摘要
目的:构建可诱导表达野生型或突变型第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)的慢病毒表达体系,为进一步研究PTEN与肿瘤间的关系提供理想的实验工具。方法:提取甲状腺癌、肺癌及其癌旁组织的RNA,经反转录后扩增PTEN片段,将其连接入慢病毒载体,与病毒包装质粒共转染人胚肾T细胞(293T)细胞,获得病毒。将其感染MDA-MB-231细胞,用嘌呤霉素筛选,获得稳定表达的细胞株。采用蛋白印迹法检测稳定转染的细胞株中PTEN基因的表达情况,用软琼脂克隆形成实验检测细胞株的克隆形成能力。结果:在甲状腺癌和肺癌中发现PTEN突变体;蛋白印迹检测结果证实野生型和突变型PTEN的慢病毒表达体系构建成功。突变型PTEN的克隆形成能力强于野生型PTEN。结论:发现了PTEN突变体,并成功构建了野生型和突变型PTEN基因的可诱导慢病毒表达体系,突变型PTEN不但有抑癌作用,还有致癌作用。
Objective To construct inducible lentiviral vector containing wild-type or mutant PTEN gene, which provides an ideal model for further study of the relationship between cancer and PTEN gene. Methods Human PTEN gene fragment from thyroid cancer, lung cancer and their adjacent tissues was amplified by RT-PCR, and then was cloned into pCR2.1TOPO vector. After sequencing, the fragment was subcloned into the lentiviral vector, and co-transfected into 293T cells with the virus package plasmid to obtain the virus. Then the virus was used to infect 231 cells to obtain stably expressing cell lines. Western blot was used to detect the expression of the PTEN gene. Soft agarose colony formation assay was used to detect the colony forming ability of cell lines. Results Mutant PTEN was discovered in thyroid cancer and lung cancer. Western blot results confirmed that the wild-type or mutant PTEN lentiviral expression system was successfully constructed. Colony forming ability of the mutant PTEN was stronger than that of the wild-type PTEN. Conclusions Mutant PTEN gene is discovered. The recombinant inducible lentiviral vector containing PTEN gene is successfully constructed. Mutant PTEN has not only the antitumor effect, but also the carcinogenic effect.
出处
《诊断学理论与实践》
2013年第2期189-193,共5页
Journal of Diagnostics Concepts & Practice
