摘要
背景:自身免疫性疾病主要是由于胸腺内自身组织特异性抗原持续表达缺失而引起强烈免疫应答的一类疾病,而胸腺功能减退和胸腺内组织特异性抗原的不稳定表达会限制治疗效果。胸腺组织主要由胸腺上皮细胞组成,但胸腺内成熟胸腺上皮细胞和胸腺上皮祖细胞有限的数量来源极大限制了相关研究。目的:研究小鼠胚胎干细胞向胸腺上皮祖细胞分化过程中自身免疫调节因子的表达变化。方法:采用两步分化方法定向诱导小鼠胚胎干细胞分化为内胚层再分化为胸腺上皮祖细胞,分别收集定向诱导分化第0,3,13天细胞,采用细胞免疫荧光、流式细胞术、Western blot、Real-Time PCR检测相关基因及蛋白的表达变化。结果与结论:①诱导分化第0天,免疫荧光检测OCT4、SSEA1表达阳性;诱导分化第3天,免疫荧光检测SOX17、FoxA2呈双阳性表达;诱导分化第13天,流式细胞术检测EpCAM1、K5、K8表达阳性;②Real-time PCR检测小鼠胚胎干细胞定向分化过程中PAX1、PAX9、FOXN1、PLET1基因表达逐渐增高;③Real-time PCR检测分化第0,3,13天AIRE基因表达升高,INS2、GAD67基因表达也升高;④Western blot检测分化第0,3,13天AIRE蛋白表达降低,胰岛素蛋白、GAD67蛋白均无表达;⑤结果表明,小鼠胚胎干细胞成功分化为胸腺上皮祖细胞,且分化而来的胸腺上皮祖细胞中AIRE基因表达很高,促进了INS2、GAD67基因的表达,为细胞移植治疗自身免疫性疾病提供评价依据。
BACKGROUND:Autoimmune diseases are a class of diseases that cause a strong immune response to the continuous lack of self-tissue-specific antigens in the thymus.Hypothyroidism and unstable expression of tissue-specific antigens in the thymus can limit the therapeutic effect.The thymus is mainly composed of thymic epithelial cells,but the limited number of mature thymic epithelial cells and thymic epithelial progenitor cells in the thymus has greatly limited related research.OBJECTIVE:To detect the expression of autoimmune regulator(AIRE)when mouse embryonic stem cells were transformed into thymic epithelial progenitor cells.METHODS:A two-step differentiation method was used to induce the differentiation of mouse embryonic stem cells into endoderm and then into thymic epithelial progenitor cells.The cells were collected at 0,3,and 13 days of induced differentiation.Immunofluorescence,flow cytometry,western blot and real-time PCR were used to detect the expression of cell-associated genes and proteins.RESULTS AND CONCLUSION:Positive expression of OCT4 and SSEA1 was detected by immunofluorescence at 0 day of induction.The double positive expression of SOX17 and FoxA2 was measured by immunofluorescence at 3 days of induction.The positive expression of EpCAM,K5 and K8 were analyzed by flow cytometry at 13 days of induction.During the directional differentiation of mouse embryonic stem cells,real-time PCR indicated that the expression of PAX1,PAX9,FOXN1 and PLET1 showed an increasing trend.The expression of AIRE gene increased significantly at 0,3,and 13 days of induction.At the same time,the expression of INS2 gene and GAD67 gene also increased.Western blot assay showed that the expression of AIRE protein gradually decreased at 0,3,and 13 days of induction;however,insulin protein and GAD67 protein were not detected.Overall findings indicate that mouse embryonic stem cells can successfully differentiate into thymic epithelial progenitor cells with highly expressed AIRE gene,which promotes the expression of INS2 and GAD67 genes,and provides an evaluation basis for cell transplantation in the treatment of autoimmune diseases.
作者
马静怡
杨文江
李远迪
黄佑娇
高杰
李红
胡蓉
苏敏
Ma Jingyi;Yang Wenjiang;Li Yuandi;Huang Youjiao;Gao Jie;Li Hong;Hu Rong;Su Min(Department of Histology and Embryology,College of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2020年第25期3994-3999,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81660033),项目负责人:胡蓉
贵州省科技支撑计划(黔科合支撑[2017]2970号),项目负责人:苏敏
贵州省普通高校科技拔尖人才支持计划(黔教合KY字[2017]071),项目负责人:胡蓉
贵州省科技合作计划项目(黔科合LH字[2016]7363),项目负责人:李红。
