脂多糖通过调节iNOS表达影响人卵巢癌SKOV3/DDP细胞顺铂耐药性的研究

Study on lipopolysaccharides influencing the cisplatin resistance of SKOV3/DDP cells through regulating the expression levels of iNOS

目的观察脂多糖诱导的SKOV3/DDP细胞内分泌型一氧化氮合酶(iNOS)表达变化对人卵巢癌SKOV3/DDP细胞株顺铂耐药性的影响,并初步探讨机制。方法体外培养SKOV3/DDP细胞,分为对照组、顺铂组、脂多糖(Lipopolysaccharides,LPS)组和联合用药组共4组,对照组给予常规不加血清培养基,顺铂组给予10μg/ml顺铂,LPS组给予浓度为1μg/ml的LPS,联合用药组给予10μg/ml顺铂和1μg/ml LPS,均作用24 h。MTT法检测各组细胞活力;蛋白免疫印迹法观察各组iNOS表达;流式细胞术检测各组细胞凋亡情况;间接免疫荧光法观察各组细胞激活型Caspase-3表达。结果联合用药组细胞活力明显低于顺铂组(P<0.05);顺铂组与对照组iNOS表达差异无统计学意义(P>0.05),LPS组和联合用药组iNOS表达水平明显高于对照组(P<0.05),且联合用药组高于LPS组(P<0.05);流式细胞术结果显示联合用药组细胞凋亡率明显高于顺铂组(P<0.05),且通过间接免疫荧光法显示联合用药组细胞激活型Caspase-3表达高于顺铂组(P<0.05)。结论 LPS能够降低人卵巢癌SKOV3/DDP细胞顺铂耐药性,这可能与其提高SKOV3/DDP细胞内iNOS表达水平有关。

Objective To observe the influences of lippolysaccharides （LPS） on cisplatin resistance in SKOV3/DDP ceils through regulating the expression levels of iNOS, and to explore its possible mechanism. Methods SKOV3/DDP cells were cultured in vitro, which were divided into control group, cisplatin group, LPS group and drug combination group （cisplatin plus LPS）randomly. The cisplatin was given to SKOV3/DDP cells in the corresponding group with the dose of 10 μg/ml, and LPS with the dose of 1 μg/ml. All groups were treated for 24 h. The cells viability was measured by MTT method . The protein expression levels of iNOS in all groups were detected by Western blotting method, and cell apoptosis was measured by flow cytometry. The expression of apoptosis related protein Active Caspase-3 was detected by indirect immunofluorescence method . Result Compared with the cisplatin group, the cell viability in drug combination group was significantly decreased（P 〈 0. 05）. No significant difference of the expression level of iNOS between the eisplatin group and the control group was found（P 〉 0.05） , and the levels of that in drug combination group and LPS group were significantly increased compared with the control group（P 〈 0.05 ）. The result of flow cytometry showed that the cell apoptosis rate in drug combination group was higher than that in the eisplatin group（ P 〈 0. 05 ） , and the expression of active Caspase-3 increased significantly in drug combination group compared with the other three groups（P 〈 0. 05）. Conclusion The cisplatin resistance of SKOV3/DDP cells was reduced by LPS, and this might through regulating the expression levels of iNOS.