利用尿液细胞建立脓毒症脑损伤患者来源的多能干细胞株实验  

Generation of sepsis encephalopathy patient-specific inducible pluripotent stem cells with urine cells

作　　者：邱欣良 潘烨 赵宁[1] 钱克俭[1] 詹以安[1] Qiu Xinliang;Pan Ye;Zhao Ning;Qian Kejian;Zhan Yian(Department of Critical Care Medicine,the First Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi,China)

机构地区：[1]南昌大学第一附属医院重症医学科,330006

出　　处：《中华危重病急救医学》2019年第12期1445-1450,共6页Chinese Critical Care Medicine

基　　金：国家自然科学基金(81660314);江西省自然科学基金(20192BAB205057)。

摘　　要：目的将脓毒症脑损伤(SE)患者中的尿液细胞重组编程为诱导性多能干细胞(iPSC)株,为探讨SE患者的神经细胞损害机制及治疗方法提供特异性的细胞模型.方法分离SE患者晨间尿液细胞进行原代培养后,通过Millipore公司的慢病毒重编程试剂盒(STEMCCA^TM试剂盒)进行慢病毒重编程,把含有OCT4、Klf4、Sox2、c-Myc 4个转录因子组合(OKSM)的慢病毒载体转染至SE患者尿液细胞中,获得SE患者来源的iPSC.使用碱性磷酸酶(AKP)染色和免疫荧光染色、裂解细胞提取后实时荧光定量反转录-聚合酶链反应(RT-qPCR)、畸胎瘤成瘤等方法判定尿液细胞来源iPSC的全能性,抑制转化生长因子β(TGF-β)信号通路,诱导iPSC转化为神经细胞.结果镜下观察iPSC呈扁平椭圆或圆形、分界及边缘清晰、排列紧凑的类人胚胎干细胞样集落群生长,AKP染色呈紫红色,荧光染色显示,干细胞多能性分子标志物OCT4、阶段性特异性胚胎抗原SSEA4、胚胎干细胞标志物TRA1-81呈高度表达;iPSC的胚胎干细胞相关因子NANOG、REX1、OCT4和Sox2的mRNA表达水平显著高于初始尿液细胞的表达量,差异有统计学意义〔NANOG mRNA(2^-ΔΔCt):1.153±0.142比0.126±0.024,t=-10.688;REX1 mRNA(2-ΔΔCt):1.419±0.206比0.103±0.066,t=-14.245;OCT4 mRNA(2^-ΔΔCt):1.233±0.176比0.201±0.022,t=-9.028;Sox2 mRNA(2^-ΔΔCt):1.334±0.119比0.159±0.017,t=-12.653,均P<0.01〕,接种至裸鼠体内形成典型三胚层结构特点的畸胎瘤,证实尿液细胞源性的iPSC具备同样的全能性,并能诱导分化为神经细胞.结论诱导分化出的SE患者尿液细胞来源的iPSC株具有多能干细胞特性,可成功诱导为人体的神经细胞,为SE的发病机制研究和探索临床治疗方法提供了理想的细胞模型.Objective To recombine the induced pluripotent stem cells(iPSC)derived from the urine of septic encephalopathy(SE)patients,and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients.Methods Urine of SE patient was collected,and tubular epithelial cells were isolated and cultured from the urine.iPSC were derived from SE patient by introducing 4 transcription factors OCT4,Klf4,Sox2,c-Myc(OKSM)into patient-specific urine cells by Millipore's Human STEMCCA^TM Constitutive Polycistronic(OKSM)Lentivirus Kit.Colony morphology,alkaline phosphatase(AKP)activity,immunofluorescence staining,quantitative reverse transcription-polymerase chain reaction(RT-qPCR),and differentiation ability were used to identify the pluripetency of these iPSC lines.In addition,neurons were derived from these iPSC by inhibiting transforming growth factor-β(TGF-β)pathway.Results The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell(hES),showed positivity for AKP by histochemical staining,and expressed embryonic stem cell(ESC)marker genes.There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells[NANOG mRNA(2^-ΔΔCt):1.153±0.142 vs.0.126±0.024,t=-10.688;REX1 mRNA(2^-ΔΔCt):1.419±0.206 vs.0.103±0.066,t=-14.245;OCT4 mRNA(2^-ΔΔCt):1.233±0.176 vs.0.201±0.022,t=-9.028;Sox2 mRNA(2^-ΔΔCt):1.334±0.119 vs.0.159±0.017,t=-12.653,all P<0.01].Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers.Furthermore,neurons were successfully induced from SE-iPSC.Conclusion The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons,furthermore,the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.

关 键 词：脓毒症脑损伤 尿液细胞 诱导性多能干细胞 

分 类 号：R45[医药卫生—治疗学]