文献类型：期刊文献

中文题名：骨髓间充质干细胞的培养及其体外标记跟踪的实验研究

英文题名：Biological Characteristics and Optimized Tracing of Rat Bone Marrow Mesenchymal Stem Cell Cultured in Vitro

作者：成忠阳[1];雷栓虎[2];董晓丽[3];郭丽[4];黄良增[2];岳海原[2];张成俊[2];郝俊龙[2];汪玉良[2];夏亚一[2];张海鸿[2];付廷军[1];刘京升[2]

第一作者：成忠阳

机构：[1]甘肃省康泰医院,兰州730000;[2]兰州大学第二医院骨科/甘肃省骨关节疾病重点实验室,兰州730000;[3]甘肃中医学院生理教研室,兰州730000;[4]甘肃省妇幼保健医院儿科,兰州730000

第一机构：甘肃省康泰医院,兰州730000

年份：2015

卷号：37

期号：2

起止页码：155

中文期刊名：世界科技研究与发展

外文期刊名：World Sci-Tech R&D

收录：CSTPCD;;CSCD:【CSCD_E2015_2016】；

基金：甘肃省自然科学研究基金计划(1308RJZA239);陇原青年创新人才扶持计划资助

语种：中文

中文关键词：流式细胞仪;表面标志物;细胞分离;细胞染色;骨髓间充质干细胞

外文关键词：flow cytometry; biological markers; cell separation; cell staining; BMSCs

摘要：目的研究大鼠骨髓间充质干细胞(BMSCs)的体外分离培养;观察Brdu、DAPI以及Hoechst33342体外染色的合适时间和最佳剂量,比较三种方法的优缺点。方法 BMSCs取自SD雄性大鼠的股骨和胫骨,然后采用贴壁分离培养法对BMSCs进行分离培养,当BMSCs培养至第三代时,采用流式细胞仪对其表面抗原CD34、CD44、CD45进行测定;同时分别用Brdu、DAPI以及Hoechst33342对BMSCs进行染色标记,Brdu标记效果用免疫细胞化学方法检测,DAPI和Hoechst33342的标记率用荧光显微镜观测,最后用MTT检测BMSCs的细胞增值率,观察三种不同方法体外染色的合适时间和最佳剂量,同时比较其优缺点。结果流式细胞仪检测发现BMSCs表达CD44阳性,CD34和CD45阴性;BMSCs染色前后其表面标志表达无明显统计学差异(P<0.05);Brdu染色标记BMSCs的最佳浓度和时间是10μmol/L、48小时;DAPI染色标记BMSCs的最佳浓度和时间是20μg/ml、30分钟;Hoechst33342染色标记BMSCs的最佳浓度和时间是5μg/ml、1小时。结论采用粘附贴壁分离培养法能够有效获取高纯度的BMSCs;运用Brdu、DAPI以及Hoechst33342三种方法标记BMSCs效果良好,方法可靠、合理,为研究BMSCs体内追踪打下了基础。
Objective To establish a method of isolation and cultivation of the rat mesenchymal stem cells（ MSCs）; to investigate the optimal dosage and timing for 5＇-bromo-2＇-deoxyuridine（ Brd U）,4＇,6-Diamidino-2-phenylindole dihydrochloride（ DAPI） and hoechst 33342 labeling of rat bonenlarrow mesenchymal stem cells（ BMSCs） in vitro and compare advantages and disadvantages of these three methods. Methods BMSCs were isolated from the marrow of the tibia and the femur of male Sprague Dawley rat,the specific surface antigens（ CD34,CD44,and CD45） of the 3rdgeneration BMSCs were identified by flow cytometry and BMSCs were labelled with Brd U,DAPI and hoechst 33342 respectively. The label efficiency for Brd U was assessed with immunocytochemistry and that of DAPI and hoechst 33342 were observed under fluorescence microscope. Cell proliferation was evaluated by using MTT. The advantages and disadvantages of the three methods were analyzed. ResultsFlow cytometry showed that BMSCs expressed CD44 but not CD34 or CD45 and there was no significant difference in the expression of stem cell specific surface antigen between before labeling and after labeling. The optimal concentration and timing of the BMSCs with Brd U labeling were respectively 10 μmol / L and 48 hours and that of DAPI labeling were 20 μg / ml and 30 min and 5 μg / ml,1 h with hoechst 33342. Conclusion High-purified BMSCs can be obtained by adherent method;the results suggest that the three different ways of labeling（ Brd U,DAPI and hoechst 33342） provide a stable,reliable and feasible means for a dynamic observation of the implanted BMSCs in vivo.