兔动员外周血间充质干细胞集落形成能力与神经元诱导分化研究  

Colony forming capacity and neuron differentiation of rabbit peripheral blood-derived mesenchymal stem cells after G-CSF mobilization

作　　者：刘小慧[1] 章涛[1] 张潜[2] 刘金伟[1] 刘祖林[1] 汤宁宁[1] 

机构地区：[1]遵义医学院附属医院贵州省细胞工程重点实验室,贵州遵义563099 [2]遵义医学院人体解剖学教研室,贵州遵义563003

出　　处：《西安交通大学学报（医学版）》2014年第1期26-29,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.81060148)~~

摘　　要：目的研究动员后兔外周血间充质干细胞(PBMSCs)的存在频率,并对PBMSCs进行神经元诱导分化鉴定。方法采用粒细胞集落刺激因子(G-CSF)皮下注射动员,结合密度梯度离心和贴壁培养法分离、培养兔外周血样品中的PBMSCs,利用集落形成率分析动员兔PBMSCs的存在频率,并对PBMSCs的神经元分化能力进行体外诱导鉴定。结果动员后的PBMSCs原代培养24h后,可见较多短梭形及多角形贴壁细胞,3～4d后出现集落样生长,传代培养的PBMSCs形态均一、呈长梭形漩涡状生长;未动员的外周血样品中的贴壁细胞少,集落形成少,且易老化,无法传代。集落形成率实验显示,动员后的PBMSCs在每百万外周血单个核细胞中有2.8～10.8个;而未动员组PBMSCs仅有0～3个。免疫细胞化学染色显示,PBMSCs经神经诱导7d后其神经元特异性烯醇化酶(NSE)、神经元特性核蛋白(NeuN)染色阳性。结论动员剂可提高兔外周血中的PBMSCs含量,所获取的PBMSCs具备神经元分化能力。Objective To study the frequency of peripheral blood-derived mesenchymal stem cells （PBMSCs） in rabbits after mobilization and determine the isolated PBMSCs＇ differentiation into neurons. Methods After mobilization by subcutaneous injection of granulocyte colony stimulating factor （G-CSF）, the PBMSCs were isolated from rabbit peripheral blood samples by combination of density gradient centrifugation and adhesive culture. The frequency of rabbit PBMSCs was determined by colony forming efficiency assay. In vitro neuron differentiation capacity of isolated PBMSCs was also evaluated. Results The mobilized PBMSCs were spindle- or polygon-shaped in morphology after primary culture for 24 hours, and had a colony-like growth at days 3--4. The morphology of subcultured PBMSCs was uniform in swirling growth. However, the adhesive cells and colonies were few and vulnerable to aging and unable to subculture for PBMSCs isolated from non-mobilization rabbits. Colony forming efficiency assay detected the frequency of mobilized PBMSCs ranging from 2.8 to 10.8 per million mononuclear cells in the peripheral blood as compared with 0 to 3 in non-mobilized group. Immunocytochemical staining showed positive expression of neuron specific markers such as neuron-specific enolase （NSE） and neuron-specific nuclear protein （NeuN） for PBMSCs after 7 days of neuron differentiation induction. Conclusion PBMSCs can be well isolated and expanded from rabbits peripheral blood after G-CSF mobilization, and the cells can be differentiated into neurons under proper induction condition.

关 键 词：兔 周血间充质干细胞 集落形成率 神经元 诱导分化 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]