摘要
目的探讨人胚胎干细胞(hESCs)定向分化为晶状体小体(LB)过程中非经典Wnt信号通路配体Wnt5a对晶状体发育的作用。方法选取人H9胚胎干细胞系,采用mTeSR培养基进行干细胞培养。“三阶段法”诱导hESCs分化为LB。培养至18 d,将细胞分为对照组和Wnt5a处理组,其中Wnt5a处理组培养基中添加500 ng/ml Wnt5a。培养至35 d,在倒置显微镜下观察细胞形态变化和LB的大小并提取对照组和Wnt5a处理组的总RNA进行转录组测序(RNA-Sequence)检测,表达差异大于1.5倍且P≤0.05被定义为差异表达基因(DEGs),进行生物信息学分析。结果培养结束时发现,与对照组相比,Wnt5a处理组细胞形成的LB面积明显变大。转录组测序结果显示,与对照组相比,Wnt5a处理组中478个基因表达下调,201个基因表达上调,其中Wnt5a可使晶状体细胞分化及晶状体特异基因的表达上调。生物信息学分析结果显示,Wnt5a主要参与了细胞外基质(ECM)改变的过程,提示在晶状体细胞分化中,Wnt5a可改变ECM;同时,富集结果还显示经Wnt5a处理,上皮-间充质转化过程相关过程受抑制,提示Wnt5a还可抑制晶状体细胞,特别是晶状体上皮细胞的异常分化;此外,Wnt5a还可影响晶状体细胞骨架重塑。结论Wnt5a可能通过上调MAPK/ERK级联信号调整ECM的结构成分,影响晶状体细胞骨架重塑,从而促进晶状体细胞的分化。
Objective To investigate the mechanism of Wnt5a in lentoid body(LB)induction from human embryonic stem cells(hESCs).Methods A“three-stage”protocol was used for LB differentiation from hESCs in vitro,and Wnt5a level was modified by adding exogenous 500 ng/ml Wnt5a on day 18 as Wnt5a treatment group.Cells of control group and Wnt5a treatment group were collected on day 35.Cells were photographed by using the Zeiss Axio Observer Z1 microscope.Transcriptome sequencing was applied by Illumina.Genes with P value≤0.05 and fold change≥1.5 were identified as differentially expressed genes(DEGs).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were used to determine the biological functions of DEGs.Results Compared with the control group,larger lentoid bodies were obtained in the Wnt5a treatment group.Transcriptome sequencing result showed that 478 genes were down-regulated and 201 genes were up-regulated in the Wnt5a treatment group compared with the control group,and Wnt5a up-regulated both lens cell differentiation and lens specific gene expression.Bioinformatics analysis result showed that most DEGs were involved in extracellular matrix remodeling,suggesting that Wnt5a regulated extracellular matrix remodeling during lens cell differentiation.The enrichment analysis result also showed that epithelial-to-mesenchymal transformation related processes were inhibited after Wnt5a treatment,suggesting that Wnt5a inhibited the abnormal differentiation of lens cells(especially lens epithelial cells)during lens cell differentiation.Wnt5a influenced the processes related to cytoskeleton remodeling.Conclusions Wnt5a may act in lens cells through MAPK/ERK signaling pathways to affect ECM and cell cytoskeletal organization,which provides a new direction for studying lens development.
作者
陈奕嘉
李金燕
欧阳帅
罗莉霞
Chen Yijia;Li Jinyan;Ouyang Shuai;Luo Lixia(State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center,Sun Yat-sen University,Guangzhou 510623,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2020年第10期837-844,共8页
Chinese Journal Of Experimental Ophthalmology
基金
广东省自然科学基金项目(2019A1515011452)。
作者简介
通信作者:罗莉霞,Email:luolixia@gzzoc.com。
