摘要
目的:利用神经诱导液体外诱导大鼠脂肪来源干细胞(adipose-derived stem cells,ASCs)神经分化,探索对ASCs体外成神经分化的最佳毛喉素浓度,并研究该浓度的培养液对ASCs体外成神经分化的作用.方法:酶消化法获得原代ASCs,通过流式细胞术和细胞染色鉴定ASCs的表型和多向分化能力.体外用含不同浓度毛喉素的神经诱导条件培养液进行神经诱导分化,采用免疫荧光染色技术筛选最佳浓度.采用含最佳浓度毛喉素的神经诱导条件培养液诱导ASCs,镜下观察诱导前后细胞的形态学特征,采用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和免疫荧光检测诱导前后细胞中神经分化标志物的mRNA和蛋白表达变化情况.结果:神经诱导条件培养液中毛喉素的最佳浓度为 10 μmol/L,含该浓度毛喉素的神经诱导条件培养液诱导的细胞,能够迅速诱导ASCs向成神经方向分化.诱导后,细胞形态类似神经细胞,神经细胞标志物抗β-Ⅲ微管蛋白(class Ⅲ β-tubulin,Tuj1)、神经丝蛋白(neurofilament-M,NF-M)、巢蛋白(nestin)抗体和S100β的mRNA水平和蛋白水平均表达增加(P<0.05).结论:含毛喉素浓度为10 μmol/L的诱导培养液能诱导ASCs神经分化,诱导细胞有望成为周围神经种子细胞.
Abstract
Objective:To explore the neural differentiation of adipose-derived stem cells(ASCs)induced by neural-induced conditional medium in vitro,and to study the optimal concentration of forskolin and the effect of optimal concentration on neurogenic differentiation of ASCs in vitro.Methods:ASCs were isolated by enzyme digestion method.The phenotype and differentiation ability of stem cells are identified by flow cytometry and cell staining.Nerve induction differentiation was performed in vitro by neural-induced conditional medium containing different concentrations of forskolin,and the optimal concentration was screened by immunofluorescence staining.ASCs were induced by neural-induced conditional medium containing the optimal concentration of forskolin and the morphological changes of cells were observed by microscope before induction,6 h after induction and 24 h after induction.Effects of induction on rat ASCs were analyzed by real-time quantitative polymerase chain reaction(RT-qPCR)and immunofluorescence.Results:The optimal concentration of forskolin in neural-induced conditional medium was 10 µmol/L,and the cells induced by neural-induced conditional medium containing 10 µmol/L forskolin could rapidly induce ASCs to differentiate in the direction of nerve formation.After induction,the cell morphology was similar to nerve cells.Results of RT-qPCR and immunofluorescence indicated that the expressions of anti-class Ⅲβ-tubulin(Tuj1),neurofilament-M(NF-M),nestin and S100 β at the mRNA level and protein level were increased after induction(P<0.05).Conclusion:Nerve differentiation of ASCs can be induced by conditional medium containing 10 µmol/L forskolin,and the induced cells are expected to become seed cells of facial nerve conduits.
基金项目
国家自然科学基金(82071160)
国家自然科学基金(82370933)
上海市高水平地方高校创新团队项目(SSMU-ZDCX20180900)
国家科技期刊平台
NSTL
万方数据