NF-κB信号通路介导结肠癌多细胞耐药的作用研究  

The mechanism of multicellular resistance mediated by NF-κB signaling pathway in colonic carcinoma cells

作　　者：李建军[1] 潘凤[1] 黄海辉[1] 胡绍毅[1] 边志衡[1] 梁后杰[1] 

机构地区：[1]第三军医大学西南医院肿瘤科,重庆400038

出　　处：《解放军医学杂志》2008年第10期1205-1208,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30171067);重庆市科技攻关计划项目(9398)

摘　　要：目的探讨NF-κB信号通路在结肠癌HT-29细胞多细胞耐药中的作用及可能机制。方法采用液体重叠培养系统和常规贴壁法对HT-29细胞进行三维(3D)和单层(2D)培养。采用倒置相差显微镜、扫描电镜观察3D培养的HT-29细胞形态,并采用免疫组化法检测增殖细胞核抗原(PCNA)的表达。电泳迁移率改变测定法(EMSA)测定3D和2D培养细胞中NF-κB的活性差异。将3D培养细胞分为两组:3D组(培养液中不加干扰因素)和3D+SN50组(培养液中加入50μg/ml SN50处理24h),采用EMSA测定两组NF-κB活性差异,TUNEL法检测细胞凋亡,Western blotting检测凋亡相关蛋白(Caspase-3、Bcl-2和Bax)的表达并对蛋白条带的光密度值进行半定量分析,外生半径测定法测定3D培养细胞对氟尿嘧啶的敏感性。结果3D培养的HT-29细胞悬浮生长,细胞相互聚集增殖形成多细胞球,中心为坏死区,周围由多层异型性细胞组成,外围细胞PCNA阳性染色明显。与2D培养细胞相比,3D细胞中NF-κB活性条带颜色较深。EMSA显示3D+SN50组NF-κB活性较3D组低,TUNEL法显示3D组细胞凋亡率(8.71%±0.73%)较3D+SN50组(15.75%±1.02%)明显降低(P<0.01)。3D+SN50组和3D组中,Caspase-3活化片段光密度值分别为126.79±13.48和87.24±10.68(P<0.01),Bax蛋白为129.73±15.28和89.26±14.31(P<0.01),Bcl-2蛋白为97.27±12.63和131.24±14.53(P<0.01)。结论NF-κB活性升高是导致结肠癌HT-29细胞多细胞耐药的重要原因之一,其机制可能与凋亡相关蛋白的表达调控有关。Objectives To explore the effect of NF-κB signaling pathway on multicellular resistance （MCR） to 5 Fu in colon carcinoma cells. Methods The multicellular spheroids （MCS） of colon carcinoma HT29 cell line were cultured by using liquid overlay tech nique, and the monolayer cells by using routine procedure. The morphology of MCS was observed with light microscope and scanning elec tronmicroscope （SEM）. The expression of proliferating cell nuclear antigen （PCNA） was determined by immunohistochemistry. 24 hours after adding 50μg/ml SN50 to spheroids cells, the 50% inhibiting concentration （K150） for 5-Fu was determined by using radial outgrowth assay. The activity of NF-κB was measured by using electrophoretic mobility shift assay （EMSA） and the specific bands were observed. Apoptosis of spheroids cells, which were pretreated with 5μg/ml 5-Fu for 24 hours, was identified by TUNEL, the apoptosis related pro teins caspase-3, Bcl 2 and 13ax were detected by Western blotting and analyzed by semi-quantitative method. Results A necrotic core formed in the centre of MCS, and the peripheral cells were actively proliferating. Compared with the monolayer cultured cells, the activity of NF-κB was increased in MCS. When the activity of NF-κB in spheroid cells was inhibited by SN50, 5 Fu readily induced cells to apopto sis （15. 75%±1. 02% vs 8. 71%±0. 73%, P〈0.01）, and promote the activity of caspase-3 （OD, 126. 79±13.48 vs 87. 24±10. 68, P〈 0.01）. Meanwhile the expression of Bcl-2 was dowmregulated （OD, 97.27±12. 63 vs 131.24±14. 53, P〈0. 01）, and of Bax raised （OD, 129. 73±15.28 vs 89.26±14. 31, P〈0. 01）. The IC50 value for 5-Fu in spheroid cells declined to 12. 99±0. 37μg/ml （vs 18. 74±0. 97μg/ml, P〈0. 01）. Conclusion Activation of NF-κB can induce MCR to 5 Fu in colon carcinoma HT29 cells, and it might be in volved in the mediation of apoptotic proteins.

关 键 词：结肠肿瘤 NF-ΚB 多药耐药相关蛋白质类 细胞凋亡 

分 类 号：R735.35[医药卫生—肿瘤]