稳定表达Wnt3a基因的猪胰腺干细胞株的建立  

Establishment of Pocine Pancreatic Stem Cells Strain Stably Expressing Wnt3a

作　　者：彭莎[1,2] 张军林[1,2] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]陕西省干细胞工程技术研究中心陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2012年第11期1300-1305,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(NSFC,31101775);高等学校博士学科点专项科研基金(SRFDP,20100204120020)

摘　　要：Wnt3a是经典Wnt通路上游的一个重要分泌蛋白,Wnt3a蛋白的活化可促进多种细胞的增殖和分裂。为了揭示Wnt3a在猪胰腺干细胞(PSC)增殖及分化中的调控机理,采用RT-PCR从pGKS2P(+)-Wnt3a质粒中扩增出Wnt3a全长片段,将测序正确的Wnt3a连接到带有水母绿色荧光蛋白(AcGFP1)报告基因的真核表达载体pIRES2-AcGFP1中,构建重组质粒。经EcoRⅠ/BamHⅠ酶切鉴定后,用脂质体转染至猪PSC。用抗生素G418筛选3周后获得稳转pIRES2-AcGFP1-Wnt3a的细胞株。荧光显微镜下观察到几乎所有细胞表达绿色荧光蛋白。RT-PCR和Western blot的结果显示,稳转Wnt3a组的猪PSC中Wnt3a mRNA及蛋白表达水平比对照组明显升高。这些结果表明,稳定表达Wnt3a的猪胰腺干细胞株成功建立,为进一步研究Wnt3a在猪PSC增殖及分化中的作用机理提供了基础资料。Wnt3a is an important secreted protein of the canonical Wnt pathway and its activation can promote cell proliferation and division. In order to reveal the roles of Wnt3a in regulating the proliferation and differentiation towards insulin-secreting cells of porcine pancreatic stem cells （PSC）, the full length of Wnt3a cDNA fragment was amplified from pGKS2P （＋）-Wnt3a plasmid by RT-PCR. The recombinant plasmid plRES2-AcGFP1-Wnt3a was constructed by inserting Wnt3a fragment sequenced correctly into eukaryotic expression vector plRES2-AcGFP1. After identifying with double digestion of EcoR I /BamH I , we transfected plRES2-AcGFP1-Wnt3a into porcine PSC by lipofectamin 2000. We obtained the stably transfected pocine PSC strain after screening culture by G418 for 3 weeks. Almost all cells expressing green fluorescence protein were observed by fluorescence microscope. The expression of Wnt3a mRNA and protein obviously increased in stable transfection group of Wnt3a compared to control group when detected by RT-PCR and Western blot. These results indicated that we successfully established the porcine PSC strain stably expressing Wnt3a. This study provide basal data for further exploring the mechanism of Wnt3a in porcine PSC proliferation and differentiation.

关 键 词：WNT3A pIRES2-AcGFP1 重组质粒 胰腺干细胞(PSC) 

分 类 号：Q813[生物学—生物工程]