高渗环境调控钙黏蛋白表达并影响拟胚体分化  

Hypertonic environment regulates cadherin expression and affects em bryoid body differentiation

作　　者：许健宜 吴引弟 方丽君 蒋虹婧 孙盱衡 刘青 肖聪 林展翼 XU Jianyi;WU Yindi;FANG Lijun;JIANG Hongjing;SUN Xuheng;LIU Qing;XIAO Cong;LING Zhanyi(School of Medicine,South China University of Technology,Guangzhou 51006,China;Guangdong Provincial People's Hospital(Guangdong Academy of Medical Sciences),Southern Medical University,Guangzhou 510180,China)

机构地区：[1]华南理工大学医学院,广东广州510006 [2]南方医科大学附属广东省人民医院(广东省医学科学院),广东广州510180

出　　处：《中国病理生理杂志》2024年第3期511-520,共10页Chinese Journal of Pathophysiology

基　　金：先进制造科学与技术广东省实验室(季华实验室)科研项目(No.X210111TD210)。

摘　　要：目的:渗透压对拟胚体(embryoid body,EB)分化的确切影响尚不明确,本研究旨在探讨增加渗透压对EB分化的影响,及该过程与钙黏蛋白之间潜在的联系。方法:本研究使用聚乙二醇300来增加培养液的渗透压,在高渗透压和常规培养液下培养EB。实验设计包括对照组、实验组(高渗组)、不同浓度的聚乙二醇300处理组和高渗+抑制剂组,采用Western blot、RT-qPCR、AM/PI染色、CCK-8、免疫细胞化学染色等检测方法,对EB的细胞活力及上皮钙黏蛋白(E-cadherin,CDH1)和神经钙黏蛋白(N-cadherin,CDH2)、三胚层标志物以及多能性标志物的表达进行了分析。结果:高渗不影响EB的细胞活力。在实验组和对照组的EB中,钙黏蛋白CDH1和CDH2的表达显示出显著差异,但无明显的上皮间质转化转变趋势。实验组中CDH2的表达显著下调,并且与渗透压变化呈现明显相关性。此外,在高渗组中,相较于对照组,EB的多能性标志物在第2~5天表达显著提高。同时,高渗组中胚层标志物的表达量明显增加,而外胚层标志物呈现下调趋势。本研究使用SB431542特异性抑制TGF-β信号上调CDH2表达,结果显示高渗组EB的中胚层和外胚层标志物的表达趋势发生了逆转。结论:当渗透压较高时,会促进EB中胚层的分化效率,这一过程可能与渗透压引起的CDH1和CDH2变化相关。AIM:Given the uncertain impact of osmotic pressure on embryoid body(EB)differentiation,this study aimed to investigate the effects of increased osmotic pressure on EB differentiation and explore the potential relation ship between this process and cadherin.METHODS:Polhethylene glycol 300(PEG 300)was used to increase the os motic pressure of the culture medium used for cultivating EBs under both high osmotic pressure and standard culture condi tions.The experimental design included a control group,an experimental group(hypertonic group),groups treated with varying concentrations of PEG 300,and an experimental group treated with an inhibitor.Western blot,RT-qPCR,AM/PI staining,CCK-8,and immunocytochemical staining was used to analyze the cell viability and the expression of CDH1 and CDH2 markers of the three germ layers,and pluripotency markers within the EBs.RESULTS:Hypertonicity did not af fect cell viability.Significant differences were observed in the expression of the cadherin proteins CDH1 and CDH2 in EBs between the experimental and control groups;however,no cleartrend towards an EMT shift was observed.Specifically,CDH2 expression was significantly down-regulated in experimental group,showing a clear correlation with variations in os motic pressure.Moreover,compared with control group,pluripotency markers in the EBs from experimental group exhibited significantly higher expression levels from the 2nd day to the 5th day.A substantial increase in the expression of mesoder mal markers was also observed;however,a downward trend was observed for ectodermal markers in experimental group.Intervention using SB431542,which up-regulates CDH2 expression by affecting TGF-βsignaling,reversed the expression trend of mesodermal and ectodermal markers in experimental group.CONCLUSION:Elevated osmotic pressure appears to enhance the mesodermal differentiation efficiency in EBs,possibly correlating with CDH1 and CDH2 changes induced by osmotic pressure.Therefore,this study emphasizes the significant role of osmotic pressure

关 键 词：渗透压 钙黏蛋白 细胞分化 拟胚体 中胚层 

分 类 号：R321.4[医药卫生—人体解剖和组织胚胎学] Q254[医药卫生—基础医学] R363.2[生物学—细胞生物学]