小鼠胚胎干细胞移植后两种示踪方法的比较  

Comparison of Two Tracing Method of Transplanted Mouse Embryonic Stem Cell

作　　者：关云谦[1,2] 谢淑[3] 孙静敏[4] 邹春林[1,2] 陈凌[1,2] 张愚[1,2] 

机构地区：[1]首都医科大学宣武医院老年病研究所细胞治疗室,北京100053 [2]教育部神经变性病重点实验室,北京100053 [3]国家人类基因组北方研究中心,北京100176 [4]空军第三通信修理所卫生所,北京100075

出　　处：《中国医学科学院学报》2010年第4期393-396,482-483,共6页Acta Academiae Medicinae Sinicae

基　　金：国家自然科学基金(30940039;30970939);国家重点基础研究发展计划(973计划)(2006CB943703);北京市科委科技计划(H020220010290)~~

摘　　要：目的观察小鼠胚胎干细胞(ES)移植入大鼠脑内后绿色荧光蛋白(GFP)质粒和Thy-1抗体在指示细胞及细胞分化方面的特点。方法标记方法一:将p-EGFP-N1质粒转入ES细胞,连续10代抗生素筛选表达GFP的GFP-ES,并将GFP-ES移植入活体大鼠脑内。取材后冰冻切片,在荧光显微镜下观察切片上的绿色荧光。标记方法二:直接移植胚胎干细胞后取材,用特异性抗小鼠Thy-1抗体作移植细胞的免疫组织化学染色,荧光显微镜下观察。另外,两种标记方法均做神经元和胶质细胞的特异抗体神经细胞核抗体(NeuN)和胶质原纤维酸性蛋白(GFAP)的免疫组织化学染色,观察是否与GFP或Thy-1抗体双标记,以此判定细胞分化情况。结果 GFP质粒转化后的ES细胞团和单细胞均表达亮绿色的GFP,转化效率为30%;移植21d后,大鼠脑内存活的移植细胞仍表达GFP,但不能和NeuN、GFAP的染色双标记。大量Thy-1阳性的植入细胞和NeuN、GFAP双标记。结论 GFP质粒标记胚胎干细胞后移植可以较好地显示移植细胞,但不能观察细胞分化;而Thy-1抗体不仅在显示移植细胞上有较好的效果,还可以准确地标记分化细胞。Objective To trace the embryonic stem(ES)cells transplanted into rat brain by labeling the cells with green fluorescent protein(GFP)and by mouse neuronal specific antibody Thy-1 and compare their features.Methods For GFP labeling,transfect pEGFP-N1 plasmid containing GFP and anti-neomycin sequences into embryonic stem cell and add neomycin for more than 10 passages.To test the GFP expression in vivo,the GFP-ES was transplanted into healthy rat brain,and the frozen sectioned slides were observed under fluorescence microscope and laser con-focal microscope 21 days later.For the amtibody labeling,embryonic stem cells were directly transplanted into the rat brain.The specific mouse thy-1 antibody was used in immunostaining of transplanted cells.For both of the two labeling method,the slides were also examined by double labeling with the antibodies,neuronal nuclei(NeuN)or glial fibrillary acidic protein(GFAP)to identify the differentiation of transplanted cells.Results Both single ES cell and cell pellets expressed bright green fluorescence the day after plasmid transfection,and more than 30% ES cells were labeled.The GFP-labeled cells could still be found gathered around the infusion channel at least 21 days later,but the GFP fluorescent could not be overlapped with NeuN or GFAP staining.On the contrary,Thy-1 antibody overlapped well with NeuN or GFAP staining.Conclusions Liposome-helped plasmid GPF transfection is effective in labeling mouse embryonic stem cell in vivo,but is not effective in showing the differentiated cells.On the contrary,Thy-1 antibody can not only show the transplanted cells,but also trace the transplanted cells after their differentiation.

关 键 词：绿色荧光蛋白 胚胎干细胞 分化 

分 类 号：Q813[生物学—生物工程]