HLA-B2704基因型强直性脊柱炎患者来源的人诱导多能干细胞体系的建立  

Establishment of human-induced pluripotent stem cells system originated from patients with HLA-B2704 genotypic ankylosing spondylitis

作　　者：黄小龙[1] 全仁夫[1] 胡华辉[1] 李伟[1] 李强[1] 邱锐 孙亚萍[3] 杨迪生[4] HUANG Xiaolong;QUAN Renfu;HU Huahui;LI Wei;LI Qiang;QIU Rui;SUN Yaping;YANG Disheng(Xiaoshan Hospital of Chinese Medicine,Hangzhou 311201,Zhejiang,China;Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China;Zhejiang University,Hangzhou 310058,Zhejiang,China;The Second Affiliated Hospital of Zhejiang University,Hangzhou 310009,Zhejiang,China)

机构地区：[1]浙江省杭州市萧山区中医院,浙江杭州311201 [2]浙江中医药大学,浙江杭州310053 [3]浙江大学,浙江杭州310058 [4]浙江大学附属第二医院,浙江杭州310009

出　　处：《中医正骨》2017年第12期11-18,共8页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：浙江省科技计划项目(2014C03031)

摘　　要：目的:建立人类白细胞抗原(human leukocyte antigen,HLA)-B2704基因型强直性脊柱炎(ankylosing spondylitis,AS)患者来源的人诱导多能干细胞(human-induced pluripotent stem cells,hiPSCs)体系。方法:采用逆转录病毒介导的感染系统把OCT4、SOX2、c-MYC、KLF4这4个外源转录因子导入HLA-B2704基因型AS患者的尿液细胞中,重编程获得hiPSCs。通过形态观察、碱性磷酸酶染色、免疫荧光染色、内源多能性相关基因检测、体外拟胚体(embryoid bodies,EBs)分化检测、EBs三胚层基因检测以及畸胎瘤形成实验,鉴定获得的hiPSCs。结果:病毒感染尿液细胞后第4天至第5天细胞核质比增大,第14天左右出现克隆,第24天后形成与人胚胎干细胞(human embryonic stem cells,h ESCs)相似的克隆。碱性磷酸酶染色显示,hiPSCs和h ESCs-H1一样,均被染成紫红色,呈阳性。免疫荧光染色显示,h ESCs阶段特异性胚胎抗原4及ESCs转录因子OCT4、SOX2均呈阳性。hiPSCs内源多能性基因OCT4、SOX2、NANOG和REX1的表达与H1内源多能性相关基因的表达比较,差异均无统计学意义(1.006±0.208,1.000±0.340,t=0.038,P=0.972;1.061±0.140,1.000±0.387,t=0.431,P=0.689;1.386±0.354,1.000±0.032,t=1.467,P=0.303;1.280±0.283,1.000±0.013,t=1.398,P=0.235)。EBs高表达内胚层基因AFP、GATA4,中胚层基因MEX1、TBX1,外胚层基因PAX6、SOX1;EBs三胚层基因AFP、GATA4、TBX1、MSX1、PAX6、SOX1的相对表达量均高于hiPSCs(60.695±8.746,1.000±0.245,t=9.647,P=0.011;3.724±0.144,1.000±0.417,t=12.136,P=0.007;4.224±0.869,1.000±0.130,t=4.988,P=0.038;684.800±63.326,1.000±0.211,t=15.270,P=0.004;131.561±15.785,1.000±0.232,t=11.700,P=0.007;98.507±40.443,1.000±0.215,t=16.000,P=0.004)。hiPSC体内形成畸胎瘤,HE染色可见内、中、外3个胚层的组织细胞;其中内胚层为小肠上皮组织细胞,中胚层为肌肉组织细胞,外胚层为神经上皮组织细胞。结论:采用逆转录病毒介导的感染系统把OCT4、SOX2、c-MYC、KLF4这4个�Objective:To establish the human-induced pluripotent stem cells(hiPSCs)system originated from patients with human leukocyte antigen(HLA)-B2704 genotypic ankylosing spondylitis(AS).Methods:Four exogenous transcription factors including OCT4,SOX2,c-MYC and KLF4 were introduced into the urinary cells(UCs)of patients with HLA-B2704 genotypic AS by using retrovirus-mediated infection system,and the infected UCs were reprogrammed for obtaining hiPSCs.The hiPSCs were identified through morphologic observation,alkaline phosphatase staining,immunofluorescence staining,endogenous multipotent related gene detection,embryoid bodies(EBs)differentiation detection,EBs triploblastic gene detection and teratoma formation experiment.Results:The nuclear-cytoplasmic ratio increased at the 4th and 5th day after UCs were infected by virus.The clone appeared at the 14th day and it was similar to human embryonic stem cells(hESCs)at the 24th day.The result of alkaline phosphatase staining showed that both hiPSCs and hESCs-H1 were presented with prunosus positive staining.The result of immunofluorescent staining showed that the stage specific embryonic antigen 4(SSEA4)of hESCs and the transcription factor of ESCs including OCT4 and SOX2 presented with positive staining.There was no statistical difference in the expression of endogenous multipotent related gene including OCT4,SOX2,NANOG and REX1 between hiPSCs and hiPSCs-H1(1.006+/-0.208 vs 1.000+/-0.340,t=0.038,P=0.972;1.061+/-0.140 vs 1.000+/-0.387,t=0.431,P=0.689;1.386+/-0.354,1.000+/-0.032,t=1.467,P=0.303;1.280+/-0.283 vs 1.000+/-0.013,t=1.398,P=0.235).High expressions of endodermal genes including AFP and GATA4,mesodermal genes including MEX1 and TBX1 and ectodermal genes including PAX6 and SOX1 were found in EBs.The relative expression levels of triploblastic genes including AFP,GATA4,TBX1,MSX1,PAX6 and SOX1 were higher in EBs compared to hiPSCs(60.695+/-8.746 vs 1.000+/-0.245,t=9.647,P=0.011;3.724+/-0.144 vs 1.000+/-0.417,t=12.136,P=0.007;4.224+/-0.869 vs 1.000+/-0.130,t=4.988,P=0.0

关 键 词：脊柱炎 强直性 HLA抗原 尿 诱导多能干细胞 

分 类 号：R593.23[医药卫生—内科学]