新生小鼠脊髓神经干细胞体外增殖及其多潜能分化特性

Culture in vitro and multipotential differentiation of neural stem cells from the spinal cord of neonatal mice

目的:观察新生小鼠脊髓神经干细胞的体外增殖及其分化特性。方法:实验于2005-11/2006-02在安徽医科大学基础医学院生物化学与分子生物学教研室完成。①在无菌条件下,利用显微解剖分离新生小鼠(出生后2d内)脊髓,采用原代机械吹打使其成为单细胞悬液,离心,弃去上清液,加入干细胞培养液(含20μg/L碱性成纤维细胞生长因子,20μg/L表皮生长因子,B27辅助培养液),以1×108L-1密度接种于25mL培养瓶中,进行原代培养。②将原代培养7d左右的细胞再次离心,弃去上清,加入干细胞培养液,机械吹打成单细胞悬液,以5×107L-1密度进行传代培养,每六七天传代1次。③机械吹打制成的单细胞悬液,经过传代培养,重新增殖为神经球,采用巢蛋白抗体鉴定培养细胞,用体积分数为0.1的胎牛血清诱导神经干细胞分化后,用神经元和星型胶质细胞标志物微管相关蛋白,胶质纤维酸性蛋白相应抗体检测神经干细胞分化情况。结果:①脊髓源性神经干细胞形态学观察:在细胞培养第3,4天即可见培养基中出现由几个或十几个细胞组成的结构紧密、大小不等的悬浮细胞团块,称之为“神经细胞球”;随着培养时间的延长和多次传代换液,细胞增殖迅速,神经细胞球数目明显增多。②脊髓神经干细胞的分化与鉴定:将神经球转入含体积分数为0.1的胎牛血清的培养液中,数小时内迅速贴壁并开始分化,5d后分化的细胞呈现出3种典型的神经细胞形态;免疫细胞化学检测神经细胞球巢蛋白染色阳性及其分化的神经元和星型胶质细胞微管相关蛋白、胶质纤维酸性蛋白分别染色阳性。结论:新生小鼠脊髓存在具有多分化潜能的神经干细胞,它们可以在体外大量增殖,经过诱导可朝神经元和胶质细胞的方向分化。

AIM： To explore the in vitro culture and differentiation of neural stent cells （NSCs） from the neonatal mice spinal cord. METHODS： The experiment was carried out in the Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Anhui Medical University from November 2005 to February 2006.（1）Spinal cord of neonatal mice （within postnatal 2 days） were dissociated through microscopic dissection in sterile condition, and single cell suspensions were mechanically acquired in primary culture. After centrifugation and supernatant deletion, cells were resuspended in DMEM/F12 serum-free medium containing 20 μg/L basic fibroblast growth factor （bFGF）, 20 μg/L epidermal growth factor （EGF） and B27 supplement, and then the ceils were planted in 25 mL culture bottles at the density of 1 ×l0^8 L^-1 for primary culture. （2）Seven days later, ceils were centrifuged again and resuspended in the same medium. The ceils were subcultured once every 6-7 days at the density of 5×10^7 L^-1. （3）The single cell suspensions were made by beating upon mechanically followed by subculture, then the ceils proliferated into cell bails again, Nidogen antibodies were used to identify the cultured ceils. After the differentiation of NSCs with fetal bovine serum in the volume fraction of 0.01, the microtubule-associated pretein-2 （MAP-2） antibody, which was marked with neurons and astrecytes, and glial fibfillary acidic protein （GFAP） antibody were used to study the differentiation of NSCs. RESULTS： （1）Morphological observations of NSCs： Three to four days after primary culture, a lot of suspension cell aggregation called neural spheres （several to one decade ceils） were found in the culture medium with tight structure and unequal size. （2） The differentiation and identification of NSCs： Several hours after the neural spheres were put into the medium containing fetal bovine serum of 0.01 volume fraction, the adhesive cell bails began to differentiate, and presented three classical neural or glial cell types 5 days later. The immunocytochemical detection showed that these neural spheres strongly expressed in nidogen, and could be further induced to express MAP-2 and GFAP, suggesting their differentiating potentials into neurons and astrecytes. CONCLUSION： A few NSCs of multipotential differentiation reside in the spinal cord of neonatal mice, and these cells have ability to culture and further differentiate into neurons and gliacytes in vitro.