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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。
Objective: To construct the whole gene expression vector of integrin β1 and investigate the effect of up-regulating the expression of integrin β1 on the biological behavior of lung cancer cells. Methods: According to the nucleotide sequence of integrin β1 gene provided by GenBank database, the whole gene expression vector of integrin β1 was constructed, and the empty vector pCDNA3.1 was used as a negative control. The vector was transformed into competent E. coli, the positive clones were picked and the recombinant vector was extracted. Two kinds of recombinant vectors were transfected into PC-9 cell line of non-small cell lung cancer. After selection with G418, the single clones were selected and amplified to obtain stable strains. Fluorescence microscopy, Real-time RT-PCR and Western blot were used to detect the expression of integrin β1 gene and protein. Cell scratch test and adhesion test compared integrin β1 cell migration and adhesion. Results: G418 cells were selected and stably transfected with the integrin β1 gene expression vector and empty vector, and named as PC-9 / D6 and PC-9 / PCD respectively. Fluorescence microscopy showed full-field cells with green fluorescence. The mRNA and protein expression of integrin β1 in PC-9 / D6 cells was significantly higher than that in PC-9 / PCD cells and PC-9 cells. Scratch test and adhesion test showed that the integrin β1 over-expression of cell lines migration and adhesion was significantly improved. CONCLUSION: Transfection and screening of integrin β1-overexpressing cell lines shows that migration and adhesion of integrin β1-overexpressing cell lines are significantly increased.