干细胞脑内移植有效标记研究:绿色荧光蛋白质粒标记的应用价值

Effective labeling of stem cell transplantation in vivo:the applicat ion value of green fluorescent protein plasmid labeling

目的:观察绿色荧光蛋白(greenfluorescentprotein,GFP)质粒转化小鼠胚胎干细胞(embryonicstemcell,ESC)的效果,以及将转化的胚胎干细胞移植入正常大鼠纹状体后,GFP质粒作为细胞存活情况示踪剂的效果。方法:首先利用感受态大肠杆菌提取大量高纯度GFP质粒DNA,然后将GFP质粒与脂质体孵育形成的转化复合物和小鼠胚胎干细胞共同孵育,使GFP质粒转入胚胎干细胞;筛选表达GFP的胚胎干细胞。将筛选后表达GFP的ESC移植入活体大鼠纹状体内,移植21d后,观察表达绿色荧光蛋白的移植细胞的存活情况。结果:GFP质粒转化以后的ES细胞团和单细胞都表达亮绿色的GFP,细胞打散计数证实大约60%的细胞携带GFP,移植21d后,大鼠脑内可见大量表达GFP的移植细胞。结论:脂质体可以将GFP质粒转入鼠的胚胎干细胞,携带有GFP的ES移植后21d,GFP可以作为示踪剂观察移植细胞的存活状况。脂质体辅助的GFP质粒转化胚胎干细胞是移植示踪的较好方法。

AIM:To observe the effect of green fluorescent protein(GFP) plasmid in the tra nsformation of mouse embryonic stem cell(ESC) and to observe the effect of CFP a s a tracer for cell survival and differentiation after transformed ESCs were tra nsplanted into striatum of normal rats. METHODS:Firstly,great amounts of high-purity GFP plasmid DNA were extracted f rom competence Escherichia coli,and then the transformed compound of CFP plasmid and liposome was co-incubated with ESC so as to transfer GFP plasmid into ESC and screen CFP-expressed ESC.Twenty-one days after GFP-expressed ESC was tran splanted into the striatum of the living rats,the combination of GFP labeling an d red fluorescence staining of NeuN and glial fibrillary acidic protein(GFAP) wa s observed. RESULTS:Both single ES cell and cell aggregates expressed bright-green GFP af ter he transformation,and about 60%of ESCs were labeled with GFP.The GFP-label ed cells could still be found 21 days later,some of which were differentiated in to NeuN-expressed neurons or GFAP-expressed glial cells. CONCLUSION:Liposome transfers GFP plasmids into rat ESC.CFP can be used as the tracer for observing cell survival and differentiation of transplanted cells in to neurons and glial cells 21 days after the transplantation of GFP-expressed E SC.That liposome aids the transformation of GFP plasmid into ESC is a better way in tracing transplantation.