GFP转基因小鼠骨髓间充质干细胞纯化方法的研究

Study on the Purification of GFP Transgenic Murine Bone Marrow Mesenchymal Stem Cells.

目的：应用两种不同方法培养纯化绿荧光蛋白（GFP）转基因小鼠骨髓间充质干细胞,以探求提高GFP小鼠骨髓间充质干细胞纯化效率的方法。方法：取4~6周龄GFP小鼠股骨全骨髓,采用贴壁法进行体外培养。待细胞融合至约80%,将细胞分为两组,分别采用常规法和改良法进行消化传代。荧光显微镜下观察各组细胞荧光状态及形态学特征,MTT法绘制细胞生长曲线,流式细胞术检测细胞表面标志物及绿荧光蛋白表达率,体外诱导观察其成骨、成脂能力。结果：贴壁法获得数量较多,形态多样的原代细胞。随着传代次数的增加,细胞形态一致性不断增高。与传统方法相比,改良法在不影响细胞增殖、分化潜能,不影响其GFP表达率的前提下,可以更早获得纯度较高的细胞。结论：改良法可提高纯化小鼠骨髓间充质干细胞效率,为其用于进一步研究工作提供基础。

Objective：To observe and compare two methods to purify bone marrow mesenchymal stem cells（BMSCs）of green fluorescent protein（GFP）transgenic mice and study the feasibility to use green fluorescence protein（GFP）-BMSCs as a labeling method in transplantation experiment.Methods：Bone marrow cells isolated from 4to6 weeks old GFP transgenic mice were directly planted in culture flask.Cells were trypsinized on attaining 80%confluency in different methods：（A）Regular method（B）Modified method.Morphological changes and intensity of the green fluorescent protein were observed under fluorescence microscope.Activity of BMSCs was identified according to the growth curve and multi-differentiation functions of BMSCs.Furthermore,the expression of some surface markers and green fluorescent protein was investigated by flow cytometry.Results：Because of the unwanted growth of hematopoietic cells and non-MSCs,lots of polymorphic cells were isolated from mouse via plastic adherent cultures.Compared with traditional way,the modified method could purify cells more efficiently.The subcultured cells maintained the capacity for amplification and were capable of differentiating into osteocytes and adipocytes.Their fluorescence was not diminished after several passages and could thus be used as a labeling method in vivo.Conclusion：The modified method is an effective method for purifying BMSCs of GFP transgenic mice.