DcR3-RNAi-SW480结肠癌稳转细胞模型的建立及方法优化

Establishment of DcR3-RNAi-SW480 stably transfected cell line of colon cancer and optimization of the methods

目的建立靶向沉默诱骗受体3(decoy receptor 3,DcR3)基因表达的DcR3-RNAi-SW480稳转细胞系,并对其条件及方法进行优化。方法构建靶向沉默DcR3基因的siRNA质粒表达载体,转染SW480细胞,并以G418及流式细胞仪筛选稳转细胞。实验分为3组:DcR3-RNAi稳转组、阴性对照组、空白对照组。蛋白质印记法(Western-blot)检测DcR3蛋白沉默情况。分别应用琼脂糖凝胶电泳、甲基噻唑基四唑(MTT)比色法、流式细胞术等方法对脂质体与质粒的比例、G418的筛选浓度、筛选的最佳方式等进行优化。结果 DcR3-RNAi稳转细胞组DcR3表达水平较对照组明显降低;在本实验室条件下,脂质体(μl)与DcR3-siRNA质粒(μg)最佳比例为3.5∶1;G418筛选浓度为800mg/L;G418联合流式细胞仪双筛的稳转细胞比例明显高于仅用G418单筛。结论成功建立特异性沉默DcR3基因表达的稳转DcR3-RNAi-SW480细胞系;G418联合流式是筛选稳转细胞较高效的方法;对转染及筛选的条件及方法进行优化,可提高转染与筛选效率。

Objective To establish the decoy receptor 3（DcR3） gene silencing and stable transfected DcR3-RNAi-SW480 cell line,and to optimize the conditions and methods.Methods The siRNA expression vector of DcR3 was constructed,and the vector was transfected into SW480 cells.Then,positive cells were screened by G418 and flow cytometer.The experiment included three groups ：the DcR3-RNAi stably transfected group,the negative control group and the blank control group.DcR3 protein expression was detected by Western-blot.Agarose gel electrophoresis,MTT colorimetric assay and flow cytometry were used respectively to optimize the proportion of liposome and plasmid,the G418 minimum concentration,the best method of screening.Results Compared with that of control group,DcR3 expression level in stably transfected DcR3-RNAi-SW480 cells was reduced significantly;In our laboratory conditions,the best proportion of liposome（μl） and DcR3-RNAi plasmid（μg） was 3.5：1;G418 minimum concentration for selection was 800 mg/L;The rate of stable transfected cells selectioned by G418 combined with flow cytometer was significantly higher than that only by G418.Conclusion The DcR3 gene silencing and stable transfected DcR3-RNAi-SW480 cell line have been established successfully;It is a highly effective method to select the transfected cells using both G418 and flow cytometry;It can improve the transfecting and selection efficiencies by optimizing the conditions and methods.