摘要
目的:研究人参皂苷对体外氧糖剥夺/再灌注(OGD/R)神经干细胞(NSCs)自我更新、增殖和分化的影响及其机制。方法:分离胎鼠海马神经干细胞,采用MTT法测定不同时间点人参皂苷对神经干细胞增殖分化的影响,并筛选出人参皂苷促进神经干细胞增殖分化的最佳浓度和最佳作用时间窗。建立神经干细胞缺氧缺糖/再灌注(OGD/R)的体外模型,并给予人参皂苷治疗,通过ELISA和Western blot方法检测培养液中血管内皮生长因子(VEGF)蛋白及细胞核中HIF-1α蛋白的表达量。采用免疫荧光双标记方法检测各组神经干细胞特异性标志物巢蛋白(Nestin)、神经干细胞增殖标记物(Brdu)、神经元祖细胞特异性标记物(Tuj-1)及星形胶质细胞祖细胞标记物(Vimentin)的表达,统计各组阳性细胞的个数、面密度值和光密度值,研究验证人参皂苷对神经干细胞的增殖与分化的影响及可能的机制。结果:筛选出人参皂苷促进神经干细胞增殖分化的最佳浓度为1μg/mL,作用的最佳时间点为24h。结果发现,与正常组比较,模型组HIF-1α蛋白和VEGF蛋白含量增加,阳性细胞的细胞个数、光密度和面密度减少,提示缺血再灌注后,导致了神经干细胞的损害,神经干细胞细胞核中HIF-1α基因转录翻译增强,促进了其下游靶基因VEGF的转录翻译增强,使HIF-1α蛋白与VEGF蛋白表达均增加,从而启动了神经干细胞增殖和分化的神经修复过程;与模型组比较,人参皂苷治疗组HIF-1α蛋白和VEGF蛋白含量增加,且阳性细胞的细胞个数、光密度和面密度比模型组明显增加(P<0.05)。结论:人参皂苷可促进缺血再灌注后HIF-1α蛋白和VEGF蛋白含量进一步增加,通过促进神经干细胞的增殖与分化而促进脑损伤结构和功能的修复。
Objective: To study the effects of ginsenoside on self-renewal, proliferation and differentiation ofneural stem cells(NSCs) undergone oxygen-glucose deprivation/reperfusion(OGD/R) in vitro and its mechanism. Methods: The NSCs were isolated from the hippocampus of the E17 embryo rat. MTT assay was used to determine the effect of ginsenoside on the proliferation and differentiation of NSCs at different time points. The best concentration and best time window of ginsenoside to promote the differentiation and differentiation of NSCs were screened. An in vitro model of NSCs subjected to OGD/R was established, and different dosages of ginsenoside were given to the model. The expressions of vascular endothelial growth factor(VEGF) protein and hypoxia-inducible factor(HIF-1α) in nucleus were detected by ELISA and Western blot. The expressions of the specific marker of NSCs(Nestin), the proliferation marker of NSCs(Brdu), the specific marker of neuronal progenitor cell(Tuj-1) and the marker of astrocyte progenitor cell(Vimentin) were detected by immunofluorescence double labeling method. The number of positive cells, the surface density and the optical density were calculated. The effect of ginsenoside on the proliferation and differentiation of NSCs and the possible mechanism were investigated. Results: The best concentration of ginsenoside to promote the proliferation and differentiation of NSCs was 1μg/mL, and the best time point was 24 h. The results showed that compared with the normal group, the HIF-1α protein and VEGF protein in the model group increased, and the number of positive cells, the optical density and the surface density decreased, which suggested that the damage of NSCs were caused by ischemia and reperfusion, and the transcription and translation of HIF-1α gene in the nucleus of NSCs were enhanced, promoting thetranscription and translation of downstream target gene VEGF. The protein expressions of HIF-1α and VEGF were increased, which initiated the neural repair process of proliferation and differentiation of NSCs. Compared with the model group, the contents of HIF-1α protein and VEGF protein in the ginsenoside treatment group were increased, and the number of positive cells, the optical density and the surface density were significantly higher than those in the model group(P<0.05). Conclusion: Ginsenoside can promote the increase of HIF-1αand VEGF protein after ischemia and reperfusion, and promote the repair of brain injury structure and function by promoting the proliferation and differentiation of NSCs.
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2017年第5期2291-2297,共7页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金面上项目(No.81373830)~~
