华通胶间充质干细胞低温保存后生物学特性研究  

Biological Characteristics of Wharton′s Jelly Derived Mesenchymal Stem Cells after Cryopreservation

作　　者：沈建良[1] 宫立众[1] 岑坚[1] 刘毅[1] 王立新[1] 尹文杰[1] 赵德峰[1] 马巍娜[1] 黄友章[1] 

机构地区：[1]海军总医院血液科,北京100048

出　　处：《中国实验血液学杂志》2013年第1期181-187,共7页Journal of Experimental Hematology

摘　　要：本研究旨在探讨低温保存对华通胶间充质干细胞(WJ-MSC)生物学特性的影响,为低温保存后WJ-MSC的临床应用及建立WJ-MSC库提供实验依据。通过脐带组织块培养制备WJ-MSC,将第5代WJ-MSC加入低温保护剂,-80℃冰箱降温,液氮保存。细胞复温后行培养和传代,低温保存组第1代(PP1)传代获得PP2-PP15。非低温保存组第6代为对照组第1代(CP1),传代获得CP2-CP15。比较对照组与低温保存组WJ-MSC的有核细胞回收率、台盼蓝拒染率、CCK-8活性、凋亡率、贴壁率、增殖指数、细胞表面抗原、细胞周期和向脂肪细胞、成骨细胞及神经元细胞诱导分化能力。结果表明,WJ-MSC经低温保存,有核细胞回收率为98.2%,台盼蓝拒染率为94.3%,CCK-8活性为91.4%,凋亡率为3.9%,贴壁率为92.6%;PP1较CP1增殖指数明显降低(P<0.05),PP2-PP15与相应对照组增殖指数的差异均无统计学显著性;WJ-MSC高表达CD29、CD90、CD44、CD71、CD73、CD105、CD166和HLA-ABC,低表达CD34、CD45和HLA-DR,两组之间差异均无统计学显著性;增殖潜伏期和对数增殖期两组之间差异也无统计学意义;向脂肪细胞、成骨细胞和神经元细胞诱导分化后分别进行油红O、碱性磷酸酶(ALP)和神经元特异性烯醇化酶(NSE)染色,两组间阳性强度肉眼观察无明显差异;细胞内甘油三酯、ALP和NSE含量两组间也无显著差异。结论:WJ-MSC低温保存后少量细胞(<10%)受到损伤,传代培养表明其生物学特性基本保持不变。Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton＇s jelly derived mesenchymal stem cells（ WJ-MSC）, and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80℃ refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage （PP1）. Thus, PP2 -PP15 WJ-MSC were obtaned by continuous cell subculture, The 1st control passage （CP1） to 15th passage （CP15） represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated ceils, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92. 6%. There was a statistically significant difference in proliferation index between PP1 and CP1 （ P 〈 0.05 ）, but there were no statistically significant differences between PP2 -PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not differe

关 键 词：华通胶 间充质干细胞 低温保存 生物学特性 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]