摘要
目的:探讨骨髓基质细胞诱导分化为神经干细胞的更佳培养液组成。方法:实验于2005-10/2006-08在省级实验室北华大学医学院实验中心完成。①实验材料:体质量120～160g的Wistar大鼠,由北华大学动物中心提供。②实验方法:取120～160g大鼠3只,体外分离培养大鼠骨髓基质细胞。培养约14d,待原代细胞贴满培养瓶,呈成纤维细胞样生长,用0.25%胰蛋白酶消化原代细胞约3min,在倒置显微镜下见贴壁细胞基本收缩成类圆形后,加入含胎牛血清的DMEM培养液终止消化,并将细胞液吹打均匀,以1×107L-1接种于2个细胞培养瓶中进行1∶2传代扩增培养。在传代培养细胞培养液中加入神经细胞诱导剂,选取生长良好的2,3,4代细胞进行实验。取第5代培养细胞,以8×103/cm2接种于预铺有盖玻片3.5cm培养皿中制备细胞爬片,每孔加入含体积分数为0.1胎牛血清的DMEM培养液2mL,达80%融合时更换新鲜培养液,并在培养液中加入1mmol/Lβ-巯基乙醇,诱导24h,磷酸盐缓冲液洗涤,换成含1mmol/Lβ-巯基乙醇的无血清培养液,5h后更换为改良神经细胞培养液,并设立未加入含β-巯基乙醇培养液的为对照。利用免疫组织化学方法检测神经元特有巢蛋白。结果:①培养的骨髓基质细胞形态学变化:接种初期,细胞贴壁生长是以分散的、克隆集落方式增殖,细胞呈圆形漂浮于培养液中,培养10d后,细胞形态基本为纺锤形的成纤维细胞样,偶有宽大扁平的多边形细胞,细胞逐渐长出小突起,出现出芽现象,14～20d后,突起细胞增多,突起延长并相互连接成网,具有神经细胞形态特征。②免疫组织化学染色检测神经元特有巢蛋白:1周左右,细胞可表达巢蛋白阳性,细胞大而圆,呈棕褐色,经培养诱导分化2周后的长突起细胞,表达神经元特异性抗原巢蛋白阳性细胞互相成网状连接。实验组第4代90%细胞巢蛋白阳性,在荧光显微镜下细胞发黄绿荧光,对�
AIM： To investigate the best culture fluid for the differentiation of neural stem cells from bone marrow stromal cells. METHODS： Between October 2005 and August 2006, the experiment was conducted in the Experimental Center of Beihua University Medical College. The bone marrow stromal calls were isolated from three Wistar rats of 120-160 g provided by the animal canter of Beihua University, and cultured for 14 days in vitro. After already attaching the culture bottle, and growing like fibroblasts, the primary cells were digested with 0.25% trypsin for about 3 minutes; when the attached calls shrank to alike round shape under inverted microscope, DMEM culture solution containing fetal bovine serum was added to stop the digestion, and the call liquid was blown and beaten homogeneously. The calls of 1：2 with 1×10^7/L were inoculated in 2 culture bottles to passage. The neural stem calls were added into the culture fluid, and the well-growing second, third and fourth generation cells were chosen for the following experiment. The fifth generation cells of 8 ×10^3/cm^2 were selected and seeded onto the 3.5 cm culture dish covered with the coverslip to prepare call attachment sections; every well was added in 2 mL DMEM containing volume fraction 0.1 fetal bovine serum, and the fresh culture solution was changed when the cells grew to 80% fusion, and 1 mmol/L 13 mercaptoethanol was added to induce the calls for 24 hours. After washing with phosphate buffer, the culture solution was replaced by the serum free culture solution containing 1 mmol/L β mercaptoethanol; 5 hours later, the culture solution was changed by the nerve call solution, and that with the culture solution with no （3 mercaptoethanol served as control. Immunohistochemistry was employed to detect the special protein of nerve, namely, Nestin. RESULTS： （1）At the primary stage of incubation, the cells attached the wall in a scattered and colony patterns, they were round and floating in the culture solution; 10 days later, the calls changed to s
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第20期3991-3993,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
吉林市科委立项题目(吉市科卫2005-03)~~
