摘要
背景：培养脐带源间充质干细胞过程中,细胞形态常易变得宽大不规则,传代不易贴壁,死亡率高,找到一个维持细胞稳定的方法是有必要的。目的：拟采用组织块培养法从人脐带Wharton’s jelly中分离培养间充质干细胞,观察碱性成纤维细胞生长因子对其生物学特性的影响。方法：采用组织块法分离培养和扩增脐带组织间充质干细胞,对照组用DMEM/F12培养基＋体积分数为10%胎牛血清培养,实验组用DMEM/F12培养基＋体积分数为10%胎牛血清＋20μg/L碱性成纤维细胞生长因子培养。观察组织块游出细胞的时间和细胞形态,每隔3d或4d更换培养液,待细胞达到80%~90%融合时,用0.25%胰酶消化后,按1：2或1：3的比例传代。结果与结论：倒置显微镜下观察对照组中从8~10d开始有细胞从组织块四周游出贴壁,实验组中从6~8d开始有细胞从组织块四周游出贴壁,1周后可见多数呈长梭形或扁平形的成纤维样细胞,围绕组织块成旋涡状,前3代细胞形态及传代间隔时间两组基本相同;3代以后实验组细胞较对照组易于贴壁,传代死亡率低,生长曲线提示增殖能力强。流式细胞术分析两组细胞周期显示第3,6代70%以上的细胞处于G0/G1期,且第3,6代细胞均阳性表达CD29,CD44,弱表达HLA-ABC,不表达CD34、CD45和HLA-DR。结果提示采用组织块培养法可从人脐带Wharton’s jelly中分离出具有间充质干细胞特性的细胞,且碱性成纤维细胞生长因子能使细胞从组织块游出时间提前和有助于促使细胞贴壁,一定程度上维持细胞形态稳定性,提高增殖能力,并且不改变细胞的表面标志物表达。
BACKGROUND：During culture of mesenchymal stem cells（MSCs） derived from Wharton＇s jelly of human umbilical cord.MSC morphology tends to become hypertrophic and irregular.MSCs were found to have higher rate of death and not be adapt to be adherent after passage.It is necessary to find a method to maintain its stability.OBJECTIVE：To isolate MSCs from human umbilical cord wharton＇s jelly by tissue block method,and to investigate basic fibroblast growth factor（bFGF） effects on biological characteristics of MSCs.METHODS：Human umbilical cord mesenchymal stem cells（hUCMSCs） were separated by tissue block method.Tissue fragments in control group were cultured in growth medium consisting of Dulbecco＇s Modified Essential /F12 Media（DMEM/F12） and 10% volume fraction of fetal bovine serum.Tissue fragments in experiment group were cultured in growth medium including 20 μg/L bFGF besides these in control group.Time of tissue blocks emigrating from cells and morphology of cells were observed.The medium was changed every 3-4 days.When the cells reached 80%-90% confluency,they were detached with 0.25% trypsin and were passaged at a ratio of 1：2 or 1：3.RESULTS AND CONCLUSION：A few long spindle or flat fibroblast-like cells were presented firstly after 8-10 days of incubation in control group,while in experiment group it took 6-8 days.One week later,lots of long spindle or flat fibroblast-like cells like whirlpool around micro-mass were presented in two groups.In the first 3 passages,cell morphous were similar and cells were passaged at approximately equivalent time.After passage 3,cells in experiment group were easier to be adherent,lower rate of death and better proliferation ability（shown by growth curve） in comparison with these in control group.Flow cytometry revealed that cell cycle at the passages 3 and 6 showed the percentage of G0/G1 was more than 70% respectively.CD44 and CD29 were highly expressed on the surface of passages 3 and 6 cells,whereas the expression of HLA-ABC was less pos
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第19期3513-3517,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
郑州大学"211工程"三期建设项目
江苏泰州医药科技公共服务平台(BM2008146)~~
