兔骨髓间充质干细胞表面抗原检测及其变化特点  

Detection and characteristics of surface antigen of bone marrow mesenchymal stem cells in rabbits

作　　者：杨亚冬[1] 张文元[1] 房国坚[1] 陈勇[1] 

机构地区：[1]浙江省医学科学院生物工程研究所,浙江省杭州市310013

出　　处：《中国组织工程研究与临床康复》2007年第3期455-458,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：浙江省科技计划重大项目(2004F11018)~~

摘　　要：目的:应用流式细胞仪对兔骨髓间充质干细胞的表面抗原进行检测,观察骨髓间充质干细胞传代次数、克隆纯化与骨髓间充质干细胞表面抗原表达之间的关系。方法:实验于2004-01/2006-08在浙江省医学科学院生物工程所完成。取3月龄新西兰大白兔用于骨髓间充质干细胞的分离培养。兔骨髓间充质干细胞的克隆化:将铺满培养瓶底的原代骨髓间充质干细胞,用D-Hanks液小心的洗1次,加入0.25%胰蛋白酶消化约4min,弃消化液,加LG-DMEM培养液收集细胞,1000r/min,离心10min,然后用LG-DMEM培养液充分混匀沉淀细胞。细胞计数后以10倍递减稀释至细胞密度为103个/mL。取0.1mL稀释后的细胞悬液加入10mL培养液,使最终细胞密度为10个/mL。将细胞悬液加入96孔培养板,每孔100μL。置37℃、体积分数为0.05的CO2饱和湿度条件下培养,每天观察克隆细胞的增殖生长状况。待克隆细胞生长至60%～80%融合时,逐步扩大培养,在液氮冻存保种的同时进行细胞连续传代。将体外普通法分离的骨髓间充质干细胞第5代、第7代、第13代、第16代、第21代、第22代及克隆纯化的骨髓间充质干细胞第3代、第5代、第15代、第26代均标记上CD14-FITC及CD44-PE,通过流式细胞仪检测其阴性率及阳性率。结果:①普通法分离的骨髓间充质干细胞各代的CD44表达呈阳性,且随着培养代次的增加,其表达的阳性率逐渐增强,到P16代以后又呈下降趋势;各代骨髓间充质干细胞表面抗原CD14出现了微弱阳性,但随着培养代次增加,其阳性率呈下降趋势。②克隆纯化的骨髓间充质干细胞其CD44呈现阳性,表达在80%以上,至P26代时CD44表达下降到70.49%;其CD14基本呈阴性。结论:兔骨髓间充质干细胞其CD44呈阳性表达,CD14呈阴性表达。随着传代代数增加CD14阴性符合率逐渐提高,CD44阳性表达率也提高。普通法分离的骨髓间充质干细胞较克隆纯化的骨髓间充�AIM： To detect the surface antigen of rabbit bone marrow mesenchymal stem cells （MSCs） by flow cytometer, and observe the relationship of the expression of surface antigen with MSCs＇ culture passage and clonal purification. METHODS： From January 2004 to August 2006, the experiment was completed in the Institute of Bioengineering, Zhejiang Academy of Medical Sciences. New Zealand rabbits were adopted in this study for the isolation and culture of MSCs, aging 3 months. Primary MSCs covering the bottom of culture bottle were washed once with D-Hanks liquid, and digested for 4 minutes with 0.25% trypsin. Then LG-DMEM was used to collect and deposit the cells at 1 000 r/min for 10-minute centrifugation. After the cells were counted, their density was diluted to 103 cells/mL in a progressive decrease tendency. Then the diluted cell suspension of 0.1 mL adding culture medium of 10 mL declined the density into 10 cells/mL. Afterward the cells were cultured on 96-well plate containing 100 μL in each well at 37 ℃ of CO2 with 0.05 volume fraction. Everyday we observed the proliferation of clonal cells, which were re-cultured in 60%-80% confluence and continued to passage. MSCs at passages 5, 7, 13, 16, 21 and 22, which were isolated in vitro, and MSCs at passages 3, 5, 15 and 26, which were cloned and purified, were both labeled with CD14-FITC and CD44-PE. Then the flow cytometer was used to detect the negative and positive rates. RESULTS： （1）The isolated MSCs were positive for CD44, and the positive rate turned down with the increase of culture passages.（2）Over 80% of MSCs after clonal purification were positive for CD44, but the positive rate of MSCs at passage 26 declined to 62.84%, whereas the purified MSCs were almost negative for CD14. CONCLUSION： Rabbit bone MSCs express positively for CD44 and negatively for CD14. And the rates of CD14-negative and CD44-positive expressions are both rose in accordance with culture passage. Compared with the MSCs after clonal purification, the isolated MSCs p

关 键 词：间质干细胞 细胞学 抗原 表面 抗原 CD44 抗原 CD14/血液 

分 类 号：R394.2[医药卫生—医学遗传学]