生长因子分步诱导法在大鼠脂肪干细胞向神经细胞分化中的应用观察

摘要

Objective To investigate the effect of rat adipose tissue-derived cells ( ADSCs) into neural cells with a growth factor two-step induction method .Methods ADSCs were isolated from the fat pads in inguinal and retroperitoneal of Sprague Dawley rats , and the third generation cells were cultured .First, the cells were induced with DMEM/F12+2%B27+20 ng/mL EGF+20 ng/mL bFGF for 7 days, and then DMEM/F12+1μmol/L RA for another 7 days.Immunocyto-chemistry was taked to identify the expression of the neuron specific markers Nestin , MAP2, NeuN and GFAP.Results ADSCs were reaped from rat fat tissue , and could passage in a considerable large amount .ADSCs could strongly express CD73(99.17%), CD90(98.61%), CD105(96.20%)and lowy expressed CD34(0.36%).The ADSCs induced for 7 days aggregated as floating cell spheres and positive for Nestin , then strongly express MAP2 and NeuN, and lowy expressed GFAP after 14 days.Conclusion The growth factor two-step induction method can efficiently induce ADSCs to differenti-ate into nerve cells .%目的：观察生长因子分步诱导法在大鼠脂肪干细胞（ADSCs）向神经细胞分化中的应用效果。方法取SD大鼠腹股沟及腹膜后脂肪，体外分离培养ADSCs，取消化、离心的第3代ADSCs，首先以DMEM／F12＋2％B27＋20 ng／mL EGF＋20 ng／mL bFGF的神经干细胞培养基诱导7 d，后以DMEM／F12＋1μmol／L RA神经诱导培养基诱导7 d。免疫荧光法鉴定分化后神经细胞特异标志物中神经细胞特异标志物巢蛋白（ Nestin ）、微管相关蛋白2（MAP2）、神经元核抗原抗体（NeuN）、神经胶质纤维酸性蛋白（GFAP）。结果从大鼠脂肪组织中成功提取AD-SCs，其能够连续传代且稳定增殖；流式细胞检测显示CD34表达率为0．36％，CD73为99．17％，CD90为98．61％，CD105为96．20％。经生长因子诱导7 d后细胞聚集成球，Nestin高表达；14 d后MAP2、NeuN高表达，而GFAP低表达。结论 ADSCs能够稳定增殖传代，生长因子分步诱导可高效诱导ADSCs定向分化为神经细胞。