1．21）％和（57．63±7．38）％，与对照组比较，30、50mg／L姜黄素组的差异有统计学意义（P〈0．05）。30、50mg／L的姜黄素均可下调NF—κB和CyclinD1的表达，同时能够降低bcl-2、升高bax的表达，而10mg／L姜黄素对上述基因表达无明显影响。结论姜黄素不仅能够通过下调NF—κB与CyclinDI的表达，还能降低bel-2的mRNA水平、升高bax的mRNA水平导致bel-2／bax的比值下降，从而有效地抑制胆囊癌GBC-SD细胞的增殖、诱导细胞凋亡。

Objective To explore the anti-proliferation effect of curcumin on human gallbladder carcinoma GBC-SD cells and its mechanism. Methods The growth inhibition of GBC-SD cells treated with curcumin of 0, 10, 30, 50, and 70 mg/L was detected by using methyl thiazol tetrazolium （MTT） assay. The growth inhibition of GBC-SD cells treated with curcumin of 30 mg/L at 0, 12, 24, 48 and 72 h was detected. Control group, 10 mg/L curcumin group, 30 mg/L curcumin group and 50 mg/L cureumin group were set up. The apoptosis rate was tested by using flow cytometry. After curcumin treatment for 24 h, the expression levels of nuclear factor-KB （NF-KB） and Cyclin D1 were detected by using Western blotting and reverse transcriptase-polymerase chain reaction （RT-PCR） , respectively. The mRNA levels of bax and B lymphocytes/leukemia-2 （bcl-2） were examined by using real-time reverse transcription polymerase chain reaction. Results Curcumin could significantly inhibit the viability of GBC-SD cells in a time- and dose-dependent manner. After treatment with curcumin （10, 30, and 50 mg/L）, the survival rate was （95.34±9.21 ）% , （70. 63±11.09）% and （50. 58±6. 31 ）% , respectively. There was significant difference between control group and 30 mg/L eurcumin group or 50 mg/L curcumin group （ P 〈 0. 05 ） ; The apoptosis rate was （9. 83± 1.98） % , （37.25 ±1.21 ） % and （57.63 ± 7.38 ） %, respectively. There was significant difference between control group and 30 mg/L eurcumin group or 50 mg/L curcumin group （ P 〈 0. 05 ）. 30 mg/L and 50 mg/L curcumin could down-regulate the expression of NF-KB and CyelinD1, reduce the expression of bcl-2 and enhance the expression of bax. However, lO mg/L curcumin had no evi- dent effect on genes mentioned above. Conclusion Curcumin could not only down-regnlate the expression of NF-KB and Cyclin DI, but also reduce the mRNA level of bcl-2 and up-regulate the mRNA level of bax, causing the reduced bcl-2/bax ratio, which could effectively inhibit the proliferation of gallbladder carcino- ma GBC-SD ceils and induce cell apoptosis.