摘要
目的探讨P13K的调节亚基p55γN端24个氨基酸-N24p55γP13K对胃癌细胞系MKN-28增殖及细胞周期的影响。方法通过免疫沉淀及Western blot检测胃癌细胞系MKN-28中Rb家族蛋白(p130/107,p110)和p55γP13K蛋白的表达。采用脂质体转染的方法,将质粒N24p55-GFP导入MKN-28,通过流式细胞仪分析细胞周期,通过BrdU掺入法,激光共聚焦及流式检测Cyclin D1,Cyclin A的表达以观察其对细胞增殖的影响。结果胃癌细胞系MKN-28含有Rb家族蛋白p130/107而无p110,也含p55γP13K蛋白,且p130/107与p55γP13K相结合。与对照质粒pEGFP-C1相比,转染了N24p55-GFP的MKN-28细胞增殖受抑制,G_0/G_1期增加,S期减少,Cy- clin D1和Cyclin A的表达明显降低。结论这种新的N24P55γP13K调节亚单位可通过p130/p107途径阻滞细胞周期进程,抑制细胞增殖,从而为胃癌的基因治疗提供了一个新的分子靶点。
Objective To study whether the eDNA encoding N-terminal 24 amino acids of P55γ subunit in Phosphoinositide-3-kinasc (PI3K) inhibit proliferation and arrest cell cycle progression in gastric cancer. Methods Immunoprecipitation and western blotting were used to detect the proteins of pRb and PI3K in gastric cancer MKN-28 cells. After transfected plasmid N-24p55-GFP into cells with lipid transfection reagent,the cell proliferation was determined by cell cycle,incorporated BrdU and expression of cyclinD1, cyclin A through flow cytometry and laser scanning confocal microscopy. Results Our data showed Rb family members p130/p107 protein but not p110 and p55γPI3K protein exist in MKN-28 cells,moreover,p55γPI3K bound with p130/p107 ,the plasmid N24p55-GFP containing unique N-terminal 24 amino acids of p55γPI3K induced G0/G1 phase cells increased and S phase cells decreased,the level of cyclinD1 and cyclin A reduced. Conclusion In MKN-28 cells, the proliferation was inhibited and the cell cycle can be arrested by p130/p107-E2F pathway, which provided a new molecular target for gene therapy in gastric cancer.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2006年第10期1221-1223,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(30300338)
国家重点基础研究发展规划"973"项目(2002CB513100-2)
教育部留学回国人员启动基金教外司(2003-14)