成人脂肪基质细胞体外诱导星形胶质细胞的形态学和超微结构研究

Adult adipose-derived stem cells differentiation into a astrocyte cells morphology and ultrastructure in vitro

目的 研究成人脂肪基质细胞（ADSC）诱导分化前、后细胞的生物学特性和超微结构特征.方法 体外分离、培养ADSC,应用化学诱导剂对其进行诱导,倒置显微镜下观察ADSC及其诱导分化细胞的形态;免疫细胞化学检测神经巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）的表达情况;Real-time PCR检测ADSC诱导分化前、后Nestin、GFAP基因mRNA的表达情况;透射电镜观察诱导分化细胞的超微结构.结果 体外培养的ADSC形态类似成纤维细胞,可在体外稳定扩增10代以上.诱导分化的细胞具有星形胶质细胞的超微结构特征;Nestin在细胞质中呈阳性表达,GFAP在细胞质及细胞核中均呈阳性表达.两种蛋白的阳性表达率在诱导14d时达到高峰分别为：Nestin（14.4 ±3.6）%,GFAP（87.3±5.3）%,此后两种蛋白的阳性表达率保持不变.Nestin及GFAP基因mRNA在诱导前及诱导后（1 d、7d、14d、20d）各时间点均有表达,且mRNA的平均相对浓度随着诱导时间的延长逐渐增高,Nestin在诱导后14d达到最高水平,GFAP在诱导后20d达到最高水平（P〈0.05）.结论 成人脂肪基质细胞的胞质中具有神经干细胞标志物Nestin的基因遗传物质,可将其诱导分化为具有成熟星形胶质细胞超微结构及GFAP蛋白、基因表达的细胞.

Objective To induce adult adult adipose-derived stem cells（ADSCs） in vitro to differentiate into neuronal-like cells,and to analyze the features of their cell morphology and ultrastructure. Methods Adipose stromal cells were obtained and amplified in vitro. Then make use of chemical induction to induce them. Observed ADSC and differentiation of cells in morphology and ultrastructure under inverted microscope and transmission electron microscopy. Immunocytochemistry to detection of Nestin, Neuron Specific Endolase（ NSE） ,and glial fibrillary acidic protein（GFAP） expression in cells. Used Real-time PCR to detection of Nestin,GFAP gene mRNA expression before and after induction in ADSC. To observe the morphology and ultrastructure of the cells prior to and after induction under microscope and electron microscope. Results The morphology of ADSC was similar to fibroblasts ,and could be amplified stability within 10 passages in vitro. Some of the cells induced display a typical astrocyte-like cells in ultrastructure. Followed neuronal induction,astrocyte-like cells began to stain brightly for CFAP, Nestin. GFAP stained in both the cytoplasm and nuclei of astrocyte-like cells, but Nestin only stained in the cytoplasm. The peak positive expression rate within 14d following neuronal induction. The rate of positive expression cells was（ 14.4 ± 3. 6） % for Nestin, （87. 3 ± 5. 3 ） % for GFAP. Then two kinds of protein expression remained the similar rate. The average relative concentration of GFAP and Nestin gene mRNA have significant statistical difference between ADSC and differented cells analyzed by Real-time PCR （P〈0.05）.The peak concentration of GFAP was within 20 d after induction,and GFAP was within 14 d after induction. Conclusion In the cytoplasm of adult adipose-derived cells possess Nestin genetic material,which is the marker of neural stem cell. The differential astrocyte-like cells have the typical morphology, ultrastructure and GFAP phenotype of mature astrocytes.