利用CRISPR/Cas9技术构建人TCRP1基因敲除慢性髓系白血病K562细胞系及其生物学功能检测  

Construction of human TCRP1 gene knockout chronic myeloid leukemia K562 cell line using CRISPR/Cas9 technology and its biological function detection

作　　者：刘孝荣[1] 陈妍 辛泽锋 钟惠锋 蔡思宇 陈运生[3] Liu Xiaorong;Chen Yan;Xin Zefeng;Zhong Huifeng;Cai Siyu;Chen Yunsheng(Department of Clinical Laboratory,Shenzhen Children′s Hospital,Shenzhen 518038,China;Institute of Pediatrics,Shenzhen Children′s Hospital,Shenzhen 518038,China;Department of Hematology and Oncology,Shenzhen Children′s Hospital,Shenzhen 518038,China)

机构地区：[1]深圳市儿童医院检验科,深圳518038 [2]深圳市儿童医院儿研所,深圳518038 [3]深圳市儿童医院血液肿瘤科,深圳518038

出　　处：《中国医师杂志》2023年第8期1187-1193,共7页Journal of Chinese Physician

基　　金：广东省基础与应用基础研究基金(2020A1515010246);深圳市科技计划项目(JCYJ20210324135414039)。

摘　　要：目的选取人慢性髓系白血病(CML)细胞K562为实验对象,利用慢病毒介导的CRISPR/Cas9基因编辑技术构建稳定敲除舌癌耐药相关蛋白1(TCRP1)基因的CML细胞系K562/TCRP1-KO,通过细胞增殖、细胞凋亡、药物敏感性等功能试验比较K562/TCRP1-KO与对照细胞(K562/cas9-CTL)的表型差异,初步探讨TCRP1基因参与CML发病的可能机制。方法在特定位点设计针对TCRP1的小向导RNA(sgRNA),寡核苷酸片段退火后与线性化的Cas9表达载体重组,慢病毒包装系统转染293T细胞,收集纯化病毒感染K562细胞,嘌呤霉素加压筛选阳性多克隆,有限稀释法进一步筛选出单克隆K562/TCRP1-KO,Sanger测序和Western blot检测成功敲除的稳定细胞株,同时构建转染lentiCRISPR载体的K562细胞作为对照细胞株(K562/cas9-CTL),采用细胞计数法、CCK8法及伊马替尼(IM)梯度稀释法、流式细胞术分别对K562/TCRP1-KO和K562/cas9-CTL进行细胞增殖、药物敏感性及细胞凋亡分析。结果成功构建了用于TCRP1敲除的sgRNA-Cas9重组质粒载体,转染293T细胞后,利用有限稀释法成功筛选到TCRP1敲除的单克隆细胞株。与K562/cas9-CTL细胞相比,K562/TCRP1-KO细胞的增殖能力显著减弱,IM药物敏感性显著增强,细胞凋亡进程显著加快(均P<0.05)。结论利用CRISPR/Cas9成功构建了TCRP1敲除的CML细胞株,TCRP1可能作为癌症相关基因影响CML细胞的增殖、IM耐药能力及凋亡进程。Objective To select human chronic myeloid leukemia(CML)cell line K562 as the experimental object,and use lentivirus mediated CRISPR/Cas9 gene editing technology to construct a stable CML cell line K562/TCRP1-KO that knocks out the tongue cancer resistance related protein 1(TCRP1)gene;and through functional tests such as cell proliferation,apoptosis,and drug sensitivity,compare the phenotypic differences between K562/TCRP1-KO and control cells(K562/cas9-CTL),and preliminarily explore the possible mechanism of TCRP1 gene involvement in the pathogenesis of CML.Methods The small guide RNA(sgRNA)targeting TCRP1 was designed at a specific location.After annealing,the oligonucleotide fragments were recombined with the linearized Cas9 expression vector,and the lentivirus packaging system was transfected into 293T cells.The purified virus was collected and infected with K562 cells.Positive polyclons were screened for puromycin pressure,and monoclonal K562/TCRP1-KO was further screened by limited dilution method.Stable cell lines were successfully knocked out by sanger sequencing and Western blot detection;Simultaneously,K562 cells transfected with lentiCRISPR vector were constructed as control cell lines(K562/cas9-CTL);Using cell counting method,cell counting kit 8(CCK8)method,imatinib(IM)gradient dilution method,and flow cytometry cell proliferation,drug sensitivity,and apoptosis analysis were performed on K562/TCRP1-KO and K562/cas9-CTL,respectively.Results The sgRNA-Cas9 recombinant plasmid vector for TCRP1 knockout was successfully constructed,and after transfection into 293T cells,TCRP1 knockout monoclonal cell lines were successfully screened using limited dilution method.Compared with K562/cas9-CTL cells,the proliferation ability of K562/TCRP1-KO cells was significantly reduced,IM drug sensitivity was significantly enhanced,and the process of cell apoptosis was significantly accelerated(all P<0.05).Conclusions A CML cell line with TCRP1 knockout was successfully constructed using CRISPR/Cas9.TCRP1 may act as a canc

关 键 词：K562细胞 白血病 髓系 慢性 基因编辑 基因 TCRP1 

分 类 号：R733.72[医药卫生—肿瘤]