稳定过表达犬EGFR蛋白的MDCK细胞的建立及其对凋亡和细胞周期的影响  

Establishment of MDCK Cells with Stable Overexpression of Canine EGFR Protein and Its Effect on Apoptosis and Cell Cycle

作　　者：杨迪[1,2,3,4] 黄玲巍 杨雅雯 乔自林 王家敏[1,3] YANG Di;HUANG Lingwei;YANG Yawen;QIAO Zilin;WANG Jiamin(Engineering Research Center of Key Technology and Industrialization of Cell-based Vaccine,Ministry of Education,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Gansu Tech Innovation Center of Animal Cell,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Department of Experiment&Teaching,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学生物医学研究中心,细胞基质疫苗关键技术与产业化教育部工程研究中心,甘肃兰州730030 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]西北民族大学生物医学研究中心,甘肃省动物细胞技术创新中心,甘肃兰州730030 [4]西北民族大学实验教学部,甘肃兰州730030

出　　处：《华北农学报》2024年第4期223-230,共8页Acta Agriculturae Boreali-Sinica

基　　金：甘肃省科技计划项目(22JR11RA239;23YFFA0071);中央高校基本科研业务费资金专项(31920230001)。

摘　　要：表皮生长因子受体(EGFR)在许多实体肿瘤中过度表达,建立稳定过表达犬EGFR蛋白的MDCK细胞株,有利于为研究犬或人类肿瘤疾病提供良好的细胞模型和研究基础。首先制备目的片段以构建EGFR基因过表达载体,与慢病毒包装载体共同转染至293T细胞中,48 h后提取上清液经纯化获得EGFR基因过表达慢病毒液。将其转染至MDCK细胞中,经嘌呤霉素筛选和单细胞克隆后观察感染效率,RT-qPCR和Western Blot、间接免疫荧光分别检测基因及蛋白的表达,建立EGFR蛋白过表达MDCK细胞株。使用流式细胞仪分别检测过表达及对照细胞株的细胞凋亡和细胞周期。犬EGFR基因位于18号染色体,由编码跨膜糖蛋白的30个外显子组成。经反应体系,PCR产物交换入线性化表达载体,获得了阳性转化子8个,大小为738 bp。所获EGFR基因过表达慢病毒滴度为3.5×10^(8 )TU/mL。转染MDCK细胞后在荧光显微镜下观察荧光效果显著,经检测EGFR基因及其蛋白在细胞中的表达量显著升高。间接免疫荧光试验显示,EGFR在细胞中表达明显增强。过表达细胞的早期凋亡率和晚期凋亡率显著上升,且在G2/M期发生阻滞。成功建立一株稳定的犬EGFR过表达MDCK细胞株,犬EGFR的过表达催化了细胞分裂,使DNA复制加快,同时刺激了细胞凋亡。Epidermal growth factor receptor(EGFR)is excessively expressed in numerous solid tumours,establishing a stable over-expressing canine EGFR protein MDCK cell line will prove beneficial in providing an exceptional cellular model and research foundation for the study of canine or human tumour diseases.Firstly,the target fragment was prepared for construction of an overexpression vector of the EGFR gene.It was co-transfected with a lentivirus packaging vector into 293T cells.After 48 hours of transfection,the supernatant was extracted and purified to obtain a lentivirus containing the overexpressed EGFR gene.This virus was then transduced into MDCK cells,following puromycin selection and single-cell cloning.The infectivity efficiency was observed,expressing gene and protein levels were detected through RT-qPCR,Western Blot,and indirect immunofluorescence assays,and an EGFR protein overexpression MDCK cell line was established.Using flow cytometry,apoptosis and cell cycle of overexpression and control cell lines were separately determined.The canine EGFR gene was located on chromosome 18 and consisted of 30 exons encoding a transmembrane glycoprotein.Through the reaction system,PCR products were ligated into linearized expression vectors,generating 8 positive transformants with a size of 738 bp.The obtained lentivirus titer of EGFR gene overexpression was 3.5×10^(8 )TU/mL.Transfected MDCK cells exhibited superior fluorescent effect was remarkable under fluorescent microscopy,resulting in a significant increase in the expression level of the EGFR gene and its protein within the cells.Indirect immunofluorescence assay revealed a substantial enhancement in the expression of EGFR in cells.In contrast,early apoptosis and late apoptosis rates among the transfected cells increased notably,with a clear arrest occurring at the G2/M phase.This research successfully developed a stable canine EGFR overexpressing MDCK cell line.Overexpression of canine EGFR stimulated cell division,elevated DNA replication,and concurrently trigge

关 键 词：犬肾细胞系 表皮生长因子受体 慢病毒载体 

分 类 号：S829.2[农业科学—畜牧学] Q78[农业科学—畜牧兽医]