肿瘤转移抑制基因nm23-H1在人肺癌细胞的表达及其检测

目的：选择已分离鉴定的人巨细胞肺癌细胞高转移亚型PGBEl，转染肿瘤转移抑制基因nm23-H1，以获得稳定表达nm23-H1基因的人肺癌细胞株。方法：采用亚克隆方法获得pcDNA3．1（-）／nm23-H1真核表达载体并进行双酶切和测序鉴定；应用脂质体转染法将重组质粒转染PGBE1细胞；用G418筛选获得阳性细胞克隆，命名为pcDNA3．1（-）／nm23-H1-PGBE1细胞株；用RT-PCR法半定量检测nm23-H1基因的mRNA表达水平和免疫组化法检测nm23-H1基因的蛋白质表达。结果：真核表达载体pcDNA3．1（-）中正确插入了具有完整阅读框的nm23-H1基因；倒置显微镜下观察到经G418筛选出的稳定转染的PGBE1细胞，形成“小岛”；RT-PCR和免疫组化显示转染的细胞中nm23-H1 mRNA和蛋白质水平表达均增高。结论：建立了稳定表达nm23-H1基因的PGBE1转染细胞株，可用于进一步研究肿瘤转移的分子机制。

Expression of metastasis-suppressor gene nm23-H1 in human lung cancer cell and its determination

Objective:To obtain the human lung cancer cell expressed stably metastasis-suppressor gene nm23-H1.nm23-H1 gene was cloned from plasmid pBV220/nm23-H1 to eukaryotic expression vector pcDNA3.1(-).Methods:Recombinant vector pcDNA3.1(-)/nm23-H1 was verified containing full length human nm23-H1 cDNA by restriction enzyme assay and sequencing.Results:This recombinant plasmid was transfected into a high metastatic human lung giant cell carcinoma(PG) by lipofectamine technique.Positive cells were screened with G418 and named pcDNA3.1(-)/nm23-H1-PGBE1 strain.High expression of nm23-H1 was identified by RT-PCR and immunohistochemistry.Conclusion:The stable pcDNA3.1(-)/nm23-H1-PGBE1 strain was constructed.This cell strain may be valuable for further study on the molecular mechanisms of cancer metastasis.