PKH26标记大鼠骨髓间质干细胞的实验研究

目的建立PKH26标记大鼠骨髓间质干细胞（MSCs）的方法，并探讨标记MSCs的基本生物学活性。方法大鼠MSCs按PKH26标记程序进行标记后培养，采用激光共聚焦显微镜、流式细胞分析仪等观察细胞生长状态和荧光标记活性变化；应用RT-PCR检测标记后MSCs的GAPDH、nucleostemin及Bmi-1基因的表达；选用碱性磷酸酶染色、Von Kossa染色及骨形成蛋白3（BMP3）基因表达分析等技术，观察标记后MSCs体外分化成骨细胞的特性。结果PKH26标记后细胞呈红色荧光，荧光的强度随着PKH26浓度的增加而递增，并与传代时间和代数相关；标记后细胞生长状态良好，基本生长特性如传代培养和生长曲线无明显改变；细胞GAPDH、nucleostemin及Bmi-1基因表达未见改变；标记MSCs诱导后ALP、Von Kossa染色阳性，呈现典型的成骨细胞形态和生物学特征，并表达BMP3基因。结论PKH26可稳定标记大鼠骨髓MSCs并能传代培养，标记后细胞形态、生长活力及多向分化潜能等无明显影响，该技术可用于追踪MSCs转归、可塑性及干细胞移植方面的实验研究。

An Experimental Study on Rat Bone Marrow Mesenchymal Stem Cells Labelled with PKH26 in Vitro

Objective:To establish a method of labelling rat bone marrow mesenchymal stem cells with PKH26 in vitro and explore the biological activity of the labelled MSCs.Methods:Rat mesenchymal stem cells were stained with red fluorescent PKH26.The growth and fluorescent intensity were observed by confocal laser microscopy and flow cytometer.The expressions of GAPDH,nucleostemin,Bmi-1 gene in the cells were detected by RT-PCR.The characteristics of labelled MSCs differentiating into osteoblasts in vitro were identified by ALP stain,Von Kossa stain and bone morphogenetic protein3(BMP3) expression.Results: The labelled cells appeared red fluorescence.The intensity of fluorescence was related to the concentration of PKH26 and the passages.The growth characteristic between the labelled and unlabelled cells was not different significantly.The differences of expression of GAPDH,nucleostemin,Bmi-1 gene between them were not observed.After being induced,the cells appeared typical cell shape and biological characteristics of osteoblasts,positive for ALP stain,Von Kossa stain and expressed BMP3 gene.Conclusions: Rat MSCs could be labelled with PKH26 stably.The labelled cells still had the abilities of self-renewal and multi-differentiation.It is suggested that the PKH26 labelling method could be used to study the homing,plasticity and transplantation of MSCs.