绿色荧光蛋白标记对大鼠骨髓间充质干细胞生长的影响

Influence of green fluorescent protein labeling on the growth of rat bone marrow mesenchymal stem cells

目的:观察腺病毒pAdEasy系统携带绿色荧光蛋白对骨髓间充质干细胞的标记效率及标记对骨髓间充质干细胞生长的影响,为进一步追踪研究骨髓间充质干细胞奠定基础。方法:实验于2006-11/2007-03在四川大学华西组织胚胎学与神经生物学实验室完成。①实验材料:成年雄性SD大鼠,体质量100～120g,由四川大学实验动物中心提供。实验过程中对动物处置符合动物伦理学标准。②实验方法:采用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓间充质干细胞,传代扩增,应用免疫细胞化学方法对培养的骨髓间充质干细胞进行鉴定。将生长到约80%融合的第3代骨髓间充质干细胞随机分为绿色荧光蛋白标记组和对照组。绿色荧光蛋白标记组加入1:100稀释的腺病毒(pAdEasy-1-pAdTrack-CMV)上清液,对照组加入磷酸盐缓冲液。③实验评估:观察绿色荧光蛋白标记对骨髓间充质干细胞生长曲线的影响。结果:体外培养的原代骨髓间充质干细胞24h内可见有少量贴壁细胞,8～10d左右达到70%～80%汇合。免疫细胞化学检测第3代骨髓间充质干细胞显示,CD44和CD90表达阳性,而CD14和CD34表达阴性。生长曲线表明,绿色荧光蛋白标记对骨髓间充质干细胞增殖无明显影响。结论:腺病毒pAdEasy系统携带绿色荧光蛋白标记的骨髓间充质干细胞效率高,标记后对细胞的生长无明显影响。

AIM： To observe the labeling efficiency of adenovirus pAdEasy system taking green fluorescence protein on bone marrow mesenchymal stem cells and the effect of the labeling on bone marrow mesenchymal stem cells so as to provide basis for follow-up study of bone marrow mesenchymal stem cells. METHODS： The experiment was conducted in the Laboratory of Histology, Embryology and Neurobiology in West China Hospital of Sichuan University from November 2006 to March 2007. ①Experimental material ：Adult male SD rats with the body mass of 100-120 g were purchased from the Experiment Animals Center of Sichuan University. All experimental procedures were conducted according to logic standard of experimental animal. ②Experimental methods：Bone marrow mesenchymal stem cells were separated and purified from the bone marrow of rats by density gradient centrifugation and by adherence to the culture plastic. After successive subculture and amplification, bone marrow mesenchymal stem cells were identified by immunocytochemical method. The rat bone mesenchymal stem cells of the third generation growing to about 80% adhesion were randomly divided into green fluorescent protein labeling group and control group. Green fluorescent protein labeling group was added adenovirus （pAdEasy-l-pAdTrack-CMV） supernate diluted with 1： 100, while the control group was added phosphate buffer saline. ③The effects of green fluorescent protein labeling on growth curve of bone marrow mesenchymal stem cells were studied. RESULTS： Primary cultured bone marrow mesenchymal stem cells adhered to plastic surface within 24 hours and reached 70%-80% confluence within 8-10 days. Immunocytochemistry showed that the 3＇d generation of these cultured cells was positive for CD44 and CD90, while negative for CD14 and CD34. Green fluorescent protein labeling had little effect on proliferation of bone marrow mesenchymal stem calls. CONCLUSION： Adenovirus pAdEasy taking green fluorescent protein labeling is highly efficient to label bone marrow mesenchymal stem cells, and it has no significant effect on cell growth.