p21^waf1-p27^kip1基因联合转染对乳腺癌细胞增殖和中心体复制的影响

Effects of transfected pIRES-p21^waf1-p27^kip1 gene on the dupfication of centrosomes and the proliferation of breast cancer cell line MCF-7

目的探讨外源性p21^waf1-p27^kip1基因联合转染对人乳腺癌细胞细胞增殖及中心体复制的影响和意义。方法应用脂质体介导法分别将构建的pIRES-p21^waf1、pIRES-p27^kip1、pIRES-p21^waf1-p27^kip1真核表达载体，转染入常规培养的人乳腺癌细胞系MCF-7，未转染组、转染空质粒组细胞做对照，Western免疫印迹法验证转染前后p21^waf1、p27^kip1基因蛋白表达情况；噻唑蓝（MTT）法绘制细胞生长曲线；流式细胞仪检测细胞周期DNA分布变化；免疫荧光技术检测转染前后中心体复制的变化情况。结果Western免疫印迹法证实p21^waf1、p27^kip1、p21^waf1-p27^kip1基因转染24h后，其蛋白表达量明显增多（P〈0.01），与对照组比较，各转染实验组细胞生长明显受抑制（P〈0.01），细胞周期大部分停滞于G1期，各实验组G1期和S期细胞比例依次为：（69.52±3.21）％、（18.38±2.51）％，（60.83±3.02）％、（24.52±1.89）％，（78.37±2.83）％、（15.27±1.74）％，对照组则为（47.28±2.25）％、（33.52±1.97）％，中心体复制异常的乳腺癌细胞数较转染前（13.47±0.33）％明显减少：（5.07±0.38）％，（6.28±0.35）％，（3.47±0.23）％，两两比较差异有统计学意义（P〈0.01）。结论外源性p21^waf1和p27^kip1基因转染可抑制人乳腺癌细胞的增殖及中心体的异常复制，二者联合转染时效果显著。提示，乳腺癌的发生、发展是多基因、多因素共同参与的过程，p21^waf1和p27^kip1在调控细胞增殖及中心体复制方面具有协同作用，对于p21^waf1、p27^kip1基因失活、低表达或不表达的乳腺癌，联合转染外源性p21^waf1、p27^kip1基因不失为一种有效的治疗手段。

Objective To explore the role of transfected pIRES-p21^waf1-p27^kip1 gene on the centrosome duplication and cell proliferation of MCF-7, a breast cancer cell line . Methods The pIRES- p21^waf1, pIRES-p27^kip1 and pIRES-p21^waf1-p27^kip1 genes were transfected into the MCF-7 ceils by lipofection. The effect on proliferation was evaluated by MTT assay and cell growth curve was drawn. The cell cycle was analyzed by flow cytometry. The centrosome duplication was detected by using indirect immunofluorescence microscopy. Results After transfected 24 hours, the p21^waf1 and p27^kip1 protein expressions were significantly increased as compared with untransfected MCF-7 cells （ P〈0.01）, and cell growth was obviously inhibited and resulted in an accumulation of cells in G1（P〈0.01）, presenting that the proportion of cells in G1 phase was obviously increased from （47.28±2.25） % to （69.52±3.21）% of p21^waf1 transfected cells, （60.83±3.02） % of p27^kip1 transfected cells, and （78.37±2.83）% of p21^waf1-p27^kip1 transfeeted cells. The proportion of cells which contained unnormal centrosomes was obviously decreased, from （13.47±0.33）% to （5.07±0.38）%, （6.28±0.35）%, （3.47±0.23）%, respectively. Conclusion The transfer of p21^waf1 wan and p27^kip1 genes could inhibit the growth of human breast carcinoma cells and the unnormal duplication of centrosomes, p21^waf1 had a really synergy with p27^kip1 in these effects, suggesting p21^waf1-p27^kip1 combined gene can inhibit the genesis and development of breast cancer and might have potential clinical significance as therapeutic agents of breast cancer.