骨髓基质细胞条件培养液通过p38信号转导通路诱导中脑神经干细胞分化  

p38 signal-transduction pathway mediates the differentiation of mesencephalic neural stem cells induced by bone marrow stromal cell-conditioned medium

作　　者：顾平[1] 温雅[1] 马芹颖[1] 王彦永[1] 崔冬生[1] 王铭维[1] 

机构地区：[1]河北医科大学第一医院河北省脑老化与认知神经科学实验室

出　　处：《中国组织工程研究与临床康复》2007年第37期7329-7332,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30400138);河北省自然科学基金资助(C2004000583)~~

摘　　要：目的:骨髓基质细胞条件培养液能够诱导中脑神经干细胞分化为高比例神经元,但具体机制尚不十分清楚。选择p38信号转导通路作为观察切入点,剖析其在诱导分化途径中所扮演的角色。方法:实验于2006-04/2007-03在河北省脑老化与认知神经科学实验室完成。①实验方法:将SD大鼠麻醉后处死,分离股骨和胫骨,冲洗骨髓腔,将洗出的细胞悬液离心,悬浮后接种,培养48h后弃去未贴壁细胞,更换新的培养基,6d左右可进行传代,约铺满85%瓶底后弃去培养液,更换为含2%B27的Neurobasal培养液,24h后经过离心的上清液即为骨髓基质细胞条件培养液。取新生SD大鼠中脑,机械分散成单细胞后离心弃上清,加入培养液分散过滤,接种时加入含2%B27的DMEM/F12培养基和20μg/L碱性成纤维细胞生长因子,7d左右的单个神经干细胞便可增殖形成球体。将第2～3代神经干细胞球种植于预先包被多聚赖氨酸的培养皿中,贴壁后更换培养液。设立对照组和抑制剂组,均加入骨髓基质细胞条件培养液,此外抑制剂组还加入p38信号转导通路抑制剂SB203580至终浓度4μmol/L。②实验评估:第7天进行免疫细胞化学染色,检测表达微管相关蛋白2的神经元与表达胶质原纤维酸性蛋白的星形胶质细胞在分化细胞中所占的比例。结果:①神经干细胞分化过程中的形态学变化及p38信号转导通路抑制剂的影响:加入抑制剂后24h,光镜下可见神经干细胞长出的细胞有两种类型:一类细胞体积较小,边缘整齐,立体感强,周围有光晕并在两极有突起,免疫荧光染色显示这类细胞微管相关蛋白2呈阳性表达,即为神经元;另一类细胞突起较粗大,不规则,多聚集在神经干细胞球的中央,呈放射状排列,免疫荧光染色表明这类细胞为胶质原纤维酸性蛋白阳性的星形胶质细胞。②神经元及星形胶质细胞在分化细胞中所占的比例:p38信号转导通路抑制剂作�AIM： To investigate the effect of p38 signal-transduction pathway on the differentiation of rat mesencephalic neural stem cells （NSCs） in the presence of bone marrow stromal cell-conditioned medium （BMSCs-CM） in vitro. METHODS： The experiment was carried out in Brain Aging and Cognitive Neuroscience Laboratory of Hebei Province from April 2006 to March 2007. ①BMSCs were collected from femur and tibia of SD rats by flushing the medullary canal after the rats were sacrificed under anesthesia. Suspension of cells was centrifuged and planted in medium. Forty-eight hours later, un-adherent cells were removed, and the medium was changed. BMSCs were passaged every 6 days. When the cells presented 85% confluence, they were plated at Neurobasal medium containing 2% B27. After 24 hours, the medium collected following centrifugation was used as BMSCs-CM. The midbrain of newborn SD rats was removed, mechanically dispersed into single cell and centrifuged. The pellets were resuspended with DMEM/F12 nutrient medium supplemented with 2% B27 and 20μg/L basic fibroblast growth factor （bFGF）. About 7 days later, the single NSC could become spherical. NSC spheres （2-3 generation） were planted to the poly-L-lysine-coated dishes. The culture fluid was changed after cell adherence. Control group and inhibitor group were set up, in which BMSCs-CM was added; in addition, SB203580 （p38 signal-transduction pathway inhibitor） was added into inhibitor group till final concentration of 4 μmol/L. ②lmmunocytochemistry was performed for the identification of different cells at the 7^th day. Percentage of the total numbers of positive cells for neuron that expressed microtubule-associated protein-2 （MAP-2） and astrocyte that expressed glial fibrillary acidic protein （GFAP） were determined by cell count. RESULTS： ①Twenty-four hours after adding SB203580, there were two types of cells migrated out from NSCs under light microscope： one type of cells with small, bright, regular edge, three-dimension, and e

关 键 词：转导 干细胞 神经元 细胞分化 

分 类 号：R394.2[医药卫生—医学遗传学]