法舒地尔干预原代神经干细胞的增殖与分化

摘要

背景:受损大脑和脊髓的自我修复能力有限, 寻找促进神经干细胞增殖与分化的潜在治疗剂, 对神经系统疾病的干细胞治疗具有重要意义.目的:探讨法舒地尔对原代神经干细胞增殖及分化的影响及其可能机制.方法:体外分离、培养15 d龄胎鼠脑组织获得神经干细胞, 免疫荧光检测细胞中Nestin的表达;以不同浓度 (50, 100, 200μmol/L) 法舒地尔干预神经干细胞24, 48, 72 h, MTT检测细胞增殖率, 流式细胞术检测细胞凋亡率;进一步应用凋亡抑制剂、铁死亡抑制剂、自噬抑制剂、坏死抑制剂、凋亡诱导剂、铁死亡诱导剂干预神经干细胞, MTT检测细胞增殖率, 生化法检测细胞丙二醛水平;最后, 应用200μmol/L法舒地尔干预神经干细胞10 d, Western blot和免疫荧光检测神经干细胞中Nestin、DCX、MAP-2、GFAP蛋白的表达.结果与结论: (1) 分离培养的原代神经干细胞表达Nestin; (2) 随着法舒地尔浓度升高及作用时间延长, 神经干细胞的增殖率逐渐增高 (P <0.05); (3) 凋亡抑制剂、铁死亡抑制剂可提高神经干细胞的增殖率 (P <0.05); (4) 法舒地尔可提高凋亡诱导剂和铁死亡诱导剂干预的神经干细胞的增殖率 (P <0.05); (5) 法舒地尔和铁死亡抑制剂均能降低神经干细胞中丙二醛水平, 铁死亡诱导剂则使神经干细胞中丙二醛水平升高 (P <0.05); (6) 经法舒地尔处理后, 神经干细胞中DCX和GFAP蛋白的表达升高, Nestin的表达降低 (P <0.05); (7) 结果表明, 法舒地尔可通过抑制凋亡与铁死亡提高神经干细胞的存活, 并能促进神经干细胞增殖及向神经元样细胞与神经胶质细胞分化.%BACKGROUND: The self-repairing ability of damaged brain and spinal cord is limited. It is important to search for potential therapeutics to promote the proliferation and differentiation of neural stem cells. OBJECTIVE: To investigate the effect of fasudil on the proliferation and differentiation of primary cultured neural stem cells and its potential mechanism. METHODS: Neural stem cells were obtained from the brain tissue of 15-day-old fetal rats in vitro. The expression of Nestin in the cells was detected by immunofluorescence. After treatment with fasudil at different concentrations (50, 100, 200 μmol/L) for 24, 48 and 72 hours, the proliferation rate of neural stem cells was detected by MTT, and the apoptosis rate was detected by flow cytometry. After further treatment with autophagy inhibitor, necrosis inhibitor, apoptosis inducer and ferroptosis inducer, the proliferation rates of neural stem cells were detected by MTT and the levels of malondialdehyde were detected by biochemical method. The expression of Nestin, doublecortin, microtubuleassociated protein and glial fibrillary acidic protein in neural stem cells were detected by western blot and immunofluorescence after treatment with 200 μmol/L fasudil for 10 days. RESULTS AND CONCLUSION: The positive expression of Nestin protein in primary cultured neural stem cells was observed. The proliferation rate of neural stem cells increased gradually with the increase of fasudil concentration as well as with the prolongation of action time (P < 0.05). Both apoptosis inhibitor and ferroptosis inhibitor can increase the proliferation rate of neural stem cells (P < 0.05). Fasudil increased the proliferation rate of neural stem cells treated by apoptosis inducer and ferroptosis inducer (P < 0.05). Fasudil and ferroptosis inhibitors both decreased the level of malondialdehyde in neural stem cells, while ferroptosis inducers increased the level of malondialdehyde in neural stem cells (P < 0.05). After treatment with fasudil, the expression of doublecortin and glial fibrillary acidic protein protein in neural stem cells increased, and the expression of Nestin decreased (P < 0.05). To conclude, fasudil can improve the survival of neural stem cells by inhibiting apoptosis and ferroptosis, and moreover, it can promote the proliferation and differentiation of neural stem cells into neuron-like cells and glial cells.