背景:脊髓腹侧根性撕脱导致运动神经元大量变性,预后差,费用高。在这项研究中,我们比较了碱性成纤维细胞生长因子2(FGF2)的不同亚型,在稳定转染的人胚胎干细胞(hESC)中过度表达,在运动神经根撕脱和用异源纤维蛋白生物聚合物(HFB)修复后。
方法:在目前的工作中,将生物工程化以过表达FGF2的18、23和31kD同工型的hESC与使用HFB的撕毁根的再植入组合使用。采用GraphPadPrism软件进行单因素或双因素方差分析,其次是Tukey或Dunnett的多重比较测试。显著性设定为*p<0.05、**p<0.01、***p<0.001和****p<0.0001。
结果:对于第一组实验,通过局部施用HFB和植入表达上述FGF2同种型的hESC,对大鼠进行腹根撕脱。运动神经元生存分析,神经胶质反应,和突触覆盖,病变两周后,表明使用过表达31kDFGF2的hESC治疗是最有效的。因此,第二组实验是用这种同工型进行的,因此,腹侧根性撕脱后直接植入脊髓。运动神经元的生存,神经胶质反应,突触覆盖,损伤后2周进行基因表达分析;同时通过步行道测试和vonFrey测试评估功能恢复12周。我们表明hESCs的移植导致显著的神经保护,再加上免疫调节,星形胶质增生的减弱,以及保存获救运动神经元的输入。行为上,31kDFGF2-hESC治疗可增强运动和感觉恢复。
结论:转基因hESCs是一种有效的神经营养因子传递平台,在近端脊髓根损伤后挽救轴突切除的运动神经元并调节神经胶质反应,而FGF2的31kD同种型除了具有显着的功能恢复外,还显示出比其他同种型优越的再生特性。
BACKGROUND: Spinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study, we compared different isoforms of basic fibroblast growth factor 2 (FGF2), overexpressed in stably transfected Human embryonic stem cells (hESCs), following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB).
METHODS: In the present work, hESCs bioengineered to overexpress 18, 23, and 31 kD isoforms of FGF2, were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA, followed by Tukey\'s or Dunnett\'s multiple comparison tests. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
RESULTS: For the first set of experiments, rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival, glial reaction, and synaptic coverage, two weeks after the lesion, indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently, the second set of experiments was performed with that isoform, so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival, glial reaction, synaptic coverage, and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection, coupled with immunomodulation, attenuation of astrogliosis, and preservation of inputs to the rescued
motoneurons. Behaviorally, the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery.
CONCLUSIONS: Transgenic hESCs were an effective delivery platform for neurotrophic factors, rescuing axotomized
motoneurons and modulating glial response after proximal spinal cord root injury, while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.