尿液细胞来源iPSCs-NSCs联合3D打印支架移植修复大鼠急性脊髓损伤的研究  

Three-dimensional scaffolds seeded with iPSCs-NSCs derived from urine cell for repair of acute spinal cord injury in rats

作　　者：李长明[1] 邵荣学 邓小梅 赵士杰[1] 王拓[1] 全仁夫[1] LI Changming;SHAO Rongxue;DENG Xiaomei;ZHAO Shijie;WANG Tuo;QUAN Renfu(Xiaoshan Hospital of Traditional Chinese Medicine(Jiangnan Hospital Affiliated to Zhejiang Chinese Medical University),Hangzhou311201,China)

机构地区：[1]杭州市萧山区中医院(浙江中医药大学附属江南医院)脊柱外科,311201

出　　处：《浙江医学》2021年第3期238-243,I0003,共7页Zhejiang Medical Journal

基　　金：浙江省医药卫生科技计划项目(2018248755);萧山区科技重大专项项目(2017209)。

摘　　要：目的探讨尿液细胞(UC)来源重编程诱导性多能干细胞(iPSCs)分化的神经干细胞(NSCs)联合3D打印支架移植修复大鼠急性脊髓损伤(ASCI)的作用。方法将UC诱导分化为iPSCs,通过ALP活性检测、HE染色观察畸胎瘤形成及免疫荧光检测全能性蛋白OCT4、NANOG、TRA-1-81、TRA-1-60的表达验证iPSCs全能性。将iPSCs向NSCs分化,免疫荧光染色检测巢蛋白(Nestin)、神经胶质原纤维酸性蛋白(GFAP)和β-微管蛋白(β-tubulinⅢ)的表达。制备3D打印支架。选择成年雄性SD大鼠42只,按随机数字表法分为模型组、支架模型组、iPSCs-NSCs组,每组14只。采用改良Allens法建立大鼠ASCI模型,1周后模型组于损伤处滴入DMEM培养液0.2 ml,支架模型组于损伤处植入载DMEM培养液的2~3 mm支架,iPSCs-NSCs组于损伤处植入载iPSCs-NSCs的2~3 mm支架。1、2、4、6、8周后采用开放领域运动测试(BBB)评分评价3组大鼠关节协调功能。8周后采用Tarlov和Rivlin评分评价3组大鼠后肢运动功能,生物信号采集处理系统观察大鼠运动、感觉诱发电位潜伏期和振幅;取伤段脊髓组织做病理检查,观察脊髓组织病理改变。结果成功提取UC并诱导分化为iPSCs,ALP染色呈阳性,HE染色显示畸胎瘤形成;免疫荧光染色显示全能性蛋白OCT4、NANOG、TRA-1-81、TRA-1-60表达。成功诱导iPSCs分化为NSCs,GFAP和β-tubulinⅢ表达良好表明细胞分化能力良好。成功打印3D支架,电镜显示内部呈三维立体疏松多孔结构。干预2、4、6、8周后,iPSCs-NSCs组大鼠BBB评分均高于模型组和支架模型组(均P<0.05);干预8周后,iPSCs-NSCs组大鼠Tarlov和Rivlin评分均高于模型组和支架模型组,运动和感觉诱发电位潜伏期均短于模型组和支架模型组,运动和感觉诱发电位振幅均大于模型组和支架模型组(均P<0.05)。HE染色显示模型组大鼠脊髓组织受损,组织水肿明显,细胞稀疏;支架模型组大鼠脊髓组织受损程度轻于模型组,组�Objective To investigate the efficacy of three-dimensional scaffolds seeded with induced pluripotent stem cells(iPSCs)derived from urine cell(UC)for repair of acute spinal cord injury(ASCI)in rats.Methods A new three-dimensional bio-printer was used to make bionic spinal cord scaffolds.The UC was induced to differentiate into iPSCs,the pluripotency of iPSCs was confirmed by phosphatase(ALP)activity and teratoma formation,immunofluorescence was used to detect the expression of totipotent proteins OCT4,NANOG,TRA-1-81 and TRA-1-60.The iPSCs were differentiated into neural stem cells(NSCs)and the expressions of Nestin,GFAP andβ-tubulinⅢproteins were detected by immunofluorescence.The three-dimensional scaffolds were prepared.Forty-two healthy male SD rats were randomly divided into model group,control+scaffold group,and iPSCs-NSCs group with 14 rats per group.ASCI in rats was induced using the electric controlled cortical impactor according to modified Allens method.A week later,0.2 ml of DMEM culture medium was dripped into the injured area in the model group,2-3 mm scaffold containing DMEM culture medium was implanted in the injured area in the control+scaffold group,and 2-3 mm scaffold containing iPSCs-NSCs was implanted in the iPSCs-NSCs group.The coordinated motor function was evaluated with BBB score and the motor function of hind limbs was evaluated with Tarlov and Rivlin scores at postoperative 1,2,4,6 and 8 weeks.The changes of latency and amplitude of motor and sensory evoked potentials were observed by biological signal acquisition and processing system after 8 weeks.HE staining was used to observe the changes of the injured spinal cord.Results UC was successfully induced to differentiate into iPSCs,the alkaline phosphatase staining was positive and HE staining showed teratoma formation,immunofluorescence showed the expression of totipotent proteins OCT4,NANOG,TRA-1-81 and TRA-1-60.The iPSCs were successfully induced to differentiate into NSCs with the expression of GFAP andβ-tubulinⅢ.The three-dimen

关 键 词：急性脊髓损伤 诱导性多能干细胞 神经干细胞 尿夜细胞 3D打印支架 

分 类 号：R651.2[医药卫生—外科学]