体外缺氧条件下新生鼠神经干细胞pERK1/2表达与叶酸的影响

Effects of folic acid on pERK1/2 expression of neural stem cells from neonatal rats under hypoxia condition in vitro

背景:课题组前期实验已经证实,神经干细胞在正常培养条件下,叶酸可通过丝裂原活化蛋白激酶通路激活ERK1/2的磷酸化,进而促进神经干细胞的增殖。目的:探讨叶酸在体外缺氧条件下对神经干细胞外信号调节蛋白激酶pERK1/2表达的影响。方法:采用无血清培养法体外分离培养新生鼠神经干细胞,以1×108L-1接种于培养瓶,设立4组,除正常对照组外,缺氧模型组、叶酸缺乏组、叶酸添加组细胞均于第3天放入自制缺氧装置,37℃恒温箱中缺氧培养6h,4组的叶酸含量分别为4mg/L,4mg/L,0.65mg/L,8mg/L。收集增殖6d的细胞,锥虫蓝计数细胞密度,RT-PCR法检测pERK1/2 mRNA的表达,Westernblot法检测pERK1/2蛋白的表达。结果与结论:与正常对照组比较,缺氧模型组神经干细胞增殖能力、pERK1/2 mRNA及蛋白的表达均明显降低。与缺氧模型组比较,叶酸添加组能促进缺氧条件下神经干细胞增殖以及pERK1/2 mRNA、蛋白的表达,而叶酸缺乏组则抑制缺氧条件下神经干细胞的增殖以及pERK1/2 mRNA、蛋白的表达,各组间比较差异有显著性意义(P<0.001)。证实添加叶酸后可激活ERK1/2磷酸化,进而促进缺氧条件下神经干细胞的增殖。

BACKGROUND：Previous studies have verified that under normal culture of neural stem cells （NSCs）,folic acid can accelerate proliferation of NSCs by phosphorylation of mitogen activated protein kinase path activation ERK1/2.OBJECTIVE：To investigate the effect of folic acid on NSC extracellular signal regulatory protein kinase pERK1/2 under hypoxic condition.METHODS：NSCs from Neonatal rats were cultured in vitro by serum-free culture method,and incubated in a flask at 1×108/L.Except normal control group,self-made hypoxia equipment was used in the hypoxia model,folic acid deficiency and folic acid supplemented groups at day 3.At 37 ℃,hypoxia culture was conducted in the thermostat for 6 hours.The contents of folic acid were 4 mg/L,4 mg/L,0.65 mg/L,8 mg/L in the four groups.Cells following 6 days were collected to count the density using trypan blue.RT-PCR was utilized to detect pERK1/2 mRNA expression.Western blot assay was employed to determine pERK1/2 protein expression.RESULTS AND CONCLUSION：Compared with the normal control group,the proliferation of NSCs and the expression of ERK1/2mRNA and pERK1/2 protein were decreased significantly in the hypoxia model group.Compared with the hypoxia model group,the proliferation of NSCs and the expression of ERK1/2mRNA and pERK1/2 protein were increased in the folic acid supplemented group,whereas decreased in the folic acid deficiency group.There were significant differences among groups （P 0.001）.Above-described results verified that folic acid supplementation can activate ERK1/2 phosphorylatin and accelerate proliferation of NSCs under hypoxia condition.