硒化合物对大鼠神经干细胞球分化成熟蛋白表达的影响

The Effect of Selenium Compounds on Expression of Differentiation Protein of Rat Neural Stem Cells in vitro

[目的]观察微量元素硒对神经干细胞分化成熟的影响。[方法]利用新生大鼠神经干细胞体外培养技术、小盖玻片培养法和免疫细胞化学方法,观察了无机硒(亚硒酸钠)和有机硒(硒甲基半胱氨酸)对神经干细胞向神经元分化标记蛋白NSE、星形胶质细胞分化标记蛋白GFAP、少突胶质细胞分化标记蛋白CNPase的表达状况,同时用Western-blot免疫印迹技术对神经元特异性烯醇化酶(neuronspecificenolase,NSE)、β-微管蛋白(β-tubulin)、星形胶质细胞-醋酸纤维蛋白(glialfibrillaryacidicprotein,GFAP)、环核苷酸-3’磷酸水解酶(2’,3’-cyclicnucleotide3’phosphohydrolase,CNPase)进行了半定量分析。[结果]适量浓度的硒对神经干细胞球的分化发育有良好的促进作用。在含硒的营养液中神经干细胞分化好、形态正常。神经干细胞向神经元、神经胶质细胞方向分化比例较高。其中胶质细胞分化方向GFAP+和CNPase+细胞计数,平均每高倍视野分别为5-6个和7-12个;并且分支多、细长,网络复杂。免疫印记结果发现有机硒能明显促进神经干细胞分化成熟蛋白NSE、β-tubulin、GFAP、CNPase的表达,化学发光显影明显高于对照组。[结论]适量的硒能促进神经干细胞的分化成熟。特别是硒甲基半胱氨酸是一个低毒和高生物效应的硒营养制剂,在神经干细胞球体外分化发育中有着重要的作用。

[Objective] Selenium(Se) is very important to the embryo and brain development. In recent study, a number of sele-noenzymes have been identified,which included the glutathione peroxidase(GPX) family,the iodothyronine deiodinase family,thioredosin re-ductases and selenophosphate synthetase, and some unknown function selenoprotein. This preliminary study tried to assess whether/how trace element selenium affects the development and differentiation of neural stem cells(NSCs) in vitro . [Methods] First, a method of NSCs culture from newborn rats' brain was established. Neurospheres got from primary culture and subculture and identified by a common NSCs' marker-nestin were used in the following studies. Then,some surface markers(neuron marker-NSE,β-tubulin,astrocyte marker-GFAP,oligo-dendrocyle marker-CNPase) of neural differentiation, after being treated with selenium-methyl-cysteine at a concentration gradient(0.1,0.5, 1.0 μg/ml) for 7 days, were detected by Western blot to find an appropriate concentration. Based on the former experiments, three groups (control group,0.025 μg/ml sodium-selenite group,and 0.5 μg/ml selenium-methyl-cysteine group) were set for the further study. The neurosphere morphology and neuritic outgrowth have been observed during treatment with the two kinds of selenium compounds. And 3 or 7 days,the surface markers mentioned above were detected by immunocytochemisty,based on the positive cells counted. [Results] The results of Western blot showed some varies of the surface markers expression,0.5 μg/ml group was shown as an appropriate concentration with its apparent effect. Then,in the further study,selenium-affected groups displayed more availabilities, to promote the neurosphere development, differentiation, and the cell growth,viability,with more quantity,branches and better structure of nerve net compared with the control group. And the Se stimulative expression effects of astrocyte(5-6/HP) and oligodendrocyle(7-12/HP) was more than those of neuron. Moreover selenium-methyl-cysteine showed even more promotion than sodium-selenite. [Conclusion] Selenium could promote the differentiation and development of neurosphere cells and especially glia at an appropriate concentration. The selenium-methyl-cysteine is a new selenium compound with lower toxicity and more availability. It may be even more exploited in future studies.