Fasudil促进体外培养骨髓源神经干细胞的增殖与存活

Promoting effect of Fasudil on the proliferation and survival of bone marrow-derived neural stem cells in vitro

目的探讨法舒地尔（Fasudil）对体外培养骨髓源神经干细胞（BMNSC）增殖和存活的影响。方法取3～4周龄的C57BL／6小鼠，分离骨髓基质细胞并诱导产生细胞球，用免疫荧光染色鉴定细胞类型。羧基荧光素二乙酸盐琥珀酰亚胺酯（CFSE）标记细胞，检测Fasudil促进BMNSC增殖能力。利用脂多糖（LPS）和γ干扰素（IFN-γ）联合处理BV-2小胶质细胞制备炎性条件培养液处理BMNSC，Fasudil干预，通过流式细胞术检测线粒体膜电位、碘化丙啶（PI）荧光值，探讨Fasudil对BMNSC的保护效果。结果免疫荧光染色证明骨髓源细胞球为神经上皮干细胞蛋白（nestin）阳性的神经干细胞。Fasudil处理明显降低CFSE平均荧光值。在LPS和IFN-γ联合作用下，BV-2小胶质细胞释放更多的白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）等炎性细胞因子，显著高于PBS处理组。该炎性条件培养液导致BMNSC线粒体膜电位明显降低并诱导更多的细胞死亡，而Fasudil处理则显著恢复线粒体膜电位接近正常值，减少细胞死亡，并且具有明显的剂量依赖性。结论Fasudil对体外培养BMNSC的增殖和存活具有显著的促进作用。

Objective To explore the effect of Fasudil on the proliferation and survival of bone marrow-derived neural stem cells （BMNSCs） cultured in vitro. Methods BMNSCs were derived from C57BL/6 mice aged 3-4 weeks and induced to produce cell spheres in serum-free medium. The cells were detected by immunofluorescence staining with nestin antibody. The effect of Fasudil on the proliferation of BMNSCs was detected by 5, 6-carboxyfluorescein diacetate succinimidyl ester （CFSE） labeling. BMNSCs were cultured under the inflammatory condition created by BV-2 microglia treated with lipopolysaccharide （LPS） and IFN-γ. The mitochondrial membrane potential （MMP） and propidium iodide （PI） fluorescence value of BMNSCs were determined by flow cytometry to explore the protective effect of Fasudil on BMNSCs. Results Immunofluorescence staining verified that bone marrow-derived cell spheres were nestin-positive neural stem cells. The treatment of Fasudil significantly decreased the average fluorescence value of CFSE. BV-2 microglias treated with LPS and IFN-γ released more inflammatory factors such as IL-6, TNF-α than PBS treated group. The inflammatory conditioned medium significantly decreased the MMP of BMNSCs and induced more cell death, while the treatment of Fasudil significantly restored the MMP of BMNSCs close to the normal value and reduced cell death in an obvious dose-dependent manner. Conclusion Fasudil has significantly promoting effect on the proliferation and survival of BMNSCs in vitro.