文章摘要
李真真,覃莲菊,宁 松,崔毓桂,马 翔,刘嘉茵.人胚胎干细胞向下丘脑神经祖细胞的诱导分化受高雄激素影响[J].南京医科大学学报,2020,(9):1252~1262
人胚胎干细胞向下丘脑神经祖细胞的诱导分化受高雄激素影响
Effects of hyperandrogen on human embryonic stem cells induced to differentiate into hypothalamic neural progenitors
投稿时间:2020-05-11  
DOI:10.7655/NYDXBNS20200903
中文关键词: 多囊卵巢综合征  高雄激素  人胚胎干细胞  诱导分化  下丘脑神经祖细胞
英文关键词: polycystic ovary syndrome  hyperandrogen  human embryonic stem cells  induced differentiation  hypothalamic neuronal progenitors
基金项目:国家自然科学基金(81571403,81671447)
作者单位
李真真 南京医科大学第一附属医院临床生殖医学中心生殖医学国家重点实验室江苏 南京 210029 
覃莲菊 南京医科大学第一附属医院临床生殖医学中心生殖医学国家重点实验室江苏 南京 210029 
宁 松 南京医科大学第一附属医院临床生殖医学中心生殖医学国家重点实验室江苏 南京 210029 
崔毓桂 南京医科大学第一附属医院临床生殖医学中心生殖医学国家重点实验室江苏 南京 210029 
马 翔 南京医科大学第一附属医院临床生殖医学中心生殖医学国家重点实验室江苏 南京 210029 
刘嘉茵 南京医科大学第一附属医院临床生殖医学中心生殖医学国家重点实验室江苏 南京 210029 
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中文摘要:
      目的:研究体外诱导人胚胎干细胞(human embryonic stem cell,hESC)向下丘脑神经祖细胞分化,并以此为细胞模型,观察不同浓度雄激素对下丘脑神经祖细胞分化的影响。方法:鉴定hESC细胞株CCRM22,体外诱导CCRM22向下丘脑神经祖细胞分化,对其进行初步的细胞生物学鉴定;在分化培养基中添加1×10-8mol/L、1×10-7mol/L睾酮(以无水乙醇为助溶剂对照),收集不同分化时期细胞,比较分化效率;利用RT?qPCR、流式细胞术、免疫荧光检测等方法鉴定分化细胞,并检测生殖功能调控相关基因的表达。结果:CCRM22分化为下丘脑神经祖细胞的效率超过85%,但睾酮处理降低其分化效率;分化细胞表达神经细胞标志物NESTIN以及下丘脑神经祖细胞特异性标志物NKX2.1,同时表达KISS1和雄激素受体(androgen receptor,AR),且AR的表达水平与睾酮浓度呈正相关。结论:成功诱导hESC分化形成下丘脑神经祖细胞,高浓度睾酮处理抑制hESC向下丘脑神经祖细胞分化,该细胞模型可应用于后续体外研究多囊卵巢综合征女性孕早期高雄激素环境对后代下丘脑神经细胞分化和发育的影响。
英文摘要:
      Objective:In this study,human embryonic stem cells(hESCs) were induced to differentiate into hypothalamic neural progenitor cells in vitro,and the effects of different concentrations of androgen on the differentiation of hypothalamic neural progenitor cells were compared. Methods:The quality of human embryonic stem cell line CCRM22 was identified,CCRM22 was induced to differentiate into hypothalamic neural progenitor cells in vitro,then 1×10-8 mol/L and 1×10-7 mol/L testosterone were added to the differentiation medium(anhydrous ethanol as cosolvent control). The differentiated cells were collected at different stages of differentiation,and the differentiation efficiency was compared. The differentiated cells were identified by RT?qPCR,flow cytometry and immnofluorescence,and the expression of genes related to reproductive function regulation was detected. Results:The differentiation efficiency of CCRM22 into hypothalamic neural progenitor cells was more than 85%,but testosterone treatment decreased its differentiation efficiency;differentiated cells expressed nerve cell marker NESTIN and hypothalamic neural progenitor cell specific marker NKX2.1,expressed both KISS1 and androgen receptor(AR),and the expression of AR was positively correlated with testosterone concentration. Conclusion:Human embryonic stem cells were successfully induced into hypothalamic neural progenitor cells. High concentration of testosterone inhibited the differentiation of hESCs into hypothalamic neural progenitor cells. This cell model can be used to study the effects of hyperandrogenic environment in early pregnancy on hypothalamic nerve cell differentiation and development of offspring in women with polycystic ovary syndrome in vitro.
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