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

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1×10~8 L~(-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6 h,4组的叶酸含量分别为4 mg/L,4 mg/L,0.65 mg/L,8 mg/L.收集增殖6 d的细胞,锥虫蓝计数细胞密度,RT-PCR法检测pERK1/2 mRNA的表达,Western blot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.

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

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 1×10~8/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.