CAJ | 학술논문

目的通过比较化学和生长因子两种不同诱导方法,观察评估脂肪干细胞(ADSCs)向神经元样细胞(NLCs)分化,并探讨诱导分化机制.方法取SD大鼠腹股沟和腹膜后脂肪分离培养获得ADSCs,取其第3代细胞进行流式细胞表型鉴定(CD29、CD34、CD45、CD73、CD90、CD105)并进行实验诱导.分为化学诱导组[DMEM/F12 +5 mmol/L β-巯基乙醇(β-BME)+2％二甲基亚砜(DMSO)+ 200 μmol/L丁酸酯羟基茴香醚(BHA)]和生长因子分步诱导组[第1步:DMEM/F12+20 μg/L表皮生长因子(EGF)+20 μg/L碱性成纤维细胞生长因子(bFGF) +2％ B27;第2步:DMEM/F12+ 10 μg/L脑源性神经营养因子(BDNF)+ 10 μg/L胶质细胞源性神经营养因子(GDNF)+1 μmol/L维甲酸(RA)].每天在光镜下观察其形态变化,并在第3天用免疫荧光法检测诱导后神经细胞特异标志物巢蛋白(Nestin)、神经元核蛋白(NeuN)、抗微管相关蛋白-2(MAP-2)和胶质纤维酸性蛋白(GFAP)表达,并行统计学分析.结果流式细胞仪检测结果显示,第3代ADSCs细胞表型CD34(0.57％)、CD45(0.67％)、CD29(99.38％)、CD90(96.21％)、CD73(99.80％)和CD105(97.97％).化学诱导组在诱导24 h后即出现神经元样细胞形态,表达Nestin和MAP-2,但其诱导后形态在第5天出现逆转且在第7天有部分细胞开始死亡;生长因子诱导组诱导3d时Nestin表达,1周后MAP-2、Neun和GFAP表达逐渐增加,诱导生成稳定NLCs.结论化学诱导ADSCs向神经元样细胞分化速度较快,但其性质不够稳定,形态出现逆转且伴随细胞死亡;生长因子法能够实现稳步诱导,诱导生成的NLCs更符合组织工程种子细胞的要求.
목적 통과비교화학화생장인자량충불동유도방법,관찰평고지방간세포(ADSCs)향신경원양세포(NLCs)분화,병탐토유도분화궤제.방법 취SD대서복고구화복막후지방분리배양획득ADSCs,취기제3대세포진행류식세포표형감정(CD29、CD34、CD45、CD73、CD90、CD105)병진행실험유도.분위화학유도조[DMEM/F12 +5 mmol/L β-구기을순(β-BME)+2％이갑기아풍(DMSO)+ 200 μmol/L정산지간기회향미(BHA)]화생장인자분보유도조[제1보:DMEM/F12+20 μg/L표피생장인자(EGF)+20 μg/L감성성섬유세포생장인자(bFGF) +2％ B27;제2보:DMEM/F12+ 10 μg/L뇌원성신경영양인자(BDNF)+ 10 μg/L효질세포원성신경영양인자(GDNF)+1 μmol/L유갑산(RA)].매천재광경하관찰기형태변화,병재제3천용면역형광법검측유도후신경세포특이표지물소단백(Nestin)、신경원핵단백(NeuN)、항미관상관단백-2(MAP-2)화효질섬유산성단백(GFAP)표체,병행통계학분석.결과 류식세포의검측결과현시,제3대ADSCs세포표형CD34(0.57％)、CD45(0.67％)、CD29(99.38％)、CD90(96.21％)、CD73(99.80％)화CD105(97.97％).화학유도조재유도24 h후즉출현신경원양세포형태,표체Nestin화MAP-2,단기유도후형태재제5천출현역전차재제7천유부분세포개시사망;생장인자유도조유도3d시Nestin표체,1주후MAP-2、Neun화GFAP표체축점증가,유도생성은정NLCs.결론 화학유도ADSCs향신경원양세포분화속도교쾌,단기성질불구은정,형태출현역전차반수세포사망;생장인자법능구실현은보유도,유도생성적NLCs경부합조직공정충자세포적요구.
Objective To evaluate the differentiation of adipose stem cells (ADSCs) into neuronlike cells (NLCs) by comparing chemical and growth factor induction methods,and deeply explore the induction mechanism and the problems.Methods ADSCs were isolated from the groin and retroperitoneal fat of SD rats and cultured.The ADSCs of 3rd generation were used for identification of phenotypes (CD29,CD34,CD45,CD73,CD90,CD105) by flow cytometry.The experiment was divided into chemical induction group [DMEM/F12 + 5 mmol/L β-mercaptoethanol (β-BME) + 2％ dimethylsurfoxide (DMSO) + 200 μmol/L butyrate hydroxyanisole (BHA)] and growth factor induction group [first step:DMEM/F12 + 20 μg/L epidermal growth factor (EGF) + 20 μg/L basic fibroblast growth factor (bFGF) + 2％ B27 ; second step:DMEM/F12 + 10 μg/L brain derived neurotrophic factor (BDNF) + 10 μg/L glial cell linederived neurotrophic factor (GDNF) + 1 μmol/L retinoic acid (RA)].Differentiated ADSCs were evaluated for Nestin,NeuN,microtubule-associated protein (MAP)-2 and glial fibrillary acidic protein (GFAP) expression by immunocytochemistry.The data were statistically analyzed.Results Flow cytometric analysis demonstrated that ADSCs at 3rd generation were positive for CD29 (99.38％),CD90 (96.21％),CD73 (99.80％) and CD105 (97.97％),but negative for CD34 (0.57％) and CD45 (0.67％).In chemical induction group,Nestin and MAP-2 were highly expressed at first day,but the nerve-like morphology reversed at the fifth day and some cells begin to die at the seventh day.In growth factor induction group,Nestin expression was detected on the third day,and MAP-2,NeuN and GFAP expression was gradually increased afer one week.The stable NLCs were eventually induced.Conclusion Comparative analysis showed that ADSCs differentiate into neuron-like cells more quickly by chemical induction,but their nature is not stable.Induction by growth factors spends a relatively long time,but the induced NLCs maintain longer and more stable.