兔骨髓间充质干细胞体外培养方法的比较  

Comparison of separation methods on biological characteristics of rabbit bone marrow mesenchy- mal stem cells

作　　者：张卫东 史宏灿 章方彪 谭荣邦 叶钢 李广宇 

机构地区：[1]扬州大学临床医学院,225001

出　　处：《中华实验外科杂志》2013年第7期1360-1363,共4页Chinese Journal of Experimental Surgery

基　　金：基金项目：国家自然科学基金资助项目（30672080、30972968、81170014）;江苏省“六大人才高峰”高层次人才基金资助项目（2009128）

摘　　要：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。Objective By comparing the different separation methods of rabbit bone marrow mes- enehymal 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 of 2nd, 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 immunofiuorescenee. Results BMSCs could be separated by each method. The adherent cells showed shuttle or muhiple 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 cul- tured ways of rabbit BMSCs. The 4th-generation BMSCs exerted the strongest proliferation capacity.

关 键 词：骨髓间充质干细胞 细胞培养 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]