蛋白磷酸酶2A催化亚基α在小鼠胚胎成纤维细胞迁移中的作用研究  

Effects of Catalytic Subunitαof Protein Phosphatase 2A on Cell Migration of Mouse Embryo Fibroblasts

作　　者：王庆华[1] 王生存[1] 李斌[2] 刘春[1] 吴刘成[1] 王旭[1] 邵义祥[1] 

机构地区：[1]南通大学实验动物中心,江苏南通226001 [2]南通大学附属医院儿科,江苏南通226001

出　　处：《动物医学进展》2016年第6期49-54,共6页Progress In Veterinary Medicine

基　　金：江苏省高校自然科学研究项目(11KJB180010);江苏省高校自然科学研究项目(14KJB180018);江苏省高校自然科学研究项目(15KJB180015)

摘　　要：利用体外基因敲除技术检测小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)周期和迁移能力的变化,探究蛋白磷酸酶2A的Cα亚基在MEFs细胞迁移过程中的作用。用Cαfl/fl的纯合子雄鼠与雌鼠1∶2配对,取E12.5d的胚胎制作小鼠胚胎成纤维细胞。利用表达Cre重组酶(Ad-Cre-EGFP)以及GFP荧光蛋白(Ad-EGFP)的腺病毒载体,对P3代MEFs进行感染,分别用PCR,RT-PCR和Western blot方法进行基因型鉴定。同时利用流式分选技术检测感染后MEFs的细胞周期的变化,利用细胞划痕试验检测了其细胞迁移能力的变化。结果显示,胰酶消化的方法成功获得了小鼠胚胎成纤维细胞,DNA,RNA和蛋白水平的鉴定获得了3对3的野生型和基因敲除MEFs。流式分选发现Ad-Cre处理的MEFs与Ad-EGFP处理的MEFs相比,处于G2期的细胞比例为30.8%±2.57%,高于Ad-EGFP MEFs的23.9%±2.46%,并且细胞碎片的比例也远高于后者。划痕试验表明,Cα亚基缺失后细胞迁移能力下降,12h就显著低于对照组。结果表明,腺病毒携带的Cre重组酶能够在体外有效地敲除掉MEFs中的蛋白磷酸酶2A的Cα亚基,PP2ACα亚基的缺失会导致MEFs细胞周期倾向于阻滞在G2期,并会降低细胞迁移的能力。To investigate the effects of catalytic subunitαof protein phosphatase 2A（PP2A）on cell cycle and cell migration in mouse embryo fibroblasts（MEFs）,the mouse embryos were got at E12.5by mating the heterozygotes of Cαsubunit conditional knockout male and female homozygous mice at the ratio of 1∶2.MEFs were prepared by trypsin digestion and genotyped by PCR,RT-PCR and Western blot to identify the genetic basis of each embryo.Adenovirus associated Cre recombinase and GFP immunofluorescence protein were constructed and delivered to the P3 MEFs to knockout the Cαgene,GFP was used as control.After confirming the knockout efficiency by PCR,RT-PCR and Western blot,FACs was carried out to determine the cell cycle.Cell scratch test was carried in MEFs treated with the two kinds of viruses to investigate the effects on migration.Adenovirus associated Cre recombinase could knockout the gene efficiently in vitro.The knockout MEFs at G2 stage was 30.8% ±2.57%,while the control MEFs was 23.9% ±2.46%,and had more cell debris.Loss of Cαdecreased the cell migration ability significantly from 12 hafter the scratch.Cre recombinase delivered by adenovirus could efficiently pop out double floxed gene in vitro.Disruption of Cαsubunit slightly arrested the MEFs at G2 stage,and decreased the migration ability.

关 键 词：蛋白磷酸酶2A 小鼠胚胎成纤维细胞 细胞迁移 细胞周期 

分 类 号：S852.2[农业科学—基础兽医学]