摘要
目的观察氟尿嘧啶(5-FU)刺激后PIDD蛋白在结肠癌HT29细胞中分布的改变,以及小干扰RNA(siRNA)干扰PIDD蛋白前后,HT29细胞耐药性的变化。方法用siRNA干扰HT29细胞内PIDD蛋白的表达,用5-FU刺激HT-29细胞。West-ern blotting法检测干扰前后细胞PIDD的表达,并比较5-FU刺激后干扰与未干扰HT-29细胞内PIDD在细胞中分布的变化。MTT比色法检测siRNA干扰前后细胞对5-FU的敏感性,并计算各组细胞的IC50值。结果未受5-FU刺激前,PIDD蛋白主要分布于细胞质中;受5-FU刺激后,PIDD蛋白迁移至细胞核。siRNA干扰后细胞中PIDD表达总量有所下降,胞质及胞核中的表达均降低,受5-FU刺激后PIDD的表达也未见升高,迁移至胞核的PIDD也未增加。MTT检测证明5-FU对转染组(PIDD-360转染12h后的HT29细胞)的IC50值为0.23±0.06μg/ml,与正常组(未作处理的HT29细胞,IC50为2.71±0.70μg/ml)和对照组(阴性对照试剂转染12h后的HT29细胞,IC50为2.78±1.03μg/ml)比较显著降低(P<0.05)。结论经siRNA干扰PIDD蛋白后,HT29细胞的耐药性明显下降,推测其机制与胞核中PIDD降低导致的NF-κB活性降低,细胞凋亡增多有关。
Objective To explore the distributive changes of PIDD protein in HT29 ceils stimulated by 5 fluorouracil(5-FU), and the influence of the PIDD protein interfered by small interference RNA (siRNA) on the drug resistance of HT29 cells in vitro. Methods siRNA was used to interfere the PIDD expression, and HT29 ceils were treated with 5-FU. Western hlotting was employed to detect the PIDD expression before and after interference. The distribution of PIDD in nucleus and cytoplasm after 5-FU treatment was also detected. Changes in drug sensitivity of HT-29 cells to 5-FU were determined with MTT assay and ICs0 values were evaluated. Results PIDD pro- tein was expressed mainly in the cytoplasm before 5-FU stimulation, and it migraded into the nucleus after stimulation. Western blotting showed that the total P1DD expression and the expression both in cytoplasm and nuclei decreased significantly after RNA interference, and no increase of the total PIDD and that migrated into nucleus was found even after 5-FU stimulation. It was found that in the transfection group (ceils were incubated 12h after being transfected with PIDD-360), normal group (cells without treatment) and control group (cells were incubated 12h after being transfected with negative transfection reagent), the IC50 values of cells treated with 5-FU were 0.23±0.06μg/ml, 2.71±0.70μg/ml and 2.78±1.03μg/ml, respectively. The IC50 value declined significantly in transfection group compared with that of both the normal group and control group (P〈0.05). Conclusion The drug resistance of HT29 cell to 5 FU is remarkably decreased after PIDD protein is interfered by siRNA. The mechanism involved may he related to cell apoptosis due to lowered activity of NF-κB induced by PIDD reduction.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第12期1444-1446,共3页
Medical Journal of Chinese People's Liberation Army
基金
重庆市自然科学基金资助项目(CSTC2006BB5060)