Non-small Cell Lung Cancer Cell Lines ERCC1Inhibition Gene In The Establishment Of The Model

Objectives: ERCC1establishment of non-small cell lung cancer gene model, Platinum for non-small cell lung cancer drug resistance related gene therapy study provides experimental models and methods.Method:1. Application real-timePCR and Western blot methods from44cells (A549cells, Lung adenocarcinoma cells in Gejiu, Xuanwei, Normal human bronchial epithelial cells in lung adenocarcinoma cells) filter out genes ERCC1expression;2. Antisense oligonucleotide synthesis ERCC1(ERCC1-ASODN), and go through the liposome-mediated gene ERCC1expression in cell lines; antisense oligo nucleotides inhibition of genes ERCC1expression, establishing ERCC1gene inhibition model;3. Real-timePCR detection of transfection of before and after the change of ERCC1mRNA level; Western blot detection of transfected genes ERCC1protein levels before and after change MTT colorimetric assay for detection of transfection on the growth of cell lines before and after impact determination of flow cytometry transfection on cell cycle and apoptosis rate before and after impact.Results:1. ERCC1gene high expression cell screening experiments, genes ERCC1mRNA expression and protein expression levels in A549cells high, namely twice and5.5times times that of Normal human bronchial cells, far higher than Xuanwei and Gejiu lung adenocarcinoma cells in lung adenocarcinoma cells;2. Successful build ERCC1-ASODN, and A549cells by liposome-mediated import, successful transfection experiments establishing the gene model:3. Anti-meaning oligo nucleotide suppression experimental in the, real-time PCR detection results display go dye Hou ERCC1mRNA in A549cell in the of expression appears has obvious of suppression, ERCC1-ASODN1group suppression effect most obvious, for go dye Qian of0.6times times; Western blot measuring was protein of expression also to ERCC1-ASODN1group suppression effect most obvious, for go dye Qian of0.13times times, p<0.05, has statistics differences; MTT experimental results as shown in ERCC1-ASODN on A549cell of suppression rendering out time dependency, best role time48h; flow type cell technology display ERCC1-ASODN dye to promote A549cell of apoptosis apoptosis,48hours Hou, ERCC1-ASODN group appears s period, the G2/M phase cells, increase in the number of G0/G1cells, cell cycle was blocking the G0/G1period.Conclusions:Experiment successful screening of genes ERCC1expression cell lines with the highest, ERCC1antisense oligonucleotide synthesis (ERCC1-ASODN), and successfully mediated by liposome transfer in A549cells successfully build ERCC1gene inhibition model;Transfer to inhibition of ERCC1mRNA and its protein expression, its role and time has a relationship at the time of48hours, strong and also can promote the A549cell apoptosis;By the above experiment successfully established ERCC1gene in non-small cell lung cancer cell line models. Platinum for non-small cell lung cancer drug resistance related gene therapy study provides experimental models and methods.