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目的:观察顺铂对TRAIL蛋白诱导子宫颈癌Caski细胞株凋亡的调节作用,并探讨其作用机理。方法:传代培养宫颈癌Caski细胞,依照所加药物不同,将细胞分为TRAIL组、顺铂组、TRAIL+顺铂组、TRAIL序贯顺铂组、顺铂序贯TRAIL组以及空白对照组,分别于加药后不同时间应用:(1)倒置显微镜观察细胞的生长状态;(2)MTT法检测TRAIL蛋白与顺铂单用、联用以及序贯应用对Caski细胞的生长抑制率;(3)Caspase-8活性检测试剂盒检测不同用药方式对Caski细胞Caspase-8活力的影响;(4)流式细胞术检测Caski细胞TRAIL受体DR4,DR5,DcR1,DcR2的表达,以及1.0mg/L顺铂作用细胞24h后表达量的变化;(5)半定量RT-PCR分析顺铂作用细胞8h后4种受体mRNA水平的变化。结果:(1)TRAIL蛋白和顺铂对Caski细胞有不同程度的生长抑制作用;(2)100ng/mlTRAIL蛋白和1.0mg/L顺铂单用作用24h细胞生长抑制率分别为35.44%、50.26%,联合应用后增至89.66%,联合用药与单独用药差异有统计学意义(P<0.05);(3)顺铂序贯TRAIL蛋白生长抑制率79.88%,TRAIL蛋白序贯顺铂55.73%,两实验组的差异有统计学意义(P<0.01);(4)Caspase-8活力在顺铂序贯TRAIL组的表达最强,为单用TRAIL组的1.52倍;(5)Caski细胞4种TRAIL受体均有表达,但表达丰度不同,顺铂可以上调死亡受体的表达;(6)顺铂处理细胞8h后DR4,DR5mRNA表达水平明显升高,DR4为对照组的1.40倍,DR5为对照组的1.57倍,差异有统计学意义(P<0.05);(7)用顺铂处理前后DcR1,DcR2表达差异无统计学意义(P>0.05)。结论:Caski为TRAIL蛋白敏感细胞,顺铂通过上调死亡受体4、5表达、提高Caspase-8活性,增强TRAIL蛋白抑制子宫颈癌Caski细胞生长的作用;顺铂序贯TRAIL的配伍方式具有更强的诱导Caski细胞凋亡的作用。
Objective: To observe the regulatory effect of cisplatin on the apoptosis of Caski cell line induced by TRAIL protein and to explore its mechanism. Methods: The cervical cancer Caski cells were subcultured. The cells were divided into TRAIL group, cisplatin group, TRAIL + cisplatin group, TRAIL sequential cisplatin group, cisplatin sequential TRAIL group and blank control group (2) MTT method was used to detect the inhibitory rate of TRAIL protein and cisplatin alone, in combination and sequential application on the growth of Caski cells; (3) (4) Flow cytometry was used to detect the expression of TRAIL receptor DR4, DR5, DcR1 and DcR2 in Caski cells, and the expression of DcR2 in Caski cells was detected by Caspase-8 activity assay kit (5) Semiquantitative RT-PCR analysis of cisplatin-induced cell 8h after four receptor mRNA levels change. Results: (1) TRAIL protein and cisplatin had different growth inhibitory effects on Caski cells. (2) The cell growth inhibition rates of 100ng / ml TRAIL protein and 1.0mg / L cisplatin alone for 24h were 35.44%, 50.26% (P <0.05); (3) The inhibitory rate of sequential growth of cisplatin was 79.88% and that of TRAIL protein was 55.73%, both of which were significantly higher than those of control (4) Caspase-8 activity was the strongest in cisplatin-treated TRAIL group, which was 1.52 times of that in TRAIL-treated group alone. (5) The expression of 4 TRAIL (6) cisplatin treatment of cells 8h after DR4, DR5 mRNA expression was significantly increased, DR4 was 1.40 times the control group, DR5 was (P <0.05). (7) There was no significant difference in the expression of DcR1 and DcR2 before and after treatment with cisplatin (P> 0.05). CONCLUSIONS: Caski is a TRAIL-sensitive cell. Cisplatin can enhance the activity of Caspase-8 by up-regulating the expression of death receptors 4,5 and increasing the inhibitory effect of TRAIL on the growth of cervical cancer Caski cells. Strong induced apoptosis in Caski cells.