不同培养基和不同体积分数小牛血清对许旺细胞生长状态的影响  

Effects of various mediums and calf serum of various volume fractions on growth status of Schwann’s cells

作　　者：郭涛[1] 何明生[1] 王廷华[2] 庞江霞[2] 

机构地区：[1]昆明医学院附属第三医院血液科,云南省昆明市650118 [2]昆明医学院神经科学研究所,云南省昆明市650031

出　　处：《中国组织工程研究与临床康复》2009年第23期4462-4466,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:许旺细胞目前正越来越多地被运用于神经修复、构建载体细胞等,但如何高效快速地培养获取大量许旺细胞尤为关键。传统方法存在细胞培养周期长、污染率高、生长状况不稳定等不足。目的:观察比较不同培养基和培养基中不同体积分数血清对许旺细胞的生长、增殖能力的影响。设计、时间及地点:细胞学体外对照观察,于2008-01/07在昆明医学院神经科学研究所完成。材料:SPF级新生一两天龄ICR小鼠30只,由昆明医学院动物科提供。DMEM/F12培养液、RPMI1640培养液、L-DMEM培养液为Gibco公司产品,小牛血清为Hyclone公司产品。方法:取ICR新生小鼠,无菌条件下取出坐骨神经,组织块消化法体外分离培养许旺细胞,设立4组:DMEM/F12培养液组、RPMI1640培养液组、L-DMEM培养液组、生理盐水组。每组又分为4个亚组:不含血清、含体积分数为10%,15%,20%小牛血清。各组细胞接种后进行传代培养,使用差速贴壁和阿糖胞苷处理的综合法对许旺细胞进行纯化。主要观察指标:传代培养后1,3,5,7,9,11,13d,相差显微镜下观察细胞形态及生长情况,免疫细胞化学染色检测细胞纯度,MTT法绘制细胞生长曲线。结果:①不含血清时,各组许旺细胞生长状态均较差;添加血清后各组许旺细胞消化传代后6h即大量贴壁,48h后出现聚合增殖现象。②含血清的DMEM/F12培养液组、RPMI1640培养液组、L-DMEM培养液组许旺细胞能较好地生长,纯度均达到90%以上,且DMEM/F12培养液组在细胞纯度、细胞总数方面均略优于RPMI1640培养液组及L-DMEM培养液组;生理盐水组许旺细胞生长欠佳,纯度也不甚理想。③与生理盐水组比较,DMEM/F12、RPMI1640及L-DMEM培养液组MTT吸光度值均显著升高(F=92.814,P<0.05),但此3种基础培养基之间比较无明显差异(F=0.909,P>0.05)。培养1,3,5,7,9,11,13d,DMEM/F12、RPMI1640及L-DMEM培养液组在含不同体积分数小牛血清的条件�BACKGROUND： Schwann＇s cells have been widely used for nerve repair and constructing carrier cells. However, how to effectively rapidly culture and obtain a large number of Schwann＇s cells is very important. Traditional method has disadvantages of long cell culture cycle, high pollution rate and unstable growth. OBJECTIVE： To observe effects of various mediums and various volume fraction of serum in the medium on growth and proliferation of Schwann＇s cells. DESIGN, TIME AND SETTING： The cytological, in vitro, controlled study was performed at the Institute of Neuroscience, Kunming Medical College from January to July 2008. MATERIALS： Thirty SPF ICR mice aged 1 2 days were provided by Animal Department of Kunming Medical College. DMEM/F12 RPMI 1640 and L-DMEM were all purchased in Gibco. Fresh bovine serum was bought from Hyclone. METHODS： Sciatic nerve was obtained from newborn ICR mice. Schwann＇s cells were/n vitro isolated by tissue digesting method. Schwann＇s cells were assigned into 4 groups： DMEM/F12 group, RPMI 1640 group, L-DMEM group and saline group. Each group was divided into 4 subgroups： serum-free, 10%, 15% and 20% volume fraction calf serum subgroups. Cells were incubated and passaged in each group. Schwann＇s cells were purified by differential velocity adherent technique and arabinosylcytosin treated method. MAIN OUTCOME MEASURES： 1, 3, 5, 7, 9, 11, 13 days after subculture, cell morphology and growth were observed under a phase contrast microscope. Cell purity was detected using immunocytochemical staining, Cell growth curve was drawn using MTT assay. RESULTS： Without serum, Schwann＇s cells grew poor in each group. After adding serum, Schwann＇s cells in each group adhered to wall 6 hours following passage, and proliferated 48 hours later. Schwann＇s cells in the serum DMEM/F12, RPMI 1640 and L-DMEM subgroups grew weft, with a purity of over 90%. Cell purity and cell number in the DMEM/F12 group were slightly better in the RPMI 1640 and L-DMEM groups. S

关 键 词：许旺细胞 培养基 血清 细胞培养技术 

分 类 号：R394.2[医药卫生—医学遗传学]