CAJ | 학술논문

目的比较不同的骨髓间充质干细胞(BMSCs)培养方法,寻求适宜的兔BMSCs培养方式.方法分别用全贴壁筛选培养法、半全红细胞裂解液培养法、完全红细胞裂解液培养法和Ficoll密度梯度离心法提取兔的骨髓液,于4、7、10、13d分别比较各组细胞数量；绘出所获得细胞的P2、P3、P4及P5的生长曲线,比较各代细胞的增殖特点；观察所培养细胞的形态,检测其CD44、CD34抗原的表达.结果 4种方法均能得到规则的以梭形为主的贴壁细胞,且流式细胞仪测得CD44抗原表达阳性率95.57％、CD34抗原表达阳性率0.99％；其中全贴壁筛选培养法获得的贴壁细胞数量在不同的时间点均明显多于其他培养方法(P＜0.05)；P4代细胞的增殖活性优于其他代数细胞(P＜0.05).结论全贴壁筛选培养法是兔BMSCs最适宜的培养方法,P4代细胞是BMSCs增殖能力最强的1代细胞.
목적 비교불동적골수간충질간세포(BMSCs)배양방법,심구괄의적토BMSCs배양방식.방법 분별용전첩벽사선배양법、반전홍세포렬해액배양법、완전홍세포렬해액배양법화Ficoll밀도제도리심법제취토적골수액,우4、7、10、13d분별비교각조세포수량；회출소획득세포적P2、P3、P4급P5적생장곡선,비교각대세포적증식특점；관찰소배양세포적형태,검측기CD44、CD34항원적표체.결과 4충방법균능득도규칙적이사형위주적첩벽세포,차류식세포의측득CD44항원표체양성솔95.57％、CD34항원표체양성솔0.99％；기중전첩벽사선배양법획득적첩벽세포수량재불동적시간점균명현다우기타배양방법(P＜0.05)；P4대세포적증식활성우우기타대수세포(P＜0.05).결론 전첩벽사선배양법시토BMSCs최괄의적배양방법,P4대세포시BMSCs증식능력최강적1대세포.
Objective By comparing the different separation methods of rabbit bone marrow mesenchymal stem cells (BMSCs),to find suitable BMSCs culture ways.Methods BMSCs were obtained by whole bone marrow adherent culture,half red blood cell lysis,whole red blood cell lysis and Ficoll density gradient centrifugation.Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the growth of BMSCs at 4th,7th,10th,and 13th day.The growth curves of BMSCs at generation of2nd,3rd,4th and 5th were drawn by MTT.Inverted phase contrast microscope was used to observe the morphological changes of cells.CD34 and CD44 antigens of BMSCs were identified by flow cytometry and immunofluorescence.Results BMSCs could be separated by each method.The adherent cells showed shuttle or multiple angle shapes,with rich cytoplasm,and the positive rate of BMSCs labeled by CD44 antigen was 95.57％,and that by CD34 antigen was 0.99％.The more cell number,larger colonies and shortest primary culture time were presented in whole bone marrow adherent culture group (P ＜ 0.05).The 4th-generation BMSCs showed more excellent proliferative activity,higher cell growth rate and more count of cells obtained than other generations (P ＜ 0.05).Conclusion Whole bone marrow adherent culture is the best suitable cultured ways of rabbit BMSCs.The 4th-generation BMSCs exerted the strongest proliferation capacity.