p15对人胆管癌细胞增殖影响的实验研究及机理的初步分析

目的　通过构建稳定高表达抑癌基因 p15的人胆管癌细胞模型 ,研究p15对胆管癌细胞增殖的影响。 方法　将抑癌基因p15的cDNA构建到高效真核表达的质粒载体 pcDNA3 neo中的EcoRⅠ /XbaⅠ位点 ,构建成p15真核表达质粒 pcD NA3 p15 ;通过脂质体法将pcDNA3 p15质粒转染人胆管癌细胞系QBC93 9,经G 418筛选 ,获得稳定高表达 p15的人胆管癌细胞模型及相应的表达空载体的对照细胞模型 ,并经Western分子杂交分析证实。结果　与对照组相比 ,p15高表达的人胆管癌细胞QBC93 9的增殖受到明显抑制 ;流式细胞光度术表明 p15同时阻遏细胞由G1 期向S期及G2 期向M期的转换。结论　Western免疫印迹分析结果表明 ,癌基因c myc的蛋白水平表达下降可能是p15抑制细胞增殖的分子机理之一。

A PILOT STUDY ON EFFECT OF P15 ON PROLIFERATION OF CHOLANGIOCARCINOMA

Objective To study the inhibitory effect of anti-oncogene p 15 on human cholangiocarcinoma cell line. by establishing the cell model of the human cholangiocarcinoma that expressed p15 stably and high.Methods The cDNA of anti-oncogene p15 was added to the EcoRⅠ/ XbaⅠ site of the pcDNA3-neo with a high effective eukaryon expression plasmid carrier to get th e pcDNA3p15 that is a eukaryon expression plasmid containing p15. The pcDNA3 p15 was transfected into the human cholangiocarcinoma cell line QBC939 by lipofecti ne. After G418 selection, the cell model of the human cholangiocarcinoma was obt ained that expressed p15 stably and high, and the matched control cell model tha t express zero load carrier.Western molecular hybridization analysis results con firmed it. Results Compared to the matched control, the growt h of QBC939 cells that expressed p15 high was inhibited. The flow cytometry show ed p15 induced QBC939 cell both G 1 and G 2/M blockade. Conclusion Western immunoblot analysis results showed that the descenting of oncog ene c-myc protein level may be one of the molecul reason that p15 inhibit prolif eration.