槲皮素对子宫颈癌细胞中乙酰肝素酶表达的影响
发表时间：2014-02-07 浏览次数：591次
张文婷,张蔚,钟亚娟等.槲皮素对子宫颈癌细胞中乙酰肝素酶表达的影响[J].中华妇产科杂志,2013,48(3):198-203.
槲皮素; 葡糖醛酸糖苷酶; 宫颈肿瘤; HeLa细胞; 细胞系,肿瘤
槲皮素对子宫颈癌细胞中乙酰肝素酶表达的影响
430060,武汉大学人民医院妇科
2013
198-203
万方
目的 探讨槲皮素对宫颈癌细胞中乙酰肝素酶(HPA)表达的影响.方法 免疫荧光法检测宫颈癌细胞系HeLa、Caski细胞中HPA蛋白的表达.实验分为3组,槲皮素组:在HeLa、Caski细胞中加入含不同浓度(20、40、80 μmol/L)槲皮素的RPMI 1640培养基；阳性对照组:小分子RNA(siRNA)转染HeLa、Caski细胞沉默其HPA的表达后,加入RPMI 1640培养基；空白对照组:在HeLa、Caski细胞中仅加入RPMI 1640培养基.3组HeLa、Caski细胞分别培养24、48、72 h后,采用实时定量PCR技术和蛋白印迹法检测其HPA mRNA和蛋白的表达.结果 (1)免疫荧光法检测显示,HeLa和Caski细胞中均有HPA蛋白的表达,均位于细胞核与细胞质中,呈红色荧光.(2)实时定量PCR技术检测显示,槲皮素组HeLa、Caski细胞中HPA mRNA的表达水平均随槲皮素浓度(分别为20、40、80 μmol/L)的升高、处理时间(分别为24、48、72 h)的延长而明显下降(P＜0.01),呈浓度和时间依赖性.与空白对照组相比,槲皮素组中40、80 μmol/L槲皮素处理后HeLa、Caski细胞中HPA mRNA表达水平均明显下降(P＜0.05)；与阳性对照组相比,槲皮素组中80 μmol/L槲皮素处理后HeLa细胞中HPA mRNA表达水平无明显变化(P＞0.05),而Caski细胞中HPA mRNA表达水平明显升高(P＜0.01).(3)蛋白印迹法检测显示,槲皮素组HeLa、Caski细胞中HPA蛋白的相对表达水平均随槲皮素浓度(分别为20、40、80 μmol/L)的升高、处理时间(分别为24、48、72 h)的延长而明显下降(P＜0.01),呈浓度和时间依赖性.与空白对照组相比,槲皮素组中40、80 μmol/L槲皮素处理后HeLa、Caski细胞中HPA蛋白表达水平明显下降(P＜0.05)；与阳性对照组相比,槲皮素组中80 μmol/L槲皮素处理后HeLa、Caski细胞HPA蛋白表达水平无明显变化(P＞0.05).结论 槲皮素对宫颈癌细胞中HPA mRNA和蛋白的表达均有明显抑制作用,且这种抑制作用与槲皮素浓度与处理时间均呈明显依赖关系. Objective To detect the expression of heparanase (HPA) in cervical cancer cells and investigate the impact of quercetin on the expression of HPA,and the molecular mechanism that quercetin inhibits the growth of cervical cancer cells.Methods The experimental groups included cervical cancer cell lines (HeLa and Caski) exposed to different concentrations of quercetin (20,40 and 80 μmol/L) in the culture medium.The control groups included a negative control group,which was cultured with RPMI 1640 only,and a positive control group,in which cervical cancer cells were transfected with HPA small interference RNA (siRNA) to silence HPA expression.The cellular expression levels of HPA were detected with fluorescence quantitative real-time PCR and western blot analysis at 24,48 and 72 hours after treatment.Results (1) HPA was significantly expressed in both cervical cancer cell lines (HeLa and Caski),and it exists both nucleus and cytoplasm.(2)The real-time PCR shows as follows:as the quercetin concentration increased (20,40 and 80 μmol/L),the mRNA expression level of HPA decreased (P ＜0.01),in which the inhibition of HPA expression was concentration dependent.In addition,the inhibition of HPA expression was also time dependent.As time growth,the expression level of HPA mRNA (24,48 and 72 hours) in HeLa and Caski cells decreased (P ＜ 0.01).Compared with negative control group,the expression level of HPA mRNA decreased in different concentrations of quercetin (40 and 80 μmol/L) in both HeLa and Caski cells (all P ＜ 0.05) ;
