摘要
背景:目前广泛应用于骨髓间质干细胞的标记方法主要为绿色荧光蛋白标记法,但标记与固定程序复杂,操作繁琐,易出现偏差。氯甲基苯甲酰氨(Chloromethyl-benzamidodialkylcarbocyanine,CM-Dil)是亲脂性膜荧光染料,能够与含有肽和蛋白质的硫氢基结合进而标记整个细胞。目的:探讨CM-Dil对大鼠骨髓间质干细胞体内示踪的可行性。设计、时间及地点:体内外细胞学观察,于2007-05/12在中山大学干细胞与组织工程研究中心、中山大学动物实验中心完成。材料:SPF级Wistar大鼠40只,由中山大学实验动物中心提供。CM-Dil为美国Sigma公司产品。方法:在体外,以标记CM-Dil的骨髓间质干细胞作为实验组,以未标记CM-Dil的骨髓间质干细胞作为对照组,比较两组细胞生长与增殖情况,MTT法检测细胞生长增殖情况。在体内,分别将标记CM-Dil的骨髓间质干细胞经门静脉移植及肝内培养,于移植后第7,15,21,30天制备肝脏切片。主要观察指标:骨髓间质干细胞CM-Dil标记率,标记细胞在肝内定植、生长与分布。结果:体外培养结果显示,两组骨髓间质干细胞在细胞生长、增殖、分裂以及形态学上均基本相似,在绿色光激发下,CM-Dil发出红色荧光,24h后细胞标记率为100%,21d后CM-Dil标记的骨髓间质干细胞荧光开始淬灭。体内实验中,经门静脉移植的骨髓间质干细胞在肝脏内主要位于间质,呈椭圆形或不规则形的分化细胞;肝内培养的骨髓间质干细胞在培养孔内呈"贴壁生长",为椭圆形分化细胞,未发现骨髓间质干细胞进入并定植于肝组织内;CM-Dil标记的骨髓间质干细胞荧光开始淬灭的时间亦为21d。结论:CM-Dil染色简单、方便、稳定,荧光开始淬灭时间较长,可望成为大鼠骨髓间质干细胞体内示踪的新方法。
BACKGROUND： The method for labeling bone marrow mesenchymal stem cells is green fluorescent protein marking method. However, this method is complicated and easy to make a mistake. Chloromethyl-benzamidodialkylcarbocyanine （CM-Dil）, a lipophilic membrane fluorescent dye, can combine with sulfhydryl containing peptide and protein. OBJECTIVE： To study the feasibility of CM-Dil membrane dye being used for tracing rat bone marrow mesenchymal stem cells in vivo. DESIGN, TIME AND SETTING： The in vitro cytological study was performed at the Animal Experimental Center and the Center for Stem Cells and Tissue Engineering, Sun Yat-sen University from May to December 2007. MATERIALS： A total of 40 SPF Wistar rats were supplied by the Animal Experimental Center of Sun Yat-sent University. CM-Dil was purchased from Sigma, USA. METHODS： Bone marrow mesenchymal stern cells labeled With CM-Dil were considered experimental group. Bone marrow mesencbymal stem cells non-labeled with CM-Dil served as control group. Cell growth and proliferation were compared in both groups. Cell growth proliferation was measured by MTr. Bone marrow mesenchymal stem cells labeled with CM-Dil were transplanted via the portal vein or were incubated in the rat liver. Liver tissues were prepared at days 7, 15, 21 and 30 following transplantation. MAIN OUTCOME MEASURES： The labeling rate of CM-Dil, transplantation, growth and distribution of labeled cells were measured. RESULTS： In vitro culture result showed that growth, proliferation, splitting and morphology of bone marrow mesenchymal stem cells were similar between both groups. CM-Dil displayed red fluorescence. Fluorescence emerged by CM-Dil was attenuated obviously 21days after labeling. Cell labeling rate was 100% at 24 hours. In vivo culture result showed that bone marrow mesenchymal stem cells transplanted through the portal vein were found in the hepatic mesenchyme, and showed ellipse or irregular cells. Bone marrow mesenchymal stem cells were adhered to the wall. Bone marrow mesenchymal stem cells were not transplanted in the liver tissues. CM-Dil was attenuated obviously 21days after labeling. CONCLUSION： The procedure of CM-Dil labeling is simple, stable and convenience. CM-Dil was attenuated obviously 2 l days after labeling. This method can be a new method of in vivo tracing of rat bone marrow mesenchymal stem cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第51期10090-10094,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
广东省科技厅重大科技专项基金(2005A30201007)
广东省自然科学基金(06021343)
教育部博士点基金(20070558259)~~
