5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞的特点及可行性(英文)  

Characteristics and feasibility of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine

作　　者：郭启仓[1] 张宇[1] 王玉芳[1] 陈晨[1] 蒲国华[1] 李占 

机构地区：[1]清华北煤炭医学院附属医院心胸外科,河北省唐山市063000

出　　处：《中国临床康复》2006年第5期144-146,共3页Chinese Journal of Clinical Rehabilitation

摘　　要：背景:骨髓干细胞移植成功的定性指标是标记移植的细胞至组织器官后存活并发挥了正常的功能。目前细胞学研究方面采用的标记方法有酶联标记、氚胸腺嘧啶核苷标记、荧光标记和5-溴脱氧尿嘧啶核苷标记等。目的:观察5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞的特点。设计:单一样本观察。单位:华北煤炭医学院附属医院心胸外科。材料:实验于2003-07/2004-12在华北煤炭医学院中心实验室完成。取月龄3个月的日本大耳白兔6只,雌雄不限,体质量(3.00±0.25)kg。方法:①利用密度为1073g/L的Percoll分离骨髓的单核细胞,以含体积分数为0.1的胎牛血清的低糖Dulbecco’s改良的Eagle’s培养基培养与扩增间充质干细胞,纯度可达95%左右。②用5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞24,48,72,96h后的检测标记率(标记组);阴性对照为未经5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞;空白对照为以磷酸盐缓冲液或正常鼠血清替代一抗。主要观察指标:①3组各检测200个细胞,并在不同时间点观察。②5-溴脱氧尿嘧啶核苷标记的骨髓间充质干细胞免疫组织化学染色结果及细胞计数结果。结果:①标记组均见5-溴脱氧尿嘧啶核苷阳性细胞,5-溴脱氧尿嘧啶核苷阳性反应物位于细胞核,呈棕色、颗粒状或弥漫性分布;对照组未见阳性细胞。②标记组24,48,72,96h5-溴脱氧尿嘧啶核苷阳性细胞的数目分别为48±2,100±4,173±2,178±3,随着标记时间的延长,标记率逐渐增高,标记72h后标记率在85%以上。③阴性对照和空白对照组各时间点均为0。结论:①用5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞的最佳时间是72h。②标记后检测的敏感性好,在低倍镜中亦可见,适合于大块组织的定量研究。③结果表明5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞是可行的。BACKGROUND： The indicant of successful transplanted bone marrow stem cells is that the labeled transplanted cells can survive in the target organs and can their biological functions. Up to date, at cell level, there are several labeled methods such as enzyme-linked, 3H-TdR, fluorescent and 5-bromodexyuridine method, etc. OBJECTIVE： To observe the biological characteristics of bone marrowderived mesenchymal stem cells labeled with 5-bromodexyuridine. DESIGN： Observations in single kind of sample SETTING： Department of Thoracic Cardiac＇ Surgery, Affiliated Hospital of North China Coal Medical College MATERIALS： The experiment was carried out in the Experimental Center of North China Coal Medical College from July, 2003 to November, 2004 on six Japanese long-earod rabbits ,with the age of 3 months , of either gender and the body mass of （3.00±0.25）kg. METHODS：①Monocytes were isolated from the bone marrow with Percoll reagent of the concentration of 1 073 g/L. The mesenchymal stem cells were cultured and proliferated with Eagle＇s medium, which was improved by adding 10% low sugar Dulbecco＇s to fetal bovine serum, so that their purity could reach about 95%. Secondly, in the experimental groups, the percentage of bone marrow-derived mesenchymal stem cells labeled with 5- bromodexyuridine was detected after 24 hours, 48 hours, 72 hours and 96 hours, respectively. The negative-controlled group was composed of bone marrow-derived mesenchymal stem cells non-labeled with 5-bromodexyuridine while the blank-controlled group was made by substituting phosphate buffer or normal mice serum for primary antibody. MAIN OUTCOME MEASURES： In three different groups, 200 cells of each group were detected and observed at different time. Outcomes of the immunochemical stauning and counting of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were also determined. RESULTS： The positive bone marrow-derived mesenehymal stem cells labeled with 5-bromodexyuridine were observed in all the ex

关 键 词：骨髓细胞 干细胞 移植 溴脱氧尿苷 

分 类 号：R551.3[医药卫生—血液循环系统疾病]