激酶4抑制因子家族基因表达的激活对肠癌细胞增殖侵袭的影响  

Proliferative and invasive effects of inhibitors of kinase 4 （P15ink4b and P16ink4a/CDKN2） gene activation on RKO human colorectal cancer cells

作　　者：方晓明[1] 姜朝晖[1] 彭佳萍[2] 姚宁[1] 方旭东[1] 郑树[2] 

机构地区：[1]解放军第一一七医院普通外科,杭州310013 [2]浙江大学医学院肿瘤研究所,杭州310013

出　　处：《中华胃肠外科杂志》2014年第1期31-35,共5页Chinese Journal of Gastrointestinal Surgery

基　　金：基金项目：国家“973”重点基础研究发展规划项目（G1998051200）;浙江省科技计划项目（011110541）

摘　　要：目的探讨激活激酶4抑制因子（INK4）家族（P15ink4b和P16ink4a／CDKN2）基因表达对结肠癌细胞增殖和迁移能力的影响。方法应用1×10^-7、5×10^-7及1×10^-6mol／L不同浓度的特异性DNA甲基转移酶抑制剂（5-Aza-CdR）对结肠癌RKO细胞进行去甲基化处理（分别为A、B、C3个实验组），另设阳性对照组（未经处理的RKO细胞）。Western blot法检测INK4家族基因的蛋白表达：软琼脂克隆细胞集落实验评估体外致瘤情况；Transwell小室实验评估体外迁移情况。将各组RKO细胞注射BALB／c裸鼠（每组10只），通过称量瘤体的重量和体积评估体内成瘤情况。结果A、B、C3组实验组细胞中P15ink4b和P16ink4a／CDKN2蛋白表达分别为阳性对照组的1．13、1．38、1．92倍和1．11、1．45、2．14倍。体外实验显示，B组和C组的细胞集落数明显少于阳性对照组（P〈0．05）；3组实验组的细胞迁移数均明显少于阳性对照组（P〈0．05）。体内实验显示，相对于阳性对照组，3组实验组裸鼠的抑瘤率分别为2．1％、16．8％和26．2％，差异有统计学意义（P〈0．01）。结论激活INK4家族基因的表达能有效抑制结肠癌细胞的增殖和迁移。Objective To explore the proliferation and invasive effects of inhibitors of kinase 4 （INK4） （P15ink4b and P16ink4a/CDKN2） gene protein activation on RKO human eoloreetal cell in vivo and in vitro. Methods RKO human eoloreetal cell line was exposed to the specific DNA methyltransferase inhibitor 5-Aza-CdR and INK4（P15ink4b and P16ink4a/CDKN2） protein expression was detected by Western blotting. Soft agar cloning experiment and Transwell chamber assay were used to detect the proliferative and invasive ability in vitro. Tumorigenicity in nude mice was analyzed in vivo. Results INK4（P15ink4b and P16ink4a/CDKN2） protein expression of RKO human coloreetal cells after exposure to 1×10^-7, 5×10^-7 and 1×10^-6 mol/L 5-Aza-CdR concentrations（A, B, C groups） were 1.13, 1.38, 1.92 folds and 1.11, 1.45, 2.14 folds compared to positive control group respectively. Soft agar cloning experiment showed the number of cell colony significantly decreased from 36.8±5.1 （positive control group） to 32.4±7.2, 21.3±5.4 and 19.5±6.4 （3 experiment groups, all P〈0.05） respectively. Transwell chamber assay showed that migrated cell number in positive control group （67.4±7.2） was significantly higher than those of 3 experimental groups （35.3±4.6, 29.5±7.3 and 25.3±6.2, respectively）. The tumor volume of metastasis model in nude mice was inhibited in experimental groups, but not significantly lower compared to control group （P〉0.05）. There were significant differences of tumor weight and inhibition rate between control group and 3 experimental groups in nude mice respectively （all P〈0.01）.Conclusion INK4 （P15ink4b and P16ink4a/CDKN2） protein activation can inhibit tumor proliferation, migration and suppress the tumor formation ability.

关 键 词：结直肠肿瘤 激酶4抑制因子家族基因 细胞增殖 细胞迁移 

分 类 号：R735.3[医药卫生—肿瘤]