柚皮苷逆转人卵巢癌耐药SKOV3／DDP细胞耐药性及逆转机制的研究

目的 研究柚皮苷对卵巢癌顺铂（DDP）耐药细胞SKOV3／DDP体外增殖的影响及耐药逆转作用，并初步探讨相关耐药机制。方法采用MTT法测定DDP对SKOV3和SKOV3／DDP细胞的半数抑制浓度（IC₅₀），柚皮苷对SKOV3／DDP细胞的增殖抑制作用及柚皮苷联合DDP对SKOV3／DDP细胞的增殖抑制作用。筛选出非细胞毒性的柚皮苷浓度，将实验细胞分为空白对照组、2.5 μg／ml DDP组、10 μmol/L柚皮苷组、10 μmol/L柚皮苷联合2.5 μg／ml DDP组，分别采用RT-PCR和Western blotting测定各组作用48 h后耐药基因MDR1 mRNA及MRP2 mRNA的表达及其相应的表达产物P-gp、MRP2蛋白的表达水平。结果1～32 μg／ml DDP处理SKOV3及SKOV3／DDP细胞48 h后，SKOV3细胞的IC₅₀为5.48 μg／ml，SKOV3／DDP细胞为14.93 μg／ml，耐药指数为2.72。柚皮苷对SKOV3／DDP细胞有明显的增殖抑制作用，且呈剂量和时间依赖性。选取10 μmol／L柚皮苷作为逆转耐药实验浓度，10 μmol／L柚皮苷联合DDP作用48 h后，SKOV3／DDP细胞对DDP的耐药指数为1.62，逆转倍数为1.68。RT-PCR及Western blotting检测显示，柚皮苷联合DDP组与DDP单药组比较，MDR1 mRNA、MRP2 mRNA表达及P-gp、MRP2蛋白表达均明显下降，差异有统计学意义（P＜0.05）。结论柚皮苷对卵巢癌SKOV3／DDP细胞的体外增殖具有明显抑制作用，并能逆转SKOV3／DDP细胞的耐药性，其逆转耐药的机制可能与下调耐药基因MDR1 mRNA及MRP2 mRNA及相应的P-pg、MRP2蛋白的表达有关。

The study on drug reverse effect and reverse mechanism of naringin on multi-drug resistant human ovarian cell line SKOV3/DDP

Objective To investigate the effect of naringin on the proliferative inhibition of cisplatin ( DDP )-resistant ovarian cancer SKOV3/DDP cells in vitro and its reverse effect of drug resistance, as well as the possible mechanisms. Methods DDP to half maximal inhibitory concentration ( IC50 ) on SKOV3 and SKOV3/DDP cells was determined by MTT method. The proliferatiive inhibi-tion of naringin alone and naringin in combination with DDP on SKOV3/DDP cells was tested by MTT method. Non-cytotoxic naringin was selected, and SKOV3/DDP cells were divided into blank control group, 2. 5μg/ml DDP group, 10μmol/L naringin group and 10μmol/L naringin+2. 5 μg/ml DDP group. RT-PCR and Western blotting was used to detect the MDR1 and MRP2 mRNA levels and protein expressions of P-gp and MRP2. Results After treated with 1-32 μg/ml DDP for 48 h, the IC50 of SKOV3 cells and SKOV3/DDP cells were 5. 48 μg/ml and 14. 93μg/ml, and the resistance index ( RI) was 2. 72. Naringin could effectively restrain the prolif-eration of SKOV3/DDP cells in a time and dose dependent manner. The dose of 10μmol/L naringin was chosen for the further experi-ment. The RI was 1. 62 and the drug resistance reversal fold was 1. 68 times after 10μmol/L naringin+2. 5μg/ml DDP acted on SK-OV3/DDP cells. The mRNA levels of MDR1 and MRP2, as well as the protein expressions of P-gp and MRP2 decreased significantly in 10 μmol/L naringin+2. 5 μg/ml DDP group compared with 2. 5 μg/ml DDP group ( P<0. 05) . Conclusion Naringin can signifi-cantly inhibit the proliferation of SKOV3/DDP cells in vitro, and can enhance the sensitivity of SKOV3/DDP cell to DDP. The reversal mechanism of drug resistance may be related to the inhibition of MDR1 and MRP2 mRNA and related proteins.