학술논문
去泛素化酶Mysm1通过调控胶质纤维酸性蛋白的表达调节神经干细胞向星形胶质细胞分化 / Deubiquitinase Mysm1 regulates differentiation of neural stem cells into astrocytes by regulating expressions of glial fibrillary acidic protein
Document Type
Academic Journal
Author
徐振华; 秦巧臻; 李晓彤; 陈悦; 江小霞; 徐东刚; XU Zhenhua; QIN Qiaozhen; LI Xiaotong; CHEN Yue; JIANG Xiaoxia; XU Donggang
Source
军事医学 / Military Medical Sciences. 48(1):36-43
Subject
Language
Chinese
ISSN
1674-9960
Abstract
目的 研究去泛素化酶Mysm1对神经干细胞(NSC)向星形胶质细胞分化的影响及其作用机制.方法 从孕12.5 d C57BL/6胎鼠脑组织中分离NSC在体外进行培养,体外诱导NSC向星形胶质细胞分化.免疫荧光染色、实时定量PCR和蛋白质免疫印迹实验检测体外NSC向星形胶质细胞分化过程中Mysm1表达量的变化;使用慢病毒敲低NSC中Mysm1的表达量并通过实时定量PCR和蛋白质免疫印迹实验检测敲低效率;免疫荧光染色和蛋白质免疫印迹实验比较体外敲低Mysm1前后NSC向星形胶质细胞分化的变化;转录组学检测体外敲低NSC内Mysm1后差异基因情况;蛋白质免疫印迹实验验证差异基因相关蛋白的变化;Cut-Tag检测体外NSC向星形胶质细胞分化过程中Mysm1在胶质纤维酸性蛋白(GFAP)基因启动子区域富集情况;在敲低Mysm1的基础上过表达GFAP,免疫荧光染色和蛋白质免疫印迹实验检测体外过表达GFAP前后NSC向星形胶质细胞分化的变化.结果 体外诱导NSC向星形胶质细胞分化时,细胞表达Mysm1逐渐升高;Mysm1敲低会抑制体外NSC向星形胶质细胞分化;Mysm1通过调控GFAP的转录影响NSC向星形胶质细胞分化;在敲低Mysm1的基础上过表达GFAP部分挽救了NSC向星形胶质细胞分化的能力.结论 Mysm1通过表观调控GFAP的转录调节NSC向星形胶质细胞分化.
Objective To investigate the effect of Mysm1 on the differentiation of neural stem cells(NSCs)into astrocytes and the possible mechanism.Methods NSCs were prepared from E12.5 cortices of wild-type C57BL/6 mice,cultured in vitro and induced to differentiate into astrocytes.Immunofluorescence staining,real-time quantitative PCR and Western blot assay were used to detect the expressions of Mysm1 during the differentiation of NSCs into astrocytes in vitro.Lentivirus was used to knock down Mysm1 expressions in NSCs before real-time quantitative PCR and Western blot assay were used to detect the knockdown efficiency.Immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after Mysm1 knockdown in vitro.Transcriptomics was adopted to detect the differential gene after knockdown of Mysm1 in NSCs in vitro.Western blot assay was used to verify the changes of proteins associated with the differential gene.Cut-Tag was used to detect the enrichment of Mysm1 in the promoter region of glial fibrillary acidic protein(GFAP)genes during the differentiation of NSCs into astrocytes in vitro.After overexpression of GFAP following knockdown of Mysm1,immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after overexpression in vitro.Results The expression of Mysm1 was gradually increased when NSCs were induced to differentiate into astrocytes in vitro.Mysm1 knockdown inhibited the differentiation of NSCs into astrocytes in vitro.Mysm1 affected the differentiation of NSCs into astrocytes by regulating the expression of GFAP.Overexpression of GFAP after Mysm1 knockdown partially rescued the ability of NSCs to differentiate into astrocytes.Conclusion Mysm1 regulates the differentiation of NSCs into astrocytes by epigenetically controlling GFAP transcription.
Objective To investigate the effect of Mysm1 on the differentiation of neural stem cells(NSCs)into astrocytes and the possible mechanism.Methods NSCs were prepared from E12.5 cortices of wild-type C57BL/6 mice,cultured in vitro and induced to differentiate into astrocytes.Immunofluorescence staining,real-time quantitative PCR and Western blot assay were used to detect the expressions of Mysm1 during the differentiation of NSCs into astrocytes in vitro.Lentivirus was used to knock down Mysm1 expressions in NSCs before real-time quantitative PCR and Western blot assay were used to detect the knockdown efficiency.Immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after Mysm1 knockdown in vitro.Transcriptomics was adopted to detect the differential gene after knockdown of Mysm1 in NSCs in vitro.Western blot assay was used to verify the changes of proteins associated with the differential gene.Cut-Tag was used to detect the enrichment of Mysm1 in the promoter region of glial fibrillary acidic protein(GFAP)genes during the differentiation of NSCs into astrocytes in vitro.After overexpression of GFAP following knockdown of Mysm1,immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after overexpression in vitro.Results The expression of Mysm1 was gradually increased when NSCs were induced to differentiate into astrocytes in vitro.Mysm1 knockdown inhibited the differentiation of NSCs into astrocytes in vitro.Mysm1 affected the differentiation of NSCs into astrocytes by regulating the expression of GFAP.Overexpression of GFAP after Mysm1 knockdown partially rescued the ability of NSCs to differentiate into astrocytes.Conclusion Mysm1 regulates the differentiation of NSCs into astrocytes by epigenetically controlling GFAP transcription.