基于慢病毒感染技术敲低CTNNB 1基因的H1299细胞系构建  

Lentivirus Infection-based Construction of the H1299 Cell Line Knocking down CTNNB1 Gene

作　　者：沈齐 金玉玲 曹金金 张玲芳 汪洋 胡翰 刘滨磊 SHEN Qi;JIN Yuling;CAO Jinjin;ZHANG Lingfang;WANG Yang;HU Han;LIU Binlei(Biological Engineering and Food,Hubei Univ.of Tech.,Wuhan 430068,China;Wuhan Binhui Biotechnology Co.,Ltd,Wuhan 436030,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉430068 [2]武汉滨会生物科技股份有限公司,湖北武汉436030

出　　处：《湖北工业大学学报》2025年第2期55-61,共7页Journal of Hubei University of Technology

摘　　要：为构建β-连环蛋白编码基因CTNNB 1稳定敲低的肺癌H1299细胞模型,以深入探讨其在肺癌转移复发及耐药性中的作用机制。通过筛选功能性siRNA序列,构建pLKO.1-CTNNB 1-shRNA-EGFP-puro重组表达载体,基因测序证实shRNA序列正确插入载体,经293T细胞包装获得慢病毒颗粒。采用慢病毒感染H1299细胞后,通过嘌呤霉素筛选获得阳性细胞群体,结合有限稀释法成功分离单克隆细胞系。流式细胞术检测显示单克隆细胞EGFP阳性率达99%。RT-qPCR及Western blot分析表明,CTNNB 1在mRNA和蛋白表达水平分别下降至对照组的54%和60%。实验成功建立CTNNB 1基因稳定敲降的H1299细胞模型,该模型为后续研究β-catenin信号通路在肺癌恶性进展中的调控机制提供了可靠的细胞平台。This study aims to establish a lung cancer H1299 cell model with stable knockdown of theβ-cateninencoding gene CTNNB1,in order to further investigate its mechanism of action in lung cancer metastasis,recurrence,and drug resistance.By screening functional siRNA sequences,a recombinant expression vector pLKO.1-CTNNB1-shRNA-EGFP-puro was constructed.Gene sequencing confirmed that the shRNA sequence was correctly inserted into the vector,and lentiviral particles were obtained through packaging in 293T cells.After infection of H1299 cells with lentivirus,a positive cell population was obtained through puromycin selection,and a monoclonal cell line was successfully isolated using the limiting dilution method.Flow cytometry analysis showed that the positive rate of monoclonal cell EGFP was 99%.RT-qPCR and Western blot analysis showed that the mRNA and protein expression levels of CTNNB1 decreased to 54%and 60%of the control group,respectively.The experiment successfully established a H1299 cell model with stable knockdown of CTNNB1 gene,which provides a reliable cell platform for subsequent research on the regulatory mechanism ofβ-catenin signaling pathway in the malignant progression of lung cancer.

关 键 词：H1299细胞 慢病毒 CTNNB 1基因 敲低细胞系 

分 类 号：R153[医药卫生—营养与食品卫生学]