3D打印支架联合iPSCs-NSCs对大鼠脊髓损伤后神经细胞凋亡及运动功能的影响  

Effect of 3D bioprinting scaffolds with induced pluripotent stem cell-neural stem cells(iPSCs-NSCs)on neuronal apoptosis and locomotor functional recovery after spinal cord injury in rats

作　　者：李长明[1] 邓小梅[1] 周化腾 全仁夫[1] LI Chang-ming;DENG Xiao-mei;ZHOU Hua-teng;QUAN Ren-fu(Spinal Surgery,Jiangnan Hospital Affiliated to Zhejiang University of Traditional Chinese Medicine(Xiaoshan Hospital of Traditional Chinese Medicine of Hangzhou),Hangzhou,Zhejiang province,311200,China)

机构地区：[1]浙江中医药大学附属江南医院(杭州市萧山区中医院)脊柱外科,杭州311200

出　　处：《浙江中西医结合杂志》2021年第6期504-508,514,共6页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省医药卫生科技计划项目(No.2018248755)。

摘　　要：目的探讨3D打印支架载重编程诱导性多能干细胞(iPSCs)-神经干细胞(NSCs)移植对大鼠脊髓损伤的作用机制。方法制备3D脊髓支架,将人尿液细胞来源iPSCs分化为NSCs,选择成年雄性SD大鼠42只,按随机数字表法分为假手术组、模型组、iPSCs-NSCs组,每组14只。采用改良Allen’s重物打击法制作脊髓损伤大鼠模型,1周后假手术组不做处理,模型组植入DMEM培养液0.2mL,i PSCs-NSCs组植入载iPSCs-NSCs的2~3mm支架预处理。于实验干预后1、3、7、14、28天采用BBB评分评估大鼠运动能力,28天后处死大鼠,苏木精-伊红(HE)染色观察伤段脊髓组织改变;免疫组化与RT-PCR检测脊髓组织中Bax、Bcl-2、Caspase-3的表达。结果(1)BBB评分:术后7、14、28天iPSCs-NSCs组与模型组下肢功能均有恢复(P<0.05),且iPSCs-NSCs组较模型组恢复更明显[(3.76±0.65)分比(2.22±0.46)分,(7.02±0.68)分比(4.41±0.42)分,(11.72±0.81)分比(7.52±0.53)分,P<0.05]。(2)HE染色:假手术组脊神经生长良好;模型组脊神经受损,组织水肿,细胞稀疏,细胞之间的间隙增大;iPSCs-NSCs组细胞生长较模型组紧密,各组织生长间隙缩小,空泡减小,组织水肿消失。(3)免疫组化检测:iPSCs-NSCs组Bcl-2阳性细胞的表达较模型组增加[(0.32±0.02)个/mm^(2)比(0.14±0.01)个/mm^(2),P<0.05],Bax阳性细胞的表达较模型组减少[(0.08±0.00)个/mm^(2)比(0.23±0.02)个/mm^(2),P<0.05];(4)RT-PCR检测:iPSCs-NSCs组Caspase-3 mRNA表达较模型组减少[(0.96±0.07)比(1.33±0.03),P<0.05]。结论3D打印支架联合iPSCs-NSCs移植可以显著改善脊髓损伤引起的运动功能下降,其机制可能与上调Bcl-2表达及下调Bax、Caspase-3表达,降低脊髓神经元细胞凋亡相关。Objective To investigate the mechanism of three-dimensional(3D)bioprinting scaffolds with induced pluripotent stem cells-neural stem cells(i PSCs-NSCs)for spinal cord injury repair in rats.Methods The 3D scaffolds were prepared.i PSCs derived from urine cells were differentiated into NSCs.Forty-two healthy male SD rats were randomly divided into sham,model,and iPSCs-NSCs groups(n=14 per group).Spinal cord injury in rats was induced using the electric controlled cortical impactor according to modified Allen’s method.A week later,different materials were implanted into the injured rats:none into the sham group,0.2 mL of DMEM medium into the model group,and a 3D scaffold carrying i PSCs-NSCs into the iPSCs-NSCs group.At postoperative 1,3,7,14,and 28 days,neurological motor function of rats in each group was evaluated by BBB score.On Day 28,all experimental rats were euthanized.Changes in the injured spinal cord were examined by HE staining.Apoptotic biomarkers including Bax,Bcl-2,and Caspase-3 were detected by immunofluorescence staining and reverse transcriptionpolymerase chain reaction.Results Rats in the iPSCs-NSCs and model groups showed improved locomotor function on the postoperative day 7,14 and 28(P<0.05).Moreover,BBB score of rats in the iPSCs-NSCs group were significantly higher than that in the model group[D7:(3.76±0.65)vs(2.22±0.46);D14:(7.02±0.68)vs(4.41±0.42);D28:(11.72±0.81)vs(7.52±0.53);all P<0.05].HE staining showed that the spinal cord nerves grew well in the sham group(no injury),while the spinal cord nerves were injured in the model group,displaying obvious edema and sparse cells.The cells in the iPSCs-NSCs group were tighter than that in the model group,with reduced growth gap and vacuoles in the nerve tissue and disappeared tissue edema.Compared to the model group,tissue from the iPSCs-NSCs group showed an increased protein level of Bcl-2[(0.32±0.02)vs(0.14±0.01),P<0.05]and a reduced protein level of Bax[(0.08±0.00)vs(0.23±0.02),P<0.05],as well as a reduced mRNA level of caspase-3[(

关 键 词：大鼠 脊髓损伤 诱导性多能干细胞 神经干细胞 仿生支架 神经元细胞凋亡 

分 类 号：R651.2[医药卫生—外科学]