世界科技研究与发展
世界科技研究與髮展
세계과기연구여발전
World Sci-Tech R & D
2015年
2期
155-162,167
,共9页
成忠阳%雷栓虎%董晓丽%郭丽%黄良增%岳海原%张成俊%郝俊龙%汪玉良%夏亚一%张海鸿%付廷军%刘京升
成忠暘%雷栓虎%董曉麗%郭麗%黃良增%嶽海原%張成俊%郝俊龍%汪玉良%夏亞一%張海鴻%付廷軍%劉京升
성충양%뢰전호%동효려%곽려%황량증%악해원%장성준%학준룡%왕옥량%하아일%장해홍%부정군%류경승
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
流式細胞儀%錶麵標誌物%細胞分離%細胞染色%骨髓間充質榦細胞
류식세포의%표면표지물%세포분리%세포염색%골수간충질간세포
flow cytometry%biological markers%cell separation%cell staining%BMSCs
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 BM-SCs；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。
目的：研究大鼠骨髓間充質榦細胞（BMSCs）的體外分離培養；觀察 Brdu、DAPI 以及 Hoechst33342體外染色的閤適時間和最佳劑量，比較三種方法的優缺點。方法 BMSCs 取自 SD 雄性大鼠的股骨和脛骨，然後採用貼壁分離培養法對 BMSCs 進行分離培養，噹 BMSCs 培養至第三代時，採用流式細胞儀對其錶麵抗原 CD34、CD44、CD45進行測定；同時分彆用 Brdu、DAPI 以及 Hoechst33342對 BMSCs 進行染色標記，Brdu 標記效果用免疫細胞化學方法檢測，DAPI 和 Hoechst33342的標記率用熒光顯微鏡觀測，最後用 MTT 檢測 BMSCs 的細胞增值率，觀察三種不同方法體外染色的閤適時間和最佳劑量，同時比較其優缺點。結果流式細胞儀檢測髮現 BMSCs 錶達 CD44暘性，CD34和 CD45陰性；BMSCs 染色前後其錶麵標誌錶達無明顯統計學差異（P ＜0.05）；Brdu 染色標記 BMSCs 的最佳濃度和時間是10μmol／L 、48小時；DAPI 染色標記 BMSCs 的最佳濃度和時間是20μg／ml、30分鐘；Hoechst33342染色標記 BMSCs 的最佳濃度和時間是5μg／ml、1小時。結論採用粘附貼壁分離培養法能夠有效穫取高純度的 BM-SCs；運用 Brdu、DAPI 以及 Hoechst33342三種方法標記 BMSCs 效果良好，方法可靠、閤理，為研究 BMSCs 體內追蹤打下瞭基礎。
목적：연구대서골수간충질간세포（BMSCs）적체외분리배양；관찰 Brdu、DAPI 이급 Hoechst33342체외염색적합괄시간화최가제량，비교삼충방법적우결점。방법 BMSCs 취자 SD 웅성대서적고골화경골，연후채용첩벽분리배양법대 BMSCs 진행분리배양，당 BMSCs 배양지제삼대시，채용류식세포의대기표면항원 CD34、CD44、CD45진행측정；동시분별용 Brdu、DAPI 이급 Hoechst33342대 BMSCs 진행염색표기，Brdu 표기효과용면역세포화학방법검측，DAPI 화 Hoechst33342적표기솔용형광현미경관측，최후용 MTT 검측 BMSCs 적세포증치솔，관찰삼충불동방법체외염색적합괄시간화최가제량，동시비교기우결점。결과류식세포의검측발현 BMSCs 표체 CD44양성，CD34화 CD45음성；BMSCs 염색전후기표면표지표체무명현통계학차이（P ＜0.05）；Brdu 염색표기 BMSCs 적최가농도화시간시10μmol／L 、48소시；DAPI 염색표기 BMSCs 적최가농도화시간시20μg／ml、30분종；Hoechst33342염색표기 BMSCs 적최가농도화시간시5μg／ml、1소시。결론채용점부첩벽분리배양법능구유효획취고순도적 BM-SCs；운용 Brdu、DAPI 이급 Hoechst33342삼충방법표기 BMSCs 효과량호，방법가고、합리，위연구 BMSCs 체내추종타하료기출。
Objective To establish a method of isolation and cultivation of the rat mesenchymal stem cells(MSCs);to investi-gate the optimal dosage and timing for 5'-bromo-2'-deoxyuridine (BrdU),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 3 rd generation BMSCs were identified by flow cytometry and BMSCs were labelled with BrdU,DAPI and hoechst 33342 respectively.The label efficiency for BrdU 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.Results Flow cytometry showed that BMSCs expressed CD44 but not CD34 or CD45 and there was no significant difference in the ex-pression of stem cell specific surface antigen between before labeling and after labeling.The optimal concentration and tim-ing of the BMSCs with BrdU 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 (BrdU,DAPI and hoechst 33342)provide a stable,reliable and feasible means for a dynamic observation of the implanted BMSCs in vivo.
