PTEN基因转染对人膀胱癌细胞BIU-87中P13K—Akt信号通路的影响

目的研究抑癌基因PTEN转染对人膀胱癌细胞BIU-87中P13K—Akt信号通路的作用。方法将携有PTEN基因的重组真核表达质粒pBp-PTEN转化大肠杆菌DH5=并扩增，抽提纯化质粒并进行酶切鉴定，pBp-PTEN体外转染BIU-87细胞（pBp-PTEN—BIU87），筛选稳定转染的细胞并扩增培养，以转染了空质粒pBp的BIU-87细胞（pBp—BIU87）和正常BIU-87细胞为对照，用RT—PCR检测PTEN的表达情况。将PTEN基因的真核表达质粒转染膀胱癌细胞BIU一87（pBp—PTEN—BIU87），以BIU87细胞和pBp-BIU87细胞为对照，应用Wester blot方法检测三组细胞P110（P13K的催化亚单位）、磷酸化P110、Akt和磷酸化Akt表达的情况。结果3种细胞P110和Akt蛋白总水平没有变化，但磷酸化P110和磷酸化Akt在PTEN转染细胞中的表达明显弱于对照组。结论PTEN是通过对PI3K—Akt信号传导途径的负调控而抑制肿瘤的形成。PTEN—PI3K—Akt途径可能是PTEN抑癌作用的重要机制。

Effects of gene PTEN transfection on PI3K-Akt signal path of bladder cancer cell BIU-87

Objective To study the effects of anti-oncogene PTEN transfection on signal transduction path of PI3K-Akt of human bladder cancer cell BIU-87. Methods A eukaryotic expression plasmid containing PTEN and pBp-PTEN, was introduced into E. coil DH5 α and amplified. Plasmid was prepared and purified, and then identified by restriction enzyme. pBp-PTEN was transfected into BIU-87 （pBp-PTEN-BIU87）, and positive cell clones were selected and amplified. With empty plasmid transfected BIU-87 （pBp.BIU87） and normal BIU-87 as control groups, expression of PTEN was detected by RT-PCR. After PTEN eukaryotic expressing plasmid was transfected into bladder cancer cell lines BIU-87, expressions of P110 （catalyzing subunit of PI3K）, phosphorylated P110, Akt and phosphorylated Akt were examined by western blot with normal BIU-87 cell and empty plamid transfected cell as control groups. Results All cells of three groups expressed the same level of total P110 and Akt, but expressions of phosphorylated P110 and phosphorylated Akt were significantly weaker in PTEN transfected ceils than those in other two groups. Conclusion PTEN might downregulate PI3K-Akt signal path as a phosphatase; the signal transduction path, PTEN-PI3K-Akt, might be an important mechanism of PTEN functons.