海马区星形胶质细胞培养上清液体外诱导人脂肪基质细胞向神经元样细胞的分化  

Hippocampal astrocyte conditioned medium induces the differentiation of human adipose-derived adult stromal cells into neuron-like cells in vitro

作　　者：叶新春[1] 何红军[1] 杨峰[1] 赵科鹏[1] 姚君[1] 刘斌[2] 

机构地区：[1]泰兴市人民医院神经内科,江苏省泰兴市225400 [2]华北煤炭医学院附属医院神经内科,河北省唐山市063000

出　　处：《中国组织工程研究与临床康复》2007年第15期2832-2835,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察海马区星形胶质细胞培养上清液能否在体外诱导人脂肪基质细胞向神经元样细胞分化。方法:实验于2004-10/2005-06在华北煤炭医学院中心实验室完成。在无菌条件下从Wistar乳鼠分离出海马组织,从分离的海马组织中获得星形胶质细胞,并收集其培养上清液。取外科手术获得的人腹部皮下脂肪组织进行人脂肪基质细胞的原代培养。30例患者均知情同意。取第3代人脂肪基质细胞接种到培养孔中,预先放置无菌盖玻片的24孔培养板,制备细胞爬片或者接种到培养瓶中,细胞生长达50%～60%融合时,去除培养液,换为海马区星形胶质细胞培养上清诱导液进行诱导,对照组培养液为无血清培养基。倒置相差显微镜下连续观察细胞生长情况和形态变化,应用免疫细胞化学、鉴定神经前体细胞的特异性标志神经巢蛋白、神经细胞的特异性标志神经元特异性烯醇化酶、微管联合蛋白2和神经胶质细胞的特异性标志胶质纤维酸性蛋白的表达。结果:①诱导培养第3天,部分人脂肪基质细胞开始变形,从原先的细长梭状细胞变成神经元样细胞,可见细胞伸出突起,多为双极或多极细胞。②刚分离接种的人脂肪基质细胞镜下呈圆形,悬浮状态,接种后24h内贴壁,并开始伸展,多呈梭形。1周后细胞融合成单层,排列出现方向性,但有少量圆形及卵圆形细胞混杂生长。③第4,5代人脂肪基质细胞在诱导48h后形态即开始发生变化,扁平的胞体较预诱导后逐渐回缩,向外伸出突起,72h后扁平的胞浆向胞核收缩,突起继续延长,以后随时间进展,具有典型神经细胞形态特点的细胞数量逐渐增多,形成双极或多极细胞。④免疫细胞化学检测人脂肪基质细胞诱导5d后发现有(10.5±3.7)%神经巢蛋白、(38.4±5.2)%胶质纤维酸性蛋白、(15.7±2.3)%神经元特异性烯醇化酶表达,未见微管联合蛋白2的表达。结论:海马区星形�AIM： To observe whether hippocampal astrocyte conditioned medium （HCAM） can induce human adipose-derived adult stromal cells （huADASc） into neuron-like cells in vitro. METHODS： The experiment was carded out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. Hippocampal tissue of newborn Wistar rats was separated under sterile condition, astrocyte was obtained from the above tissue and was cultured, then the culture supernatant fluid was collected. Primary culture of huADASc was performed in fatty tissue of human abdominal region obtained in surgery. The 30 patients all knew and agreed to participate in the trial. The 3rd generation of huADASc was inoculated on 24-well culture plate, which was pre-put on the surface of sterile cover glass to prepare cells covering the whole slide or directly inoculate in culture bottle. When growth of cell fused about 50%-60%, culture media was gotten rid of. Cells were induced with HCAM; meanwhile, cells in the control group were cultured with serum-free media. Inverted phase contrast microscope was used to observe its growth and morphological changes. Immunocytochemistry was used to evaluate the expressions of NESTIN, neuron specific enolase （NSE）, microtubule associated protein 2 （MAP2） and glial fibrillary acidic protein （GFAP）. RESULTS： ①On the 3^rd day of culture, partial huADASc started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. ② New isolated huADASc was round, float under microscope, and then adhered to the wall within 24 hours after incubation, and began to extend, with fusiform. One week later, cells fused to monolayer, arranged directionally, but a few round and ovate cells grew. ③The fourth and fifth generations of huADASc became change 48 hours after induction. Deplanate cell body shortened, stretched out prominence outside. 72 hours later, deplanate cell body shor

关 键 词：星形胶质细胞 脂肪基质细胞 诱导分化 

分 类 号：R338.1[医药卫生—人体生理学]