利用报告基因表达系统快速鉴定骨诱导活性材料  

The reporter gene expression system for rapid identification of osteoinductive biomaterials

作　　者：李彩侠[1] 宋宏梅[1] 王刚[1] 张义正[1] 李珉[1] 童英[1] 

机构地区：[1]四川大学生命科学学院四川省分子生物学及生物技术重点实验室,成都610064

出　　处：《四川大学学报（自然科学版）》2011年第5期1174-1178,共5页Journal of Sichuan University(Natural Science Edition)

基　　金：国家重点基础研究发展计划("973")资助项目(2005CB6239)

摘　　要：通过构建真核表达质粒,使增强型绿色荧光蛋白(Enhanced Green Fluorescent Pro-tein,EGFP)报告基因的表达受骨形成相关转录因子Cbfa1基因启动子的调控,再用该质粒转染大鼠成肌细胞L6,建立稳定细胞株.在成骨培养基中诱导培养后,该细胞株呈现较明显的绿色荧光,表明细胞在经诱导的成骨分化过程中,Cbfa1基因表达增强,Cbfa1启动子驱动了EGFP的表达.将该细胞株接种到有较强骨诱导活性的羟基磷灰石/磷酸三钙(hydroxyapa-tite-tricalcium phosphate,HA/TCP)陶瓷支架材料的表面,可观察到细胞中绿色荧光的增强;而接种到不具备骨诱导活性的胶原海绵(Collagen sponge)材料表面的细胞,未观察到绿色荧光的变化.细胞绿色荧光的增强与材料的骨诱导活性相一致,表明该细胞株可用来快速鉴定生物材料的骨诱导活性.The eukaryotic plasmid was constructed by using Cbfal promoter to regulate the expression of reporter gene Enhanced Green Fluorescent Protein （EGFP）, and transfected into rat myoblast L6 to establish the stable cell line. When cultured in osteoinducible media, the stable cell line showed stronger green fluroscence, indicating that when the cells differentiated to osteoblasts, Cbfal gene expression increased, so does EGFP gene which regulated by Cbfal promoter. When the stable ceils were seeded on the surface of hydroxyapatite-tricalcium phosphate （HA/TCP） ceramic scaffolds, green fluorescence of cells increased, in accordance with the identified osteoinducible activity of HA/TCP. No change of cellular green fluorescence was observed when cells were seeded on collagen sponge, the inert material for osteoinduction. The increase of cellular green fluorescence matched with the osteoinducible activity of the materials, demonstrating the established cell line to be a useful tool for fast and accurate identification of the osteoinducible activity of biomaterials.

关 键 词：骨诱导 骨形成相关转录因子 增强型绿色荧光蛋白 启动子 报告基因 生物材料 

分 类 号：Q786[生物学—分子生物学]