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李长明.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变化[J].浙江中西医结合杂志,2021,31(6):
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变化
The effect of three-dimensional scaffolds seeded with IPSCs-NSCs apotosis and locomotor recovery in a rat model of spinal cord injury
投稿时间:2020-10-23  修订日期:2021-04-10
DOI:
中文关键词:  脊髓损伤  神经干细胞  仿生支架  尿  神经元细胞凋亡
英文关键词:Spinal cord injuries  Neural stem cells  Bionic scaffold  Urine  Neuronal apoptosis
基金项目:浙江省医药卫生科技计划项目(2018248755)
作者单位E-mail
李长明 杭州市萧山区中医院 lcmmail@126.com 
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中文摘要:
      目的:3D打印载重编程IPSCs-NSCs移植对大鼠脊髓损伤(spinal cord injurys,SCI)的作用及其相关机制。方法:制备3D脊髓支架,将UC细胞来源iPS细胞分化为神经干细胞(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,每组各14只。采用改良 Allen’s 重物打击法制作SCI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,iPSCs-NSCs 组植入载iPSCs-NSCs的2~3mm支架预处理。于实验干预后1天、3天、7天、14天、28天采用 BBB 评分评估大鼠运动能力,28天后处死大鼠取伤段脊髓组织做病理切片,HE染色观察脊髓组织改变;免疫组化与Western-blot检测脊髓组织中Bax、Bcl-2、Caspase3的表达。结果:①BBB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]。②HE染色:假手术组脊神经生长良好;模型组脊神经受到损伤,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)个比(0.14±0.01)个,P<0.05];IPSCs-NSCs组Bax阳性细胞的表达较模型组少[(0.08±0.00)个比(0.23±0.02)个,P<0.05];④RT-PCR检测:IPSCs-NSCs组Caspase-3的mRNA含量较模型组减少[(0.96±0.07)ng/L比(1.33±0.03)ng/L,P<0.05];结论:3D打印支架联合IPSCs-NSCs移植治疗SCI可以显著促进Bcl-2的表达,降低Bax、Caspase3 的表达,改善SCI引起的运动功能下降,其机制可能与上调Bcl-2的表达及下调Bax、Caspase3表达,降低脊髓神经元细胞凋亡相关。
英文摘要:
      Objective To investigate the role of three-dimensional scaffolds seeded with IPSCs-NSCs for repair of spinal cord injury in rats and investigate the underlying mechanism. Methods A new three-dimensional bio-printer was used to make bionic spinal cord scaffolds.IPS cells derived from UC cells were differentiated into neural stem cells (NSCs).Forty-two healthy male SD rats were divided into sham group, control group,IPSCs-NSCs group with 14 rats per group,according to the random number table,14 in each group.Spinal cord injury in rats was induced using the electric controlled cortical impactor a week later.Rat neurological motor function was evaluated with BBB score was evaluated with Tarlov and Rivlin scores at postoperative 1-28 days.At 28 d,all rats were killed, He staining was used to observe the changes of the injured spinal cord.Immunofluorescence staining and RT-PCR were used to detect the expression of apoptotic related proteins expression (Bcl-2, Bax, Caspase 3).Results: Both of the IPSCs-NSCs group and model group had locomotor function recovery on postoperative day 7 ,14 and 28(P<0.05),but the IPSCs-NSCs group had significantly better locomotor function recov- ery than model group did (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 cell growth in iPSCs-NSCs group was more compact than that in model group, and the growth gap and vacuoles of each tissue were reduced, and tissue edema disappeared; Compared with model group,the iPSCs-NSCs group had reduced expression of Bax(0.08±0.00 vs 0.23±0.02,P<0.05),increased expression of Bcl-2(0.32±0.02 vs 0.14±0.01,P<0.05).Compared with model group,the iPSCs-NSCs group had reduced mRNA expression of caspase-3(0.96±0.07 vs 1.33±0.03,P<0.05)Condusion ①Three-dimensional scaffolds seeded with IPSCs-NSCs for repair of spinal cord injury in rats can significantly promote the expression of Bcl-2 and decrease the expression of Bax and caspase-3.②Three-dimensional scaffolds seeded with IPSCs-NSCs for repair of spinal cord injury in rats can improve the motor function,The mechanism may be related to up regulating the expression of Bcl-2, down regulating the expression of Bax and caspase 3, and reducing the apoptosis of spinal cord neurons.
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