Wnt3a转染小鼠胸腺激酶缺陷细胞及对β-连环素蛋白亚细胞分布的影响

Transfection of mouse L-M (TK-) cells with Wnt3a and its effect on the subcellular distribution of beta-catenin

背景:Wnt信号通路是动物胚胎发育过程中重要的调控通路,建立Wnt信号通路细胞模型对于研究该通路有重要意义。目的:以Wnt3a真核表达质粒转染小鼠胸腺激酶缺陷细胞,构建能够持续激活Wnt信号通路的体外细胞模型,观察经典Wnt信号通路对β-连环素蛋白亚细胞分布的影响。方法:将扩增真核表达质粒pgk-Wnt3a-pcDNA3.0进行酶切鉴定。应用脂质体转染技术将真核表达质粒pgk-Wnt3a-pcDNA3.0和对照质粒pgk-neo-pcDNA3.0转染入胸腺激酶缺陷细胞,经过G418筛选后挑出细胞克隆,扩大培养。应用RT-PCR技术检测表达产物,应用间接免疫荧光技术检测Wnt3a对鼠胸腺激酶缺陷细胞β-catenin亚细胞分布的影响。结果与结论:Wnt3a质粒经过酶切鉴定证明所扩增的质粒为目的质粒。经过G418筛选3周后,挑选10个稳定转染的细胞克隆,进行RT-PCR检测,可见在L-Wnt3a细胞的cDNA产生1条长度为320bp亮带,表明扩增产物为所需要的目的片段,Wnt3a质粒转染胸腺激酶缺陷细胞后有Wnt3a在mRNA转录水平表达。转染对照质粒的胸腺激酶缺陷细胞cDNA未扩增出目的条带。免疫荧光检测可见Wnt3a质粒转染胸腺激酶缺陷细胞后细胞浆中有Wnt3a蛋白表达,转染对照质粒组无表达。Wnt3a质粒转染胸腺激酶缺陷细胞核中可见明亮的红色荧光,提示β-连环素蛋白在胸腺激酶缺陷细胞-Wnt3a细胞聚集并进入细胞核中。转染对照质粒的胸腺激酶缺陷细胞浆未见有明显β-连环素蛋白着色,细胞核中无β-连环素蛋白的聚集。构建了能够持续激活Wnt信号通路的体外细胞模型,真核表达Wnt3a质粒转染胸腺激酶缺陷细胞能够获得稳定表达,促进胸腺激酶缺陷细胞浆中β-连环素蛋白向细胞核转移。

BACKGROUND：Wnt signaling pathway plays an important regulative role in the embryonic development processes. Accordingly,it is of great significance to establish the cell model of Wnt signaling pathway so as to conduct study on it. OBJECTIVE：To establish Wnt signaling pathway cell model by transfecting L-M （TK-） cells with Wnt3a eukaryotic expression plasmid,and to investigate the effect of canonical Wnt signal pathway on the β-catenin subcellular distribution. METHODS：The eukaryotic expression plasmid pgk-Wnt3a-pcDNA3.0 after amplification was digested by restriction endonuclease first. Then it was transfected together with the control plasmid pgk-neo-pcDNA3.0 into L-M （TK-） cells via lipofection,after which the cell colony was screened by G418 for amplification. RT-PCR was used for detecting the expression products and the indirect immunofluorescence assay for observing the effect of Wnt3a on the β-catenin subcellular localization of L-M （TK-） cells. RESULTS AND CONCLUSION：The Wnt3a plasmid was verified by endonuclease digestion to have produced the expected plasmids after amplification. According to the RT-PCR detection to the 10 stably-transfected cell colonies achieved by 3 weeks of G418 screening,it was seen,on the L-Wnt3a cDNA,a strip of bright band of 320 bp in length,which showed that the products of amplification were exactly the expected fragments and that the Wnt3a plasmid was expressed on mRNA transcriptional level after being transfected with L-M （TK-） cells. In contrast,no expected band was found on the cDNA of L-M （TK-） cells transfecting the control plasmid. In addition,the immunofluorescence assay detection showed that the protein expression of Wnt3a was found in the cytoplasm of the L-M（TK-） cells tranfecting Wnt3a plasmid,while for those transfecting the control plasmid,it was opposite. β-catenin,as showing by bright red fluorescence,was found to concentrate and enter into the nucleus of the L-M （TK-） cells transfecting Wnt3a plasmid,while for those transfecting the control plasmid,it was opposite. Cell model with continually activated Wnt signaling pathway is established. The stable expression of Wnt3a in L-M （TK-） cells transfected with pgk-Wnt3a-pcDNA3.0 is obtained. The expression of Wnt3a is able to promote the transfer of β-catenin from cytoplasms into nucleus in L-M （TK-） cells.