顺铂增强TRAIL蛋白诱导Caski细胞凋亡机理研究  

Study of cisplatin enhancing Caski cells apoptosis induced by TRAIL protein

作　　者：李民华[1] 王惠兰[1] 丁霏[1] 高洁凡[1] 刘文利[1] 王振海[1] 

机构地区：[1]河北医科大学第二医院妇产科,石家庄050000

出　　处：《现代妇产科进展》2009年第7期490-494,共5页Progress in Obstetrics and Gynecology

基　　金：河北省科委基金资助项目(No:04276101D-22)

摘　　要：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 cisplatin regulation effect on Caski cells apoptosis induced by TNF-related apoptosis-inducing ligand （TRAIL）, furthermore discussing its mechanism. Methods：Human cervical cancer cell line Caski in vitro was treated with TRAIL or DDP only,TRAIL and DDP, DDP-sequential TRAIL, TRAIL-sequential DDP respectively. Cell morphology were observed by inverted microscope. Growth inhibition rates of cells were determined with MTT assay. Caspase-8 activity of Caski cells after treatment with TRAIL, TRAIL-sequential DDP, DDP-sequential TRAIL were detected. Expressions of TRAIL recepetors before and after DDP（1.0mg/L） treatment were determined by flow cytometer （FCM）. Expressions of TRAIL receptors DR4,DRS,DcR1 ,DcR2 mRNA before and after DDP treatment were tested by semiquantity reverse transcription-polymerase chain reaction （RT-PCR）. Results： （ 1 ） DDP and TRAIL had inhibitory effect on Caski cells;（2）Inhibitory rates of Caski cells was 35.44% after treatments with 100ng/ml TRAIL alone for 24h,45.34% with 1.0mg/L DDP alone for 24h, and the combination inhibitory rates of TRAIL and DDP increased to 89.66%, the difference between combination group with the TRAIL group or the DDP group had statistical significance （ P 〈 0.05 ） ; （3） The groups of DDP-sequential TRAIL and TRAIL-sequential DDP had different effects on growth inhibition of Caski cells, the former inhibitory rates was 79.88%, the latter was 55.73%. There was statistical significance between two experimental groups （ P 〈 0.01 ） ; （4） Caspase-8 activity of DDP-sequential TRAIL group was 1.52 times of TRAIL group ; （ 5 ） Expressions of DR4 and DR5 were upregulated obviously after DDP treatment 24h. FI rose from 2.07±0.52 to 2.35 ＋0.30 for DR4 and from 2.14±0.31 to 2.55±0.15 for DR5. Compared with the control group, the difference had statistical significance （ P 〈0.05） ; （6） mRNA expressions of DR4, DR5 in Caski cells were increased markedly after DDP treatment（P 〈 0.05

关 键 词：TRAIL蛋白 顺铂 子宫颈肿瘤 半胱氨酸天冬氨酸蛋白酶8 凋亡 

分 类 号：R737.33[医药卫生—肿瘤]