源于尿道上皮细胞的诱导多能干细胞构建阿尔茨海默病研究模型  

Potential Alzheimer disease model of induced pluripotent stem cells derived from urethral epithelial cells

作　　者：王旎 刘舜杰 孟洋阳 雷清锋[1] 韦睿[1,2] 李中 WANG Ni;LIU Shun-jie;MENG Yang-yang;LEI Qing-feng;WEI Rui;LI Zhong(Department of Neurology,The Sixth Affiliated Hospital,Sun Yat-sen University,Guangzhou 501655,China;Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences,Guangzhou 510530,China;Guangdong Provin-cal Key Laboratory of Brain Function and Disease,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China;Shenzhen Research Institute of Sun Yat-Sen University Hi-tech Industrial Park,Shenzhen,518057,China)

机构地区：[1]中山大学附属第六医院神经科,广东广州510655 [2]中国科学院广州生物医药与健康研究所,广东广州510530 [3]广东省脑功能与脑疾病重点实验室,广东广州510080 [4]中山大学深圳研究院,广东深圳518057

出　　处：《中国病理生理杂志》2022年第3期487-494,共8页Chinese Journal of Pathophysiology

基　　金：广东省自然科学基金(No.2020A1515010111)。

摘　　要：目的:阿尔茨海默病(AD)是常见的神经退行性疾病之一,目前仍未有理想的研究模型,这使得其发病机制尚不完全清楚。因此,本研究将利用非侵袭性方法收集的尿道上皮细胞重编程为诱导多能干细胞(iPSC)从而构建AD疾病研究模型。方法:本研究选取3名男性AD患者作为实验组和2名正常男性作为对照者组,通过分离实验组和对照组的尿道上皮细胞,并将其重编程成为iPSC,利用碱性磷酸酶染色、RT-qPCR及畸胎瘤形成实验等验证其干细胞特性。随后将iPSC定向神经分化成为神经细胞,采用ELISA方法验证AD特异性标志物的表达水平。同时,将BACE1基因在iPSC细胞系中过表达,采用ELISA方法验证AD特异性标志物的表达水平,以上实验每组研究对象均进行2~3次重复实验。结果:尿道上皮细胞被成功地重编程成iPSC,且具有分化为三种不同胚层来源组织的多能性。定向分化患者来源的神经细胞分泌AD标志物的水平与正常对照比较没有显著差异(P>0.05)。但是,过表达BACE1后的iPSC细胞系和分化的神经细胞,AD相关标志物表达水平较对照组显著增高(P<0.05),说明该细胞系作为AD模型的可行性。结论:本研究成功地将AD患者的尿道上皮细胞重编程为iPSC,并将其与正常来源的iPSC进行比较,观察到患者来源的iPSC具有相对正常的蛋白表达谱,提示该细胞的临床应用价值。同时,过表达BACE1基因的iPSC能检测到高表达的AD相关标志物,提示该细胞可以作为研究AD发病机制的细胞模型。AIM:Alzheimer disease(AD)is the most common neurodegenerative disorder.There is currently no effective treatment for AD,which may be attributed in part to lack of effective AD models.A non-invasive method to generate induced pluripotent stem cells(iPSC)from urethral epithelial was used in current study.METHODS:Urethral epithelial cells from three male AD patients and two male normal subjects were reprogrammed into iPSC.iPSC are tested by alkaline phosphatase(AP)staining,RT-q PCR,and teratoma.Then the iPSC was induced into neural cells.ELISA was used to test AD-associated markers secreted by neural cells.Meanwhile,BACE1 gene was over-expressed in iPSC and AD-associated marker was examined by ELISA.RESULTS:The urethral epithelial cells were successfully reprogrammed into iPSC,which can differentiate into tissues derived from three different germ layers.There is no significance in secreted AD-associated markers between neural cells and iPSC(P>0.05).However,over-expression of BACE1 in iPSC and its neural differentiation cells expressed higher level of AD-associated marker than controls(P<0.05).CONCLUSION:Urethral epithelial cells from AD patient urine samples are reprogrammed into iPSCs.Over-expression of BACE1 gene in iPSC stimulate the expression of AD-related markers,suggesting it is an ideal disease model for AD.

关 键 词：阿尔茨海默病 尿道上皮细胞 诱导多能干细胞 神经分化 BACE1基因 

分 类 号：R363.2[医药卫生—病理学] R741.02[医药卫生—基础医学]