人胚胎干细胞向肺动脉平滑肌样细胞分化的研究  

Differentiation of Human Embryonic Stem Cells into Pulmonary Artery Smooth Muscle-Like Cells in Vitro

作　　者：黄兹锐 李晓红[2] 陈景[2] 邱敏 何标川 欧艳秋[3] 周成斌[2] 庄建[2] 陈寄梅 HUANG Zi-rui;LI Xiao-hong;CHEN Jing;QIU Min;HE Biao-chuan;OU Yan-qiu;ZHOU Cheng-bin;ZHUANG Jian;CHEN Ji-mei(School of Medicine,South China University of Technology,Guangzhou 510641,China;Guangdong Provincial Key Laboratory of South China Structural Heart Disease,Guangdong Cardiovascular Institute,Guangdong Provincial People's Hospital,Guangzhou 510080,China;Epidemiology Department,Guangdong Cardiovascular Institute,Guangdong Provincial People's Hospital,Guangzhou 510080,China)

机构地区：[1]华南理工大学医学院,广州510641 [2]广东省人民医院、广东省心血管病研究所、华南结构性心脏病重点实验室,广州510080 [3]广东省人民医院、广东省心血管病研究所流行病学研究室,广州510080

出　　处：《循证医学》2020年第2期108-114,共7页The Journal of Evidence-Based Medicine

基　　金：国家重点研发计划资助项目(2018YFC1002600);广东省自然科学基金资助项目(2018A0303130266,2018A030313329);广州市科技计划资助项目(201704020126)。

摘　　要：目的肺动脉狭窄是一种常见的先天性心脏病亚型,采用生物材料扩张肺动脉是目前治疗手段之一。含有细胞的生物材料治疗效果最佳,但制备生物材料的肺动脉平滑肌细胞来源是目前亟待解决的问题。本研究采用化学因子诱导方法,探索人胚胎干细胞(human embryonic stem cell,hESC)向肺动脉平滑肌细胞分化的能力。方法常规培养hESC细胞系H9,当细胞融合度为10%时,分别加入侧板中胚层(lateral mesoderm,LM)和神经外胚层(neural ectoderm,NE)的定向分化诱导剂。通过real-time PCR、Western blot、细胞免疫荧光检测对这些诱导分化而来的细胞进行鉴定并检测其向血管平滑肌细胞(vascular smooth muscle cell,VSMC)分化的能力。结果 H9细胞呈克隆样生长,SOX2、OCT4、NANOG均为阳性表达。经诱导分化后,与对照组相比,ISL-1、NKX2.5、KDR(LM的标志基因),Nesting、PAX6、GBX2(NE细胞的标志基因)表达上调,而hESC的标志基因SOX-2、OCT-4和NANOG表达下调。这两种细胞能进一步分化成α-SMA、CNN1、MYH11、TAGLN、SMTN-A表达阳性的VSMC。结论特定的分化条件能诱导hESC向肺动脉平滑肌样细胞分化。Objective Pulmonary stenosis(PS)is one of the most common subtypes of congenital heart disease.Using bioengineered tissue materials to expand the pulmonary artery is one of the current treatments for PS.Cell-coating biomaterials have the best therapeutic effect,but it􀆳s a critical problem that the source of pulmonary artery smooth muscle cells for bioengineering tissue is rare.In this study,chemical factor induction methods were used to explore the ability of human embryonic stem cells(hESC)differentiating into pulmonary artery smooth muscle cells.Methods hESC line H9 was culture and changed to lateral mesoderm(LM)and neural ectoderm(NE)inducing medium respectively when the cell confluent reached to 10%.Real-time PCR,Western blot,and cellular immunofluorescence were used to detect the ability of H9 to differentiate into vascular smooth muscle cells(VSMC).Results H9 cells grew in a clone-like manner,SOX2,OCT4,and NANOG were all positively expressed.The expressions of ISL-1,NKX2.5,KDR(marker genes of LM),Nesting,PAX6,GBX2(marker genes of NE cells)were up-regulated while the hESC marker gene SOX-2,OCT-4 expressions were down-regulated after induction compared with control group.These two type of cells can further differentiate into VSMCs withα-SMA,CNN1,MYH11,TAGLN,and SMTN-A positive.Conclusion hESC can differentiate into pulmonary artery smooth muscle like cells under specific inducing condition.

关 键 词：胚胎干细胞 侧板中胚层 神经外胚层 平滑肌细胞 干细胞分化 

分 类 号：R321-3[医药卫生—人体解剖和组织胚胎学]