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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 a lentiviral expression system that can induce the expression of wild-type or mutant type 10 phosphatase and tensin homolog (PTEN) on chromosome 10, and provide an ideal experimental tool for further study on the relationship between PTEN and tumor. Methods: The RNA of thyroid cancer, lung cancer and its adjacent tissues was extracted and amplified by RT-PCR. The PTEN fragment was ligated into the lentiviral vector and co-transfected into the 293T cells of human embryonic kidney with virus packaging plasmid. Get the virus. The cells were infected with MDA-MB-231 cells and screened with puromycin to obtain stably expressed cell lines. The expression of PTEN gene in stable transfected cell lines was detected by Western blotting. The colony formation ability of the cell lines was tested by soft agar colony formation assay. Results: The PTEN mutant was found in thyroid cancer and lung cancer. The results of Western blotting confirmed that the lentiviral expression system of wild-type and mutant PTEN was successfully constructed. Mutant PTEN is more capable of cloning than wild-type PTEN. CONCLUSION: PTEN mutants were found and the inducible lentiviral expression system of wild-type and mutant PTEN genes was successfully constructed. Mutant PTEN not only has tumor suppressor effect but also has carcinogenic effect.