目的:明确Lhx8过表达慢病毒在海马齿状回胆碱能神经元发生中的作用。方法:构建Lhx8过表达重组质粒、进行慢病毒包装,通过颅脑立体定位和微量注射的方法,将所得慢病毒Lenti6.3-Lhx8注入SD大鼠海马齿状回中;于注射后第1、3、7、10、14天...目的:明确Lhx8过表达慢病毒在海马齿状回胆碱能神经元发生中的作用。方法:构建Lhx8过表达重组质粒、进行慢病毒包装,通过颅脑立体定位和微量注射的方法,将所得慢病毒Lenti6.3-Lhx8注入SD大鼠海马齿状回中;于注射后第1、3、7、10、14天在激光共聚焦显微镜下直接观察增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的荧光表达;检测术后第7天时EGFP/Lhx8/ChAT三标阳性细胞的数目。结果:慢病毒注射后第3天,海马齿状回中EGFP绿色荧光信号最强;此后EGFP表达下降,注射后第14天,几乎检测不到EGFP阳性细胞;术后第7天,注射Lenti6.3-Lhx8的慢病毒侧海马齿状回门区内能够检测到EGFP/Lhx8/ChAT三标阳性细胞,与阴性对照侧比较明显增多。结论:海马齿状回内注射Lhx8过表达慢病毒能够促进胆碱能神经元的发生。展开更多
Background:Cell replacement therapy has been envisioned as a promising treatment for neurodegenerative diseases.Due to the ethical concerns of ESCs-derived neural progenitor cells(NPCs)and tumorigenic potential of iPS...Background:Cell replacement therapy has been envisioned as a promising treatment for neurodegenerative diseases.Due to the ethical concerns of ESCs-derived neural progenitor cells(NPCs)and tumorigenic potential of iPSCs,reprogramming of somatic cells directly into multipotent NPCs has emerged as a preferred approach for cell transplantation.Methods:Mouse astrocytes were reprogrammed into NPCs by the overexpression of transcription factors(TFs)Foxg1,Sox2,and Brn2.The generation of subtypes of neurons was directed by the force expression of cell-type specific TFs Lhx8 or Foxa2/Lmx1a.Results:Astrocyte-derived induced NPCs(AiNPCs)share high similarities,including the expression of NPC-specific genes,DNA methylation patterns,the ability to proliferate and differentiate,with the wild type NPCs.The AiNPCs are committed to the forebrain identity and predominantly differentiated into glutamatergic and GABAergic neuronal subtypes.Interestingly,additional overexpression of TFs Lhx8 and Foxa2/Lmx1a in AiNPCs promoted cholinergic and dopaminergic neuronal differentiation,respectively.Conclusions:Our studies suggest that astrocytes can be converted into AiNPCs and lineage-committed AiNPCs can acquire differentiation potential of other lineages through forced expression of specific TFs.Understanding the impact of the TF sets on the reprogramming and differentiation into specific lineages of neurons will provide valuable strategies for astrocyte-based cell therapy in neurodegenerative diseases.展开更多
文摘目的:明确Lhx8过表达慢病毒在海马齿状回胆碱能神经元发生中的作用。方法:构建Lhx8过表达重组质粒、进行慢病毒包装,通过颅脑立体定位和微量注射的方法,将所得慢病毒Lenti6.3-Lhx8注入SD大鼠海马齿状回中;于注射后第1、3、7、10、14天在激光共聚焦显微镜下直接观察增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的荧光表达;检测术后第7天时EGFP/Lhx8/ChAT三标阳性细胞的数目。结果:慢病毒注射后第3天,海马齿状回中EGFP绿色荧光信号最强;此后EGFP表达下降,注射后第14天,几乎检测不到EGFP阳性细胞;术后第7天,注射Lenti6.3-Lhx8的慢病毒侧海马齿状回门区内能够检测到EGFP/Lhx8/ChAT三标阳性细胞,与阴性对照侧比较明显增多。结论:海马齿状回内注射Lhx8过表达慢病毒能够促进胆碱能神经元的发生。
基金This work was supported in part by research grants from the National Basic Research Program of China(973 ProgramGrant No.2014CB965001 to JZ)Innovative Research Groups of the National Natural Science Foundation of China(#81221001 to JZ)+2 种基金Joint Research Fund for Overseas Chinese,Hong Kong and Macao Young Scientists of the National Natural Science Foundation of China(#81329002 to JZ)the National Institutes of Health:2R56NS041858-15A1(JZ),1R01NS097195-01(JZ),and R03 NS094071-01(YH)the State of Nebraska,DHHS-LB606 Stem Cell 2009-10 to JZ.
文摘Background:Cell replacement therapy has been envisioned as a promising treatment for neurodegenerative diseases.Due to the ethical concerns of ESCs-derived neural progenitor cells(NPCs)and tumorigenic potential of iPSCs,reprogramming of somatic cells directly into multipotent NPCs has emerged as a preferred approach for cell transplantation.Methods:Mouse astrocytes were reprogrammed into NPCs by the overexpression of transcription factors(TFs)Foxg1,Sox2,and Brn2.The generation of subtypes of neurons was directed by the force expression of cell-type specific TFs Lhx8 or Foxa2/Lmx1a.Results:Astrocyte-derived induced NPCs(AiNPCs)share high similarities,including the expression of NPC-specific genes,DNA methylation patterns,the ability to proliferate and differentiate,with the wild type NPCs.The AiNPCs are committed to the forebrain identity and predominantly differentiated into glutamatergic and GABAergic neuronal subtypes.Interestingly,additional overexpression of TFs Lhx8 and Foxa2/Lmx1a in AiNPCs promoted cholinergic and dopaminergic neuronal differentiation,respectively.Conclusions:Our studies suggest that astrocytes can be converted into AiNPCs and lineage-committed AiNPCs can acquire differentiation potential of other lineages through forced expression of specific TFs.Understanding the impact of the TF sets on the reprogramming and differentiation into specific lineages of neurons will provide valuable strategies for astrocyte-based cell therapy in neurodegenerative diseases.
