Luciferase2/mKate2双报告基因对小鼠骨髓间充质干细胞的标记及活体光学成像研究  

Bioluminescence Imaging and Fluorescence Imaging on Murine BM-MSCs Labeled with Dual Optical Reporter Gene of Luciferase2 / mKate2

作　　者：刘静静[1,2] 胡晓俊[1,2] 李征然[1,2] 李丹[1,2] 颜荣华[1,2] 覃杰[1,2] 王劲[1,2] 单鸿[1,2] 

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 [2]中山大学介入放射学研究所,广东广州510630

出　　处：《中山大学学报（医学科学版）》2014年第3期334-339,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：NSFC-广东联合基金(U1032002);国家自然科学基金(81071206;81271621;81271562);国家自然科学基金青年基金(81101097;81101096);2010-2012卫生部临床学科重点项目(No.164)

摘　　要：【目的】Luciferase2/mKate2双报告基因标记小鼠骨髓间充质干细胞(mMSC),并进行活体生物发光和荧光双模态光学成像研究。【方法】全骨髓细胞贴壁分离法提取mMSC,进行细胞表面标志物和多向分化潜能的鉴定;CMVLuciferase2-mKate2慢病毒感染mMSC,根据细胞内远红荧光蛋白mKate2的表达水平评估转染效率并利用流式细胞仪进行纯化筛选;对筛选后的mMSC(mMSC-CMV-Luc2-mKate2,mKate2+>95%)进行活体生物发光和荧光双模态光学成像。【结果】成功分离mMSC;表型分析结果显示mMSC高表达Sca-1(94.5%)、CD44(99.6%),低表达CD11b(0.092%)、CD45(0.11%);mMSC具有成脂和成骨分化能力。CMV-Luciferase2-mKate2慢病毒感染mMSC 96 h后,细胞内mKate2的荧光表达率为22.97%,经流式细胞仪筛选后mKate2表达率大于95%。对筛选后的mMSC进行活体生物发光成像和荧光成像可探测光信号,光信号强度与细胞数量呈明显线性正相关(生物发光成像为R2=0.9893;荧光成像为R2=0.9226)。【结论】CMVLuciferase2-mKate2慢病毒转染mMSC可稳定表达双报告基因Luciferase2和mKate2,体外可根据细胞内mKate2的表达水平间接评估转染效率、进行细胞筛选,活体内可同时进行生物发光成像和荧光成像,为mMSC体内移植后生物学行为的研究提供了良好、无创的定量示踪手段。[Objective] To label murine bone marrow-derived mesenchymal stem cells （mMSC） with Luciferase2/mKate2 reporter genes and track mMSC through both bioluminescent imaging （BLI） and fluorescence imaging.[Methods] The mMSC were isolated and propagated by an adhesive screening method.Cell characteristics and multilineage differentiation capacity of mMSC were performed in vitro.mMSC were transfected with the lentivirus CMV-Luciferase2-mKate2 and positive cells were purified by fluorescence-activated cell sorting （FACS）.The purified mMSC （mMSC-CMV-Luc2-mKate2,mKate2＋ ＞95％） were tracked by in vivo BLI and fluorescence imaging.[Results] mMSC were successfully obtained from murine bone marrow and readily transfected with lentivirus CMV-Luciferase2-mKate2.The transfection efficiency was 22.97％.The FACS phenotype analysis revealed that the mMSC were positive for Sca-1 （94.5％） and CD44 （99.6％）,but negative for CD11b （0.092％） and CD45 （0.11％）.These cells readily differentiate into adipocytes and osteocytes.The transfected mMSC were purified by FACS and nearly 95％ of mMSC were mKate2 positive.Optical signal intensity of mMSC detected by BLI and fluorescence imaging significantly correlated with cell numbers in vivo （BLI：R2=0.9893; fluorescence imaging：R2 =0.9226）.[Conclusion] CMV-Luciferase2-mKate2 labeled mMSC can stably express the dual optical report gene of luciferase2 and mKate2.Transfection efficiency and cell purification could be performed in vitro based on mKate2 expression.This dual optical reporter gene system provided a good,noninvasive and quantitative tracing method for the biological behavior of mMSC after transplantation simultaneously by BLI and fluorescence imaging in vivo.

关 键 词：骨髓间充质干细胞 示踪 生物发光成像 荧光成像 

分 类 号：R237[医药卫生—中医学]