PP-1蛋白稳定高表达MC3T3-E1成骨样细胞系的建立

目的建立稳定高表达蛋白质磷酸酶-1(protein phasphatase-1,PP-1)的MC3T3-E1成骨样细胞系。方法采用RT-PCR技术从MC3T3-E1细胞中扩增蛋白质磷酸酶-1(PP-1)基因,将获得的基因定向插入pCI-neo真核表达质粒中,PCR及双酶切鉴定后用脂质体将表达载体转染入MC3T3-E1细胞,经G418加压有限稀释筛选,建立稳定高表达PP-1的MC3T3-E1成骨样细胞系,采用Western blot法检测PP-1的蛋白表达情况。结果 PCR及双酶切鉴定表明表达载体pCI-neo-PP-1构建正确;Western blot检测结果显示PP-1蛋白能在转染pCI-neo-PP-1的MC3T3-E1细胞中稳定高表达。结论成功建立了稳定高表达PP-1的MC3T3-E1成骨样细胞系,为进一步深入研究PP-1在细胞力学信号转导通路中的功能及作用机制奠定了实验基础。

Establishment of MC3T3-E1 osteoblast-like cell lines with stable high expression of phasphatase-1 protein

Objective To establish MC3T3-E1 osteoblast-like cell lines with stable high expression of PP-1 protein.Methods PP-1 gene was augmented from the MC3T3-E1 cells using RT-PCR,and was inserted directly into the pCI-neo vector.The expression vector pCI-neo-PP-1 was transfected into MC3T3-E1 cell lines with Liposomes after identification by PCR and double digestion.The positive cell clones were selected with G418.Then,the stable transfection and expression of PP-1 in MC3T3-E1 cells were detected by Western blot.R...