益气化瘀方调控胚胎干细胞BDNF/TrkB/PI3K信号通路促进脊髓损伤恢复的机制研究

In our previous studies, we have demonstrated that overexpression of Mash-1 gene promoted neuronal differentiation of embryonic stem cell differentiation after transplantation into spinal cord injury, and avoid tumor formation. However the survival of transplanted cells was still poor. We have also demonstrated that Yiqihuayu recipe can increase BDNF expression in the dorsal root ganglia and reduce neurons apoptosis. Therefore, in this study we attempt to promote BDNF expression in embryonic stem cell by treating with Yiqihuayu recipe, increase the survival of transplanted cell and promote the repair of spinal cord injury. Spinal cord injury clip-compression mouse model will be used, and then transplanted with Mash-1 overexpression embryonic stem cell and treated with Yiqihuayu recipe. Motor activity was assessed weekly with the Basso mouse scale (BMS). The injured spinal cord was then examined histologically including quantification of cavitation. The BDNF content in spinal cord was detected with Elisa. Phosphorylation of PI3K, AKT, MAPK, ERK and genes expression of Bax, Bcl-2 were analysis with Western blot and real-time PCR, respectively. We also used TrkB inhibitor to analysis the mechanism and protect effect of Yiqihuayu recipe from in vitro. This study was aimed to preliminarily explore the therapeutic mechanism of Yiqihuayu recipe combined with Mash-1 overexpression embryonic stem transplantation for spinal cord injury, which provide valuable theoretical guidance and experimental basis for traditional Chinese medicine combined with stem cells transplantation for spinal cord injury.

前期研究证实，过表达Mash-1基因可提高胚胎干细胞在脊髓损伤部位分化成神经元的比率，避免移植细胞形成肿瘤，但仍然存在细胞存活数量不足的问题。而益气化瘀方可促进背根节神经细胞表达BDNF，减少周围神经细胞凋亡。因此，本项目计划采用益气化瘀方促进BDNF表达，增加移植细胞的存活数量，更好地促进脊髓损伤修复；拟采用小鼠钳夹脊髓损伤模型，给予益气化瘀方联合Mash-1修饰胚胎干细胞移植治疗，经后肢运动功能评分，组织病理学分析，Elisa检测BDNF含量，Western blot分析PI3K、AKT、MAPK、ERK蛋白磷酸化表达，PCR检测Bax、Bcl-2等凋亡基因表达；并采用TrkB抑制剂，从体外分析益气化瘀方含药血清对胚胎干细胞的保护作用；初步阐明益气化瘀方联合Mash-1修饰胚胎干细胞移植促进脊髓损伤修复的作用机制，为中医药联合干细胞移植治疗脊髓损伤提供有价值的理论指导和实验依据。

本课题拟观察益气化瘀方联合Mash-1修饰的胚胎干细胞对小鼠脊髓损伤的修复作用。采用钳夹法建立小鼠急性脊髓损伤模型，于造模后第3天将Mash-1修饰的胚胎干细胞移植到脊髓损伤部位，并给予益气化瘀方干预。通过观察小鼠的后肢功能评分、脊髓的剩余面积以及神经营养因子、炎症因子和凋亡相关基因的表达改变；并采用免疫荧光检测OCT3/4、nestin、β-tubulin Ⅲ、GFAP的阳性细胞数目，分析移植细胞在体内的存活和分化情况。实验结果显示，与模型组比较，益气化瘀方可减少炎症因子TNF-α和凋亡基因caspase-3的表达，轻微提高小鼠运动功能。与单纯干细胞移植组比较，益气化瘀方联合Mash-1修饰胚胎干细胞移植能减少炎症因子TNF-α的基因表达水平，提高移植细胞的存活数量，进而改善小鼠的后肢运动评分。提示益气化瘀方能减轻脊髓损伤的炎症反应，增加移植细胞在脊髓损伤部位的存活数量，促进脊髓损伤的修复。