构建沉默Runx 2基因的牙龈癌细胞慢病毒载体  

Construction of lentiviral vector of gingival cancer cell silencing Runx2 gene

作　　者：胡金龙 秦晗 张勇[1] 邓晴 HU Jinlong;QIN Han;ZHANG Yong;DENG Qing(Department of Stomatology,Lianyungang Clinical College of Nanjing Medical University,Lianyungang 222002,Jiangsu,China)

机构地区：[1]南京医科大学连云港临床医学院口腔科,江苏连云港222002

出　　处：《西部医学》2023年第9期1287-1291,共5页Medical Journal of West China

基　　金：国家自然科学基金项目(81500893)。

摘　　要：目的筛选沉默成骨细胞特异性转录因子2(Runx2)基因的慢病毒载体,转染人上颌牙龈癌颈淋巴结转移癌细胞系GNM细胞,获得沉默Runx 2基因的GNM细胞,为探究Runx2在GNM细胞中的转录调控奠定实验基础。方法根据预实验结果在含HiTransG P的培养液中,病毒按照感染复数(MOI)10转染GNM细胞,将病毒按序号分为NC组:CON313;KD1组:LV-Runx2-RNAi(80978-1);KD2组:LV-Runx2-RNAi(80979-1);KD3组:LV-Runx2-RNAi(80980-1),使用荧光显微镜观察转染120 h后各组荧光标记基因的表达;实时PCR分析Runx2的转录产物水平,比较Runx 2基因的组间沉默效率;Western Blot进一步检测Runx2蛋白沉默效果。结果各组细胞经慢病毒转染,120 h后观察见细胞状态良好,其中NC组荧光表达未见;KD1组荧光表达较高;KD2组荧光表达最高;KD3组荧光表达较低。实时PCR分析显示Runx 2基因沉默效果最佳的为KD2组,沉默效率可达64.4%(P<0.05)。Western Blot结果显示各实验组中KD2组GNM细胞的Runx2蛋白表达最低(P<0.05)。结论成功筛选出特异性转染GNM细胞的慢病毒载体,同时初步确定转染实验相关适宜参数。Objective To screen lentiviral vector of runt-related transcription factor2(Runx2)gene to transfect GNM cells with cervical lymph node metastasis from human maxillary gingival carcinoma and obtain the silencing of Runx2 gene expression in GNM cell,which laid the experimental foundation for the later study on the transcriptional regulation of Runx2 in GNM cells.Methods Based on the preliminary results,in the culture medium containing Hitransg P,virus were transfected with GNM cells according to multiplicity of infection(MOI)10 and viruses were divided into four groups according to their serial number:NC:CON313;KD1:LV-Runx2-RNAi(80978-1);KD2:LV-Runx2-RNAi(80979-1);KD3:LV-RUNX2-RNAi(80980-1),fluorescence microscopy was used to observe the expression of fluorescence marker genes in each group 120 h after transfection.Real-time PCR was used to analyze the transcription product level of Runx2 and compare the silencing efficiency of Runx2 gene between groups.Western Blot further detected the protein silencing effect of Runx2 gene.Results After 120 hours of lentivirus transfection,the cells in each group were in good condition.The fluorescence expression was not observed in NC group.The fluorescence expression was higher in KD1 group.The fluorescence expression in KD2 group was the highest.The fluorescence expression was lower in the KD3 group.Real-time PCR analysis showed that the best silencing effect of Runx2 gene was in the KD2 group.The silencing efficiency was up to 64.4%(P<0.05).Western Blot results showed that Runx2 protein expression of GNM cells in the KD2 group was the lowest among all experimental groups(P<0.05).Conclusion The lentiviral vectors specifically transfected GNM cells are successfully screened out,and relevant appropriate parameters of transfection experiment are preliminarily determined.

关 键 词：成骨细胞特异性转录因子2 牙龈癌 慢病毒 转染 

分 类 号：R34[医药卫生—基础医学]