神经干细胞的单层培养及分化过程中HES1和MASH1的表达变化

Culture of Neural Stem Cell in Vitro and the Expression Changes of HES1 and MASH1 during Differentiation

【目的】建立神经干细胞(NSC)体外培养方案,使NSC的体外可以得到增殖和分化,并检测NSC分化过程中HES1和MASH1的表达变化。【方法】无菌条件下取SD大鼠胚胎端脑,无血清悬浮培养,形成P2代神经球后,将神经球消化成单细胞,分为神经球悬浮培养和单层贴壁培养,并对两种不同培养方式下的NSC进行干性鉴定和分化能力鉴定。用不同浓度(20、40、80μg/L)的脑源性神经生长因子(BDNF)诱导NSC分化,选出最合适的浓度。用BDNF最佳诱导浓度诱导NSC分化,Q-PCR检测分化过程中HES1和MASH1的表达变化。【结果】神经球悬浮培养和单层贴壁培养均可得到的NSC;分化3 d后,在不加入任何诱导因素的情况下,单层贴壁培养的NSC能更高比例[(19.55±1.09)%]地分化为β-tubulinⅢ阳性细胞;BDNF 40μg/L能诱导出更多的β-tubulinⅢ阳性细胞[(34.17±0.60)%];在分化1、3和7 d时,Q-OCR结果表明MASH1在BDNF诱导下组较NSC和对照组表达水平明显上升。【结论】单层贴壁培养的神经干细胞更利于分化为神经元,BDNF为40μg/L时能诱导出更高比例的神经元,BDNF可以诱导MASH1在NSC分化过程中表达明显上升。

[ Objective ] Culturing of NSC in vitro were investigated, in which NSC could proliferate and differentiate. The expression changes of HES1 and MASH1 were detected during the differentiation of NSC. [Methods] The cerebrums of rat embryos were separated in sterile working condition and cultured in serum-free medium. When the secondary neurospheres were formed, Accutase digested them into single cells. Then they were divided into two parts, one was cultured with suspension neurospheres, the other was plated to adherent monolayer. And the abilities of ＂stem＂ and ＂differentiation＂ in two methods were examined. NSC were induced with different concentrations （20, 40, 80 wg/L） of brain-derived neurotrophic factor （BDNF） to differentiation, the best one was determined. After induced differentiation of NSC in the presence of BDNF, the expression changes of HES1 and MASH1 were detected. [Results] NSC were obtain in both of two. β-tubulin m-positive cells was higher in adherent monolayer culture （19.55 ± 1.09）% than in suspension neurospheres without any inducing factor. NSC were more induced to β-tubulin III-positive cells within the BDNF of 40 ug/L （34.17±0.60）%. After differentiation 1, 3, and 7 d, results of Q-PCR were showed that the relative quantities of MASH1 in BDNF group were significant higher than NSC or control. [Conclusion] The adherent monolayer culture was a better method for differentiation of neurons from NSC ; BDNF of 40 ug/L was a better concentration than any others ; the expression of MASH1 could be increased by BDNF during differentiation of NSC.