黏附调节因子p120ctn 1A细胞核定向表达质粒的构建及鉴定  

Construction and identification of p120ctn 1A nucleus-localization plasmid

作　　者：唐娜[1,2] 朱华倩[3] 曹红一[1,2] 王业林[1,2] 林旭勇[1,2] 戴顺东[1,2] 

机构地区：[1]中国医科大学附属第一医院病理科 [2]中国医科大学病理学教研室,辽宁沈阳110001 [3]中国医科大学,五年制94期4班辽宁沈阳110001

出　　处：《中华肿瘤防治杂志》2012年第4期241-245,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81071717);教育部博士点基金(20102104120027)

摘　　要：目的:构建并鉴定p120ctn 1A细胞核定向表达质粒pCMV/p120ctn 1A。方法:采用PCR的方法扩增人p120ctn 1A活性片段,将其插入细胞核定向表达载体pCMV/myc/nuc/GFP,构建重组质粒pCMV/p120ctn 1A,经脂质体介导转染肺癌细胞系SPC-A-1,用荧光显微镜观察绿色荧光信号,免疫细胞化学及蛋白质印迹法鉴定其在肺癌细胞SPC-A-1中的表达与定位,用Transwell小室法监测细胞侵袭能力的变化。结果:限制性双酶切及DNA测序分析结果显示,插入pCMV/p120ctn 1A的片段为2 798bp左右,与预期p120ctn 1A基因片段大小相同。转染pCMV/p120ctn 1A质粒后,荧光显微镜观察到绿色荧光信号特异性定位于细胞核内。免疫细胞化学结果显示,SPC-A-1细胞核内p120ctn的表达量显著增强。蛋白质印迹法检测证实,转染pCMV/p120ctn 1A质粒后的p120ctn 1A蛋白表达量显著上调。体外侵袭实验结果证实,转染后SPC-A-1细胞的侵袭能力明显增强。结论:成功构建了p120ctn 1A细胞核定向表达载体pCMV/p120ctn 1A,为深入研究p120ctn的核内功能提供了有力的实验工具。OBJECTIVE： To construct p120ctn 1A nuclear target localizaiton plasmid.METHODS： p120ctn 1A cDNA was amplified by PCR and inserted to nucleus-localization expression vector pCMV/myc/nuc to construct pCMV/p120ctn 1A.Then the pCMV/p120ctn 1A was transfected into SPC cells.The expression and localization of p120ctn 1A in SPC-A-1 cells were verified by fluorescent microscope,immunocytochemistry and Western blot.Besides,the invasive ability of SPC-A-1 cells was detected by Transwell assay.RESULTS： The size of inserted fragments in the recombinant vectors pCMV/p120ctn 1A was 2 498 bp,corresponding to that of p120ctn 1A.After transfecting pCMV/M-CSF into SPC-A-1 cells by Lipofectamine 2000,pCMV/p120ctn 1A-transfected SPC-A-1 cells expressed p120ctn 1A protein in nucleus.The expression of p120ctn 1A was significantly elevated in pCMV/p120ctn 1A-transfected SPC-A-1 cells.Meanwhile,the invasive ability of SPC-A-1 cells was enhanced significantly.CONCLUSION： The nuclear localization vector pCMV/p120ctn 1A is successfully constructed.

关 键 词：P120CTN 核内定位载体 重组质粒 

分 类 号：R734.2[医药卫生—肿瘤]