重编程人成纤维细胞为诱导型类小脑颗粒样细胞的体外实验研究  

In vitro experimental study of reprogramming human fibroblasts into induced cerebellar granule cells

作　　者：沈刚 苏作鹏 全勇 徐福林 徐锋[2] SHEN Gang SU Zuo-peng QUAN Yong et al(Department of Neurosurgery, Central Hospital of Minhang District, Shanghai, Shanghai 201100, China)

机构地区：[1]上海市闵行区中心医院神经外科,201100 [2]复旦大学附属华山医院神经外科

出　　处：《临床神经外科杂志》2017年第1期30-34,共5页Journal of Clinical Neurosurgery

基　　金：国家自然科学基金(81471242)

摘　　要：目的在体外直接诱导重编程获取患者成纤维细胞来源的诱导型类小脑颗粒样细胞(hi CGCs),并研究这些细胞的生物学特征。方法筛选与小脑神经细胞发育、分化相关的诱导因子;构建携带有转录因子的表达载体;以标准皮肤细胞培养流程培养出人头皮成纤维细胞,使用慢病毒载体介导Ascl1、Sox2和OCT4三个转录因子在胞内表达,并在培养过程中加入神经营养因子:BMP4,FGF8b和Wnt3a;诱导获得具有小脑颗粒神经元特征的hi CGCs。结果经细胞免疫组织化学和电生理检测,证实该hi CGCs具有类似正常小脑颗粒神经元的生物学和生理学特征。使用Ascl1、Sox2和OCT4三个转录因子及BMP4、FGF8b和Wnt3a三种营养因子可将成人成纤维细胞直接转变成为具有小脑颗粒神经元特征的hi CGCs。结论 Hi CGCs在形态上与小脑颗粒神经元非常相似,能够表达小脑颗粒神经元的特异性蛋白,并具有类似正常小脑颗粒神经元的电生理特性。Objectives Directly reprogram patients＇ fibroblasts into human induced cerebellar granule cells（ hi CGCs） and study the biological characteristics of these cells in vitro.Methods Screening inducing factors associated with cerebellar cell development and differentiation; build expression vector with transcription factors; collect and culture patients＇ skin fibroblasts; use slow virus carrier mediated Ascl1,OCT4 and Sox2 transcription factors in intracellular expression,and add neurotrophic factor： BMP4,FGF8 b and Wnt3 a in the process of culture,get human induced cerebellar granule neurons（ hi CGCs） with characteristics of cerebellar granule neurons）. Results Confirm the hi CGCs with similar normal biology and physiology characteristics of cerebellar granule neurons via cell immunohistochemical and electrophysiological examination. By using three transcription factors Ascl1,OCT4 and Sox2 and three nutrition factors BMP4,FGF8 b and Wnt3 a,adult fibroblasts can be directly transformed into hi CGCs with the characteristics of cerebellar granule neurons. Conclusions Hi CGCs are very similar with cerebellar granule neurons in morphology,can express granular specific proteins and similar normal electrophysiological characteristics of cerebellar granule neurons.

关 键 词：人诱导型类小脑颗粒样神经细胞 再生医学 谱系重编程 

分 类 号：R394.2[医药卫生—医学遗传学] Q421[医药卫生—基础医学]