硒和B-27添加剂在神经干细胞存活与分化中的作用

Effects of selenium and B-27 supplements on viability and differentiation of neural stem cell in newborn rat

目的观察微量元素硒和神经细胞培养添加剂B-27对神经干细胞存活与分化的影响。方法采用新生Wistar大鼠神经干细胞体外分离培养技术,应用盖玻片培养法和免疫细胞化学法来观察在无血清培养液中加入添加剂B-27和不含B-27时,硒对神经干细胞存活以及神经元分化标记蛋白β-微管蛋白、星形胶质细胞标记蛋白抗胶质细胞醋酸纤维蛋白(GFAP)和少突胶质细胞标记蛋白抗环核苷酸磷酸二酯酶(CNP酶)表达的影响。结果在培养液中加入B-27添加剂同时加入硒时,神经干细胞能够存活并且分化成熟良好。在不含B-27添加剂同时也不含硒的培养液中神经干细胞不能够存活。在不含B-27添加剂而含有硒时,神经干细胞则能够存活并且能分化成神经元、星形胶质细胞和少突胶质细胞,各种细胞的形态典型。显微镜下计数发现神经干细胞在含硒的培养液中向神经胶质细胞方向分化比例较高。其中神经元分化β-微管蛋白阳性细胞每高倍视野为11.2个。胶质细胞分化方向GFAP阳性和CNP酶阳性细胞计数平均每高倍视野分别为16.1个和9.3个。结论硒对于神经干细胞体外存活和分化是必需的营养成分。在不含B-27,但有硒存在时,神经干细胞能够存活并分化成熟。

Objective To assess how trace element selenium and B27 supplements affect the neural stem cell （NSc） differentiation in vitro. Methods The development and differentiation of NSc from the newborn rat were observed with primary culture and subculture during treating by sodium-selenite, and selenium-methyl-cysteine （SMC）. The immunocytochemisty techniques were used to identify the NSc and mature protein expression with neuron marker β-tubulin, astrocyte marker GFAP, and oligodendrocyle marker CNPase. The neurosphere morphology and neurite outgrowth were observed. Results Adding the complete B-27 serum-free supplement, Selenium could promote the neurosphere viability, development and differentiation. Without selenium and B-27 , neurosphere could not survive and differentiate. Without B-27 in the medium but there containing selenium, the neurosphere could promote the viability and development into neuron, astrocyte and oligodendrocyte, as compared with the no-containing B-27 and selenium groups, these differentiated cells might have more quantity, more branches and better morphological nervenet. The count of the neuron, astrocyte and oligodendrocyte was 11.2/Hp, 16.1/Hp and 9.3/Hp. Conclusions The selenium should be very important for neural stem cells＇ survival. Selenium could promote the neurosphere cells differentiation and development.