慢病毒载体介导增强型绿色荧光蛋白筛选稳定转染的兔骨髓间充质干细胞
Lentiviral vector mediated enhanced green fluorescent protein screening for stably transfected rabbit bone marrow mesenchymal stem cells

摘要：目的 分离、培养并鉴定兔骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)，探讨慢病毒载体介导增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)感染兔BMSCs的最佳条件，筛选稳定转染的兔BMSCs。方法 通过全骨髓贴壁法获得兔BMSCs；茜素红、甲苯胺蓝及油红O染色对BMSCs成骨、成软骨及成脂分化进行鉴定；免疫荧光组化染色检测CD44及CD90的表达；不同浓度嘌呤霉素筛选BMSCs的最小致死浓度；以感染复数(multiplicity of infection, MOI)为50、100、150、200的慢病毒载体介导eGFP转染BMSCs，倒置显微镜下观察荧光表达，并用嘌呤霉素筛出稳定转染系。结果 当MOI为150，慢病毒载体介导eGFP感染兔BMSCs效率最高；嘌呤霉素筛选稳定转染兔BMSCs的最适质量浓度是1.0μg/mL。结论 成功培养了兔BMSCs，用慢病毒介导的GFP标记了兔BMSCs并筛选出了稳定转染的兔BMSCs，建立了一个简便有效的干细胞标记方法，为BMSCs在动物体内实验标记示踪奠定了基础。
ABSTRACT: Objective To isolate, culture and identify rabbit bone mesenchymal stem cells (BMSCs) so as to explore the optimal conditions for lentiviral vector-mediated enhanced green fluorescent protein (eGFP) infection in rabbit BMSCs and screen stable transfected BMSCs in rabbits. Methods BMSCs were obtained by whole bone marrow adherence method. The osteogenic, chondrogenic and adipogenic differentiation of BMSCs was made by alizarin red, toluidine blue and oil red O staining, respectively. The expressions of CD44 and CD90 were detected by immunofluorescence. The concentration of puromycin was used to screen the minimum lethal concentration of BMSCs; the lentiviral vector with multiplicity of infection (MOI) of 50, 100, 150 and 200 mediated eGFP BMSCs were infected; the fluorescence expression was observed under an inverted microscope, and the stable transformation system was screened with puromycin. Results When MOI was 150, lentiviral vector-mediated eGFP infection of rabbit BMSCs was the most efficient. The optimum concentration of puromycin for stable transfection of rabbit BMSCs was 1.0μg/mL. Conclusion Rabbit BMSCs were successfully cultured in this experiment. The stem cells were labeled with lentivirus-mediated GFP and stable transfected rabbit BMSCs were screened. A simple and effective stem cell labeling method was established to label BMSCs in vivo