蜕皮甾酮对人脐带间充质干细胞冻存复苏后生物学活性的影响  

Influence of ecdysterone on bioactivity of cryopreserved Wharton's Jelly-derived mesenchymal stem cells after being frozen and defrosted

作　　者：冯长江[1,2] 吴旭[1] 马奎[2] 杨思明[2] 李军[1] 吴霞[1] 付小兵[2] 

机构地区：[1]南方医科大学南方医院胸心血管外科,广东广州510515 [2]解放军总医院第一附属医院全军创伤修复与组织再生重点实验室,北京100048

出　　处：《感染．炎症．修复》2012年第1期18-21,F0003,共5页Infection Inflammation Repair

基　　金：广东省科技计划项目(2008B010600006);中国博士后科学基金面上资助项目(20100471820)

摘　　要：目的:研究体外条件下蜕皮甾酮(EDS)对冻存复苏后人脐带间充质干细胞(hUCMSCs)生物学活性的影响。方法:hUCMSCs采用梯度冷冻技术冻存,6个月后复苏,扩增、传代,分3组培养:空白对照组用普通培养基培养;低剂量EDS组在普通培养基中加入100μg/ml EDS;高剂量EDS组在普通培养基中加入200μg/mlEDS。显微镜下观察细胞形态,10 d时计算克隆形成能力,绘制细胞生长0～7 d的细胞生长曲线,流式细胞仪鉴定hUCMSCs的细胞表面特异标记(CD34、CD45、CD29、CD105),油红O染色及茜素红染色观察hUMMSCs向脂肪细胞、成骨细胞分化的能力。结果:两EDS组克隆形成率约为75%,空白对照组约为40%。生长曲线显示两EDS组增殖速度较空白对照组快,但两EDS组增殖速度相近。流式检测结果显示两EDS组的CD29、CD105阳性率明显高于空白对照组,CD34、CD45呈阴性表达,其表达明显低于空白对照组(P<0.05),两EDS组之间差异无统计学意义;成骨、成脂分化能力检测发现,各组细胞均能够向脂肪细胞、成骨细胞分化,而高剂量EDS组细胞在未加诱导培养基的情况下仍出现向成骨细胞分化的趋势。结论:在一定浓度范围内,EDS对细胞复苏后恢复细胞活性以及提高存活率方面具有积极作用,但是较高浓度的EDS有诱导细胞向成骨细胞方向分化的趋势。Objective： To investigate the influence of ecdysterone （EDS） on bioactivity of cryopreserved Wharton＇s Jelly derived mesenchymal stem cells （hUCMSCs） after being frozen and defrosted. Methods： After 3 passages, hUCMSCs were preserved in liquid nitrogen after a gradual lowering of the temperature. Six months lat- er, cells were defrosted and amplified ,and then they were divided into 3 groups： blank control group in which cells were cultured in common medium in low concentration of EDS group ceils were cultured in common medium with 100 μg/ml of EDS; and in high concentration of EDS group cells were cultured in common medium with 200μg/ml of EDS. The morphology was observed with light microscope, and the rate of cloning formation was compared af- ter 10 days＇cultivation. The cell growth curve of 1 - 7 days determined. The expression of cell surface antigens was analyzed by flow cytometry （CD34, CD45, CD29, CD105）. The osteogenic and adipogenic differentiation of hUCMSCs were investigated with alizarin red and Oil Red O staining respectively. Results： The rates of cloning formation were 75% and 75% in the two EDS groups and 40% in the blank control group. The analysis of cell growth curve showed that proliferation of hUCMSCs in the two EDS groups more rapid than that of the blank con- trol group, but there was no significant difference between the two EDS groups. Flow cytometry analysis showed that the positive expression rates of CD29 and CD105 were higher, and the expression rates of CD34 and CD45 were lower in the two EDS groups than those in the control group （P〉0.05）, however, there was no significant difference between the two EDS groups （P〉 0. 05 ）. Cells in all groups could differentiate into oste0genic and adipogenic direction with induction medium, while only higher EDS group could induce those cells to osteogenic differentiation without the induction medium. Conclusions：EDS to a certain concentration may have the positive effect on bioactivity and survival of cryop

关 键 词：蜕皮甾酮 脐带间充质干细胞 冻存 复苏 

分 类 号：R722.12[医药卫生—儿科]