自身免疫调节因子在小鼠胚胎干细胞向胸腺上皮祖细胞分化中的表达

文题释义：自身免疫调节因子：基因分析显示由于单基因突变引起一种自身免疫病，此基因则被命名为自身免疫调节因子即AIRE基因。AIRE基因的突变或者缺失会造成胸腺内自身组织特异性抗原转录缺失，影响阴性选择，从而致使自身反应性T细胞逃逸外周，引起自身免疫反应。AIRE基因一直是免疫学相关研究中的热点。胸腺上皮细胞：胸腺上皮细胞分为髓质胸腺上皮细胞和皮质胸腺上皮细胞，二者均来源于胸腺上皮祖细胞，据研究报道髓质胸腺上皮细胞表达的AIRE基因调控着胸腺内的阴性选择，但是由于胸腺上皮祖细胞和胸腺上皮细胞不易分离且数量少，其应用和研究一直受限。该实验在体外将胚胎干细胞分化为胸腺上皮祖细胞，可以为相关研究提供细胞来源。  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。ORCID: 0000-0001-5253-3085(胡蓉) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Expression of autoimmune regulator during differentiation of mouse embryonic stem cells into thymic epithelial progenitor cells

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.