肺芽类器官中ID2的表达促进肺血管形成  

Expression of ID2 in lung bud organoids promotes pulmonary vascular formation

作　　者：刘洪宪 杨隽 LIU Hong-xian;YANG Jun(State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005;Department of Physiology, School of Basic Medical Sciences,Zhejiang University School of Medicine, Hangzhou 310058, China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院,医学分子生物学国家重点实验室,北京100005 [2]浙江大学医学院基础医学院生理学系,浙江杭州310058

出　　处：《基础医学与临床》2021年第5期623-629,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(81870051);国家重点研发计划项目(2016YFA0102300)。

摘　　要：目的研究在肺早期发育中,肺芽中DNA结合抑制因子-2(ID2)对肺血管形成的作用。方法利用CRISPR/Cas9系统对人胚胎干细胞(hESCs)进行基因编辑,构建ID2敲除载体,将正常hESCs和ID2敲除株定向分化为内皮细胞(ECs)和肺芽类器官(LBOs);应用荧光定量PCR检测分化过程中ID2、CD31和NKX2.1等基因表达,流式细胞测量术分析分化效率;蛋白质免疫印迹检测ID2、CD31和PAX9等蛋白表达水平。通过ECs与LBOs的3D共培养,比较不同来源的LBOs对ECs成管功能的影响。结果ID2在hESCs向ECs分化过程中能够促进细胞进入中胚层阶段;而ID2缺失则导致大量成管功能缺陷ECs的形成;ID2可促进hESCs向LBOs分化;LBOs能够促进肺血管内皮细胞的成管功能,而敲除ID2使这一促进作用减弱。结论在肺发育早期,ID2的表达影响ECs和LBOs的分化效率和功能,肺芽中的ID2的缺失会导致其周围的肺血管生成减少。Objective To investigate the role of inhibitor of DNA binding-2(ID2)in pulmonary vasculature formation in early lung development.Methods CRISPR/Cas9 system was used to edit ID2 in human embryonic stem cells(hESCs)to construct ID2 knockout strains,hESCs and ID2 knockout strains were further differentiated into endothelial cells(ECs)or lung bud organoids(LBOs).Quantitative PCR was used to detect ID2,CD31,NKX2.1 and other genes expression during differentiation,flow cytometry was performed to analyze the differentiation efficiency,Western blot was applied to check ID2,CD31 and PAX9 protein expression,3D co-culture of ECs and LBOs was performed to compare the effect of LBOs derived from different hESCs on the tubular formation of ECs.ResultsID2 promoted cells to enter the mesoderm stage during the differentiation of hESCs into ECs,but ID2 deletion led to the formation of a large number of ECs with tubular defects.ID2 promoted the differentiation of hESCs into LBOs.In the 3D co-culture system,LBOs could promote ECs tube-forming ability,while the deficiency of ID2 weakened this promotion.Conclusions In early stage of pulmonary tissue development,ID2 affects the differenti-ation and function of ECs and LBOs,loss of ID2 in lung bud reduces the angiogenesis of surrounded ECs.

关 键 词：DNA结合抑制因子-2(ID2) 肺芽类器官(LBOs) 内皮细胞(ECs) 

分 类 号：Q28[生物学—细胞生物学]