兔骨髓基质干细胞成骨诱导及增强型绿色荧光蛋白基因腺相关病毒体外转染的研究  

Osteogenic induction of rabbit's bone marrow stromal cells and transfection of AAV-EGFP in vitro

作　　者：郑召民[1] 董智勇[1] 邝冠明[1] 李佛保[1] 

机构地区：[1]中山大学附属第一医院骨科,广州510080

出　　处：《中华创伤骨科杂志》2006年第10期914-918,共5页Chinese Journal of Orthopaedic Trauma

基　　金：教育部霍英东教育基金"优选资助课题"(94020);广东省自然科学基金项目(036643)

摘　　要：目的探讨重组增强型绿色荧光蛋白基因腺相关病毒（AAV—EGFP）对细胞生物学行为的影响，为组织工程寻找一种理想的病毒载体及细胞示踪标记方法。方法采用全骨髓法分离培养BMSCs，诱导培养液诱导细胞成骨转化，行细胞形态学检查、细胞表面抗原鉴定、碱性磷酸酶（ALP）染色及Von Kossa染色，评价细胞成骨情况。在此基础上转染AAV—EGFP，观察细胞形态学改变及荧光表达的时间与强度，计算转染效率，确定转染的较佳感染复数（MOI）值，MTT法描记细胞生长曲线，观察AAV载体对细胞活性的影响。结果细胞扩增迅速、形态良好、纯度较高，经诱导后呈现明显的成骨改变。实验确定AAV转染细胞的较佳MOI值为1×10^5，以此值转染AAV—EGFP后细胞荧光持续高效稳定表达（〉8周），并可随细胞有丝分裂传至子代，可以满足示踪标记的需要，AAV转染效率高，对细胞活性影响小。结论AAV长期稳定表达目的基因，是一种理想的组织工程病毒载体；基于基因转染方法高效稳定表达EGFP，可作为种子细胞良好的示踪标记方法。Objective To evaluate the effects of adeno-associated virus-enhanced green fluorescent protein （AAV-EGFP） on the biologic behavior of rabbit＇s bone marrow stromal cells （BMSCs） by means of a simple method of culturing and osteogenic induction in vitro so as to find an ideal viral vector and cell tracing mark for tissue engineering. Methods Total bone marrow culture was conducted to obtain rabbit BMSCs which were then induced in the osteogenic direction. The morphology of the cells was observed continuously, and their surface antigen and ossification were detected by alkali phosphatase stain and Von Kossa stain. On the basis of the above results, AAV-EGFP was transfected into the induced cells. The morphologic changes of the cells, the expression time and intensity of fluorescent light were observed. The transfection efficiency was detected to find the best multiplicity of infection （MOI） value. The cell growth curves were drawn to evaluate the biologic effects of AAV-EGFP on the cyto-activity. Results The morphology and purity of the rabbit BMSCs obtained were good. The ossification of the cells was significant after osteogenic induction. The best MOI value was found to be 1 × 10^5. The expression intensity of fluorescent light was strong with the expression time more than eight weeks so that the fluorescent light could be observed after cell generations. The transfection efficiency of AAV was high without significant biologic effects on the cyto-activity. Conclusions The total bone marrow culture and in vitro cell induction can satisfy the requirements for seeding cells in tissue engineering. AAV is an ideal viral vector for tissue engineering. Transfection of AAV-EGFP to cells could be an ideal method for cell tracing mark.

关 键 词：骨髓基质干细胞 成骨诱导 腺相关病毒 增强型绿色荧光蛋白 转染 

分 类 号：R318.0[医药卫生—生物医学工程]