小分子组合诱导大鼠胚胎成纤维细胞重编程为功能性神经元

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中文摘要：目的:建立通过小分子化合物将大鼠胚胎成纤维细胞(REF)重编程为化学诱导神经元(ciNC)的方法体系，为细胞移植治疗神经退行性疾病提供安全有效的供体细胞。方法:以裴钢研究团队建立的人成纤维细胞直接重编程为神经元的方法为基础，通过更换包被液、缓解氧化应激损伤、添加神经源性保护因子、调整毛喉素浓度和小分子去除实验对诱导培养基和成熟培养基进行优化，并通过免疫荧光法和蛋白质印迹法验证不同诱导阶段细胞的相关蛋白质。结果:以原方案进行诱导时，细胞存活率仅(34。24±2。77)%。包被液更换为基质胶后，细胞存活率上升到(45。41±4。27)%;添加褪黑素后细胞存活率提高至(67。95±5。61)%，(23。43±1。42)%的细胞转变为神经样细胞;添加小分子P7C3-A20后细胞存活率进一步提升至(76。27±1。41)%，(39。72±4。75)%的细胞转变为神经样细胞;毛喉素浓度提升至30 μmol/L时，神经样细胞比例达到(55。79±1。90)%;去除SP600125后，(86。96±2。15)%细胞存活，神经样细胞生成率提高至(63。43±1。60)%。以优化后的方案诱导，可经过神经前体细胞将REF诱导为ciNC。其中，神经前体细胞能够高表达神经前体细胞标志物SRY-box转录因子2、配对盒6以及神经元特异性标志物Ⅲ类β-微管蛋白，而成纤维相关蛋白波形蛋白表达量减少。神经前体细胞在成熟培养基中诱导两周后，可见大部分细胞呈神经样细胞形态，诱导后的ciNC高表达Ⅲ类β-微管蛋白和微管相关蛋白2，而成纤维相关蛋白波形蛋白表达减少。结论:优化后的小分子组合在常氧条件下能将REF重编程为ciNC。

外文标题：Experimental study on small molecule combinations inducing reprogramming of rat fibroblasts into functional neurons

外文摘要：Objective:To establish a methodological system for reprogramming rat embryonic fibroblasts(REF)into chemically induced neurons(ciNCs)via small molecule compounds to provide safe and effective donor cells for treatment of neurodegenerative diseases.Methods:Based on the method established by PEI Gang's research group to directly reprogram human fibroblasts into neurons,the induction medium and maturation medium was optimized by replacing the coating solution,mitigating oxidative stress injury,adding neurogenic protective factors,adjusting the concentration of trichothecenes,performing small-molecule removal experiments,and carrying out immunofluorescence and Western blotting on cells at different stages of induction to validate the effect of induction.Results:When the original protocol was used for induction,the cell survival rate was(34.24±2.77)%.After replacing the coating solution gelatin with matrigel,the cell survival rate increased to(45.41±4.27)%;after adding melatonin,the cell survival rate increased to(67.95±5.61)%and(23.43±1.42)%were transformed into neural-like cells;after adding the small molecule P7C3-A20,the cell survival rate was further increased to(76.27±1.41)%,and(39.72±4.75)%of the cells were transformed into neural-like cells.When the concentration of trichothecene was increased to 30 μmol/L,the proportion of neural-like cells reached(55.79±1.90)%;after the removal of SP600125,(86.96±2.15)%of the cells survived,and the rate of neural-like cell production increased to(63.43±1.60)%.With the optimized protocol,REF could be successfully induced into ciNC through the neural precursor cell stage,in which the neural precursor cells were able to highly express the neural precursor cell markers SRY-related HMG-box gene 2(Sox2)and paired box 6(Pax6)as well as neuron-specific marker tubulin 1(Tuj1),while the expression of fiber-associated protein vimentin was reduced.After two weeks of induction of neural precursor cells in a maturation medium,most cells displayed neuronal-like cell morphology.The induced ciNCs were able to highly express the mature neuronal surface markers Tuj1 and microtubule-associated protein 2(MAP2),while the expression of vimentin was reduced.Conclusion:The small molecule combinations optimized in this study can reprogram REF to ciNCs under normoxic conditions.

外文关键词：

Somatic reprogrammingSmall molecule compoundEmbryo fibroblastChemically induced neuronsRats

作者：

高群伟、代振佳、杨新康、刘长青、刘高峰

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作者单位：

蚌埠医科大学生命科学学院,安徽蚌埠 233030

蚌埠医科大学安徽省神经再生技术与医用新材料工程研究中心,安徽蚌埠 233030

关键词：

体细胞重编程小分子化合物胚胎成纤维细胞化学诱导神经元大鼠

基金：

国家自然科学基金国家自然科学基金安徽省重点研究与开发计划安徽省重点研究与开发计划安徽省自然科学基金蚌埠医学院重大科技项目孵育计划国家级大学生创新创业训练项目国家级大学生创新创业训练项目安徽省大学生创新创业项目蚌埠医学院研究生科研创新计划

项目编号：

82371382817713812022e070200302022e0702 00322308085MH2562021byfy002202210367019202310367005S202210367144Byycx22003

出版年：

2024

DOI：

10.3724/zdxbyxb-2024-0007

浙江大学学报(医学版)

浙江大学

浙江大学学报(医学版)

CSTPCD北大核心

影响因子：0.926

ISSN：1008-9292

年,卷(期)：2024.53(4)