摘要
目的建立大鼠骨髓间充质干细胞(BMSCs)的分离和培养方法 ,并探讨采用氯甲基苯甲酰胺(CM-Dil)对BMSCs进行体外标记和体内示踪的可行性。方法全骨髓贴壁法分离获取BMSCs,收集第3代细胞,流式细胞术检测CD34、CD44和CD29的表达率。加入CM-Dil对BMSCs进行体外标记,荧光倒置显微镜下观察24h、21d和30d时的标记情况,通过描绘生长曲线明确CM-Dil对体外培养BMSCs生长特性的影响。制作局灶性脑缺血大鼠模型,将CM-Dil标记的BMSCs立体定向移植至大脑纹状体区,于移植后7、14、21d取脑组织制备冰冻切片,荧光显微镜下观察标记细胞在脑内的存活及分布情况。结果 BMSCs贴壁培养至第3代后呈成纤维细胞样,形态均一、排列有序,CD34、CD44和CD29表达率分别为1.71%、80.32%和84.89%。体外CM-Dil标记24h的BMSCs发出红色荧光,标记率为100%;21d后,经传代培养的BMSCs荧光强度与24h时点相近,但30d后荧光有所淬灭。标记后细胞的生长、增殖特性未受影响,形态无改变。脑组织冰冻切片发现移植的BMSCs在7、14、21d时主要位于针道附近,并向周围扩散,数量逐渐减少,呈椭圆形或不规则形。结论通过贴壁培养可以从大鼠骨髓中分离培养出较纯的BMSCs。CM-Dil染色简单、有效、无细胞毒性,体外标记BMSCs的最长时限为21d,可作为BMSCs脑内定向移植的体内示踪方法 。
Objective To establish a method to isolate and cultivate rat bone marrow mesenchymal stem cells （BMSCs）,and explore the feasibility of labeling in vitro,and tracing in vivo,BMSCs with chloromethyl-benzamidodialkylcarbocyanine （CM-Dil）. Methods BMSCs were obtained and subsequently cultured with whole bone marrow cell culture system,and the third generation of BMSCs was harvested. The positive rates of CD34,CD44 and CD29 were detected by flow cytometry. CM-Dil was used to label BMSCs in vitro and the efficiency of labeling after 24 hours and at day 21 and 30 were examined under fluorescent microscope. Moreover,the growth curves were sketched to determine the negative effects of CM-Dil on the vitality and proliferation of BMSCs cultured in vitro. Rat model of focal cerebral ischemia was reproduced,CM-Dil labeled BMSCs were implanted into corpus striatum of rats＇ brain with computer-guided stereotaxis thereafter. Brain tissues were obtained to prepare frozen sections at 7th,14th and 21st day post-implantation,and the survival and distribution of labeled cells were observed with fluorescent microscopy. Results The third generation passage of cultured BMSCs grew orderly,with shape of desmocytes,and homogeneous in morphology. The positive rates of CD34,CD44 and CD29 expressions in BMSCs were 1.71%,80.32% and 84.89%,respectively. Red fluorescence was observed in CM-Dil labeled BMSCs in vivo 24 hours after labeling,with a positive rate of 100%. The fluorescence intensity of passage cultured BMSCs observed on day 21 was similar to that observed at 24 hours after labeling,but diminished on day 30. The growth,proliferation and morphology of BMSCs,were not influenced by CM-Dil labeling. On day 7,14 and 21 post-implantation,BMSCs decreased in quantity,appearing in oval or irregular shapes,and most of the cells were found around the needle tract,with a tendency of diffusion to peripheral area. Conclusion High purity of BMSCs may be obtained with whole bone marrow cell culture system from bone marrow of rats. CM-Dil labeling is easy to handle and effective,with no cytotoxicity to cells. The latest labeling period of CM-Dil is 21 days in vitro,therefore it seems to be an effective method for in vivo tracing of BMSCs in brain after implantation.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期946-949,953,共5页
Medical Journal of Chinese People's Liberation Army
基金
广东省自然科学基金(06301402)
教育部博士点基金(20070572004)
同济大学"中医大师传承人才培养项目"(国中医药函[2008]185号)
关键词
骨髓
间质干细胞移植
脑缺血
氯甲基苯甲酰胺
荧光染料
bone marrow
mesenchymal stem cell transplantation
brain ischemia
3
3＇-dioactadecyl-5
5＇-di（4-sulfophenyl）oxacarbocyanine
fluorescent dyes
