微载体旋转立体培养成人退变髓核细胞增殖及生长动力学研究  

Growth and proliferation in adult degenerative nucleus pulposus cells with microcarrier culture in vitro

作　　者：宁斌[1] 刘海飞 龚维明[1] 田庆芬[1] 王德春 胡有谷 

机构地区：[1]山东大学附属济南市中心医院,济南250013 [2]山东省创伤骨科研究所,青岛266016

出　　处：《中国矫形外科杂志》2012年第19期1777-1781,共5页Orthopedic Journal of China

基　　金：国家自然基金面上项目资助(编号:30672132)

摘　　要：[目的]通过单层培养和微载体旋转立体培养成人退变髓核细胞对比,研究两种培养方式对其生长活性及增殖能力的影响。[方法]本实验通过Trypan blue法计数细胞并绘制生长曲线,应用3H-TdR放射免疫法检测退变髓核细胞增殖活性,流式细胞仪检测退变髓核细胞周期,比较两种培养方式对细胞增殖的影响。[结果]微载体旋转立体旋转培养较单层培养细胞从贴壁到对数生长期开始时间(16～20 d)较长,但是严格意义的稳定生长期(3～5 d)很短;对数生长期长,一般14～17 d,期间细胞增殖活跃。两种培养方式的细胞处于稳定生长期时,细胞增殖能力没有明显区别,而处于对数生长期时,微载体培养的细胞的增殖能力明显高于单层培养组的细胞(P<0.05)。细胞周期测定显示两种细胞停滞在G0/G1期的占71%左右,培养方式对其影响不明显。而微载体培养的细胞的S期明显较单层培养的细胞为长(P<0.05)。[结论]成人退变髓核应用微载体培养后增殖能力及生长活性提高,可解决组织工程学椎间盘种子细胞难以收集的难题,为将来开展椎间盘组织工程学的研究奠定良好的基础。[ Objective] To culture the adult degenerative nucleus pulposus cells using monolayer and stiring microcarrier system to investigate the influence of both culture methods for the cells of growth activity and proliferation. [ Methods] In our study, we compared the two methods of culturing cells by drawing the growth curve with Trypan blue method, detecting the cellu- lar proliferative activity with 3H- TdR radioimmunoassay, and assaying the nucleus pulposus cell cycle with FCM. [ Results ] Microcarrier cultured cells needed 16 ~ 20 d developing from adherence to exponential phase which was longer than monolayer cultured cells. The stationary phase （average 3 ~ 5 d） was short, however, the exponential phase （average 14 -17 d） was long in which cells were actively proliferated. In the stationary phase, there was no statistically difference between the two culture methods. In the exponential phase, the cells growth ability was significantly higher in microcarrier culture system （ P 〈 0. 05 ） than in the monolayer culture system. There was no difference in G0/G1 phrase according to the cell counting （71%） by both culture systems. S phrase was longer in microcarrier culture system （P 〈 0. 05） than in monolayer culture system. [ Conclu- sion] Proliferation capacity and growth activity of adult degenerative nucleus pulposus cells is increased by microcarrier culture system. This method is potential to solve the difficulties of collecting seed cells for intervertebral disc engineering.

关 键 词：微载体 髓核细胞 细胞周期 

分 类 号：R681.53[医药卫生—骨科学]