远红荧光蛋白报告基因mKate2对肝癌细胞的标记及荧光成像  

Labeling of liver cancer cell for fluorescence imaging study by far-red fluorescence protein reporter gene mKate2

作　　者：李丹[1] 沈敏[1] 张波[1] 李征然[1] 庞鹏飞[1] 朱康顺[1] 王劲[1] 黄明声[1] 孟晓春[1] 单鸿[1] 

机构地区：[1]中山大学附属第三医院放射科分子影像学实验室中山大学介入放射学研究所,广州510630

出　　处：《中华医学杂志》2011年第19期1344-1347,共4页National Medical Journal of China

基　　金：国家自然科学基金（81071206,81070349）;NSFC-广东联合基金（U1032002）

摘　　要：目的 制备远红荧光蛋白报告基因mKate2慢病毒,用于标记人肝癌细胞株HepG2,并进行体外的细胞荧光成像,为肿瘤的活体荧光成像示踪提供基础.方法 以pmKate2-N质粒为模板,将mKate2基因克隆到慢病毒表达载体pLenti6.3/V5-DEST;pLenti6.3-mKate2表达质粒和包装质粒共转染293T细胞进行病毒包装,并测定慢病毒的活性滴度;mKate2慢病毒以病毒感染复数（MOI）=6感染HepG2 96 h后,通过荧光显微镜观察和流式细胞分析法（FACS）检测感染效率;收集2×106个mKate2-HepG2细胞,通过光学成像系统进行荧光成像,确定最佳成像参数.结果 测序结果显示,mKate2基因序列正确,无突变或缺失;mKate2慢病毒的活性滴度为1.6×106TU/ml;mKate2慢病毒感染HepG2 96 h后,荧光显微镜下观察到大量红色荧光蛋白的表达,FACS检测显示mKate2的阳性率为93.8%±0.4%;激发光（530±15）nm和发射光（710±28）am为对mKate2-HepG2细胞进行荧光成像的最佳参数.结论 慢病毒能够介导远红荧光蛋白报告基因mKate2对人肝癌细胞株HepG2的高效标记,并且mKate2标记的HepG2能够进行体外细胞荧光成像,可以用于下一步的活体肿瘤示踪研究.Objeclive To create far-red fluorescence protein reporter gene mKate2 lentivirus.label human liver cancer cell line HepG2 with lentivirus and explore the feasibility of in vitro fluorescence imaging of labeled tumor cells so as to provide experimental rationales for in vivo fluorescence tumor imaging.Methods mKate2 gene was amplified from pmKate2-N plasmid.Then the fragment was inserted into the lentivirus expression vector pLenti6.3/V5-DEST.The expression plasmids pLenti6.3-mKate2 and the packaging plasmids were cotransfected into 293T cells.The biological titer of lentivirus was determined.HepG2 cells were infected with mKate2 lentivirus at a MOI（virus multiplicitv of infeetion）of 6 for 96 hours.The infection efficiency was assayed through fluorescence microscope and fluorescent-activated cell scanning（FACS）.And 2×106 mKate2-HepG2 cells were collected for fluorescence imaging through an optical imaging system.And the optimal imaging parameters were determined.Results DNA sequencing analysis confirmed that mKate2 gene sequence was correct and there was no mutation or deletion.The biological titer of produced mKate2 lentivims was 1.6×106 TU/ml.At 96 hours after mKate2 lentivirus infection,fluorescence microscope showed that mKate2 was expressed in a large percentage of cells.FACS assay showed that the mKate2 positive rate was 93.8%±0.4%.Excitation light 530±15 nm and emission light 710±28 nm were the optimal imaging parameters for mKate2-HepG2 cells.Conclusion Lentivirus can mediate efficiently the mKate2 reporter gene labeling of human liver cancer cell line HepG2.The mKate2-labeled HepG2 cells can be detected through in vitro fluorescence imaging.Further tracing studies of in vivo tumor fluorescence imaging age technically feasible.

关 键 词：远红荧光蛋白 基因 报告 肝肿瘤 

分 类 号：R735.7[医药卫生—肿瘤]