摘要
目的:构建整合素β1的全基因表达载体,并探讨上调整合素β1蛋白表达对肺癌细胞生物学行为的影响。方法:根据GenBank数据库提供的整合素β1基因核苷酸序列,构建整合素β1的全基因表达载体,同时将空载体pCDNA3.1作为阴性对照。将载体转入感受态大肠杆菌,挑选阳性克隆,抽取重组载体。2种重组载体转染非小细胞肺癌细胞株PC-9细胞,用G418筛选后挑选单克隆并扩增获得稳定株。荧光显微镜、Real-timeRT-PCR、Western blot检测整合素β1的基因及蛋白水平的表达情况。细胞划痕试验和粘附试验比较整合素β1对细胞迁移、粘附能力的影响。结果:G418筛选出稳定转染整合素β1全基因表达载体和空载体的细胞,分别命名为PC-9/D6和PC-9/PCD。荧光显微镜见满视野带绿色荧光的细胞,PC-9/D6细胞整合素β1的mRNA、蛋白表达明显高于对照的PC-9/PCD细胞及母细胞PC-9。划痕试验和粘附试验表明整合素β1过表达的细胞株的迁移和粘附能力明显提高。结论:成功转染并筛选出整合素β1过表达细胞株,整合素β1过表达的细胞株的迁移和粘附能力明显升高。
Objectives： To construct integrin β1 whole gene expression vector, and to explore the effects of inhibition of integrin β1 protein expression on the biological behavior of lung cancer cells. Methods： Genomic sequences of integrin β1 （IGTB I） gene was retrieved from Genbank, and the cDNA was synthesized and inserted into plasmids pCDNA3.1. Recombinant plasmids were then transformed into competent E.coli. The positive clones were selected and recombinant plasmids were extracted. The integrin β1 whole gene expression vectors and vacant vectors were transfected into PC-9 by lipofectemine2000. The stably transfected cell clones were obtained after being screened with G418. Fluorescence microscope, Real-time RT-PCR and Westem blot were performed to examine the inhibitory effect at the RNA level and protein level. Then we detected the influences of integrin β1 on cell migration and adhesion capacity by cell scratch test and adhesion test. Results： The stably transfected pCDNA3.1-IGTB1 cell clones was significantly up-regulated as validated by Real-time RT-PCR and Western blot. Scratch test and adhesion test showed that the migration and adhesion capacity of integrin β1-inhibited cell line significantly reduced. Conclusion： We successfully transfected and screeened out the integrin β1 over-expressed cell lines, and the cell lines can significantly increased the migration and adhesion capability of cancer cells.
出处
《现代生物医学进展》
CAS
2009年第20期3807-3810,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金资助项目(30873023)
关键词
整合素Β1
过表达
肺癌
迁移
粘附
integrin β1
overexpression
lung cancer
migration
adhesion
