间充质干细胞源性外泌体对正己烷中毒大鼠神经组织受损轴突修复作用及机制研究

Chronic n-hexane poisoning is a serious occupational disease in China, it could induce central-peripheral neuropathy which is characterized by axonal degeneration. However, there is no effective treatment to restore its severely impaired nerve function. Stem cell-derived exosomes have the similar biological function with stem cells and could recover the axon injury. According to the literatures and previous studies, the applicant put forward the hypothesis that stem cell-derived exosomes could promote the regeneration of axon in rats with n-hexane exposure, laying the theoretical foundation for this study. In this study, we established the model of mesenchymal stem cell-derived exosomes treatment to the exposure to n-hexane in rats and primary neuron in vitro. The nerve damage detecting platform, electrophysiological technique and morphological observation were used to evaluate the recovery of nerve function and the degree of axon repair. Moreover, immunofluorescence technique was used to detect the targeted distribution of Texas-red-labeled mesenchymal stem cells exosomes in the damaged nerve tissue. Besides, the gene technique and luciferase reporter gene technique were also used to explore the mechanism that the exosomes by NGF-dependent PI3K/Akt pathway and downstream signaling promotes the axonal regeneration in n-hexane poisoning rats. This study will provide a new method for the treatment of nerve tissue damage caused by chronic exposure to n-hexane, and also provide reference for the repair of nerve tissue injury caused by exposure to other chemicals.

慢性正己烷中毒是我国严重职业病，可诱发轴索变性为特征的中枢-周围神经病。然而，尚无有效干预手段恢复其严重受损神经功能。干细胞外泌体能模拟干细胞生物学功能，对轴突损伤有修复作用。根据文献分析和前期研究，申请者提出干细胞外泌体介导NGF促进正己烷中毒大鼠轴突再生修复机制假设，为本课题立项奠定理论基础。本研究构建正己烷中毒和间充质干细胞外泌体干预大鼠模型及原代神经元模型；用神经损伤检测平台、电生理技术和形态学观察，评价神经功能恢复和轴突修复程度；用免疫荧光技术检测Texas-red标记间充质干细胞外泌体在受损神经组织靶向分布；用RNA沉默技术、荧光素酶报告基因技术等，探讨干细胞外泌体介导NGF依赖性激活PI3K/Akt通路及下游信号，促进正己烷中毒大鼠轴突再生修复作用及机制。本研究将为慢性正己烷中毒神经组织损伤修复，提供干细胞外泌体干预新方法，也为其它化学物暴露所致神经组织损伤修复提供参考依据。

正己烷是一种广泛用于电子工业的有机溶剂，常温下易挥发，主要通过呼吸道、皮肤途径进入人体。长期低剂量暴露会使机体慢性中毒，导致以轴突损伤为特征的周围神经病变。其主要临床症状为四肢肌力下降，感觉神经功能障碍，严重者下肢瘫痪。研究表明，神经系统损伤后其自我修复能力较差。因此积极探索行之有效的干预方法，促进患者神经功能恢复和改善，具有重要意义。本研究用2,5-己二酮（HD）和骨髓间充质干细胞源性外泌体（MSC-exo），构建正己烷中毒和外泌体干预大鼠模型。结果表明，MSC-exo显著改善HD暴露大鼠神经行为学和神经电生理学功能以及轴突的病理改变；MSC-exo上调HD暴露大鼠坐骨神经NGF、p-TrkA、p-Akt、p-mTOR、GAP43表达，下调miRNA Let-7家族中Let-7d-5p等多个亚型。而Let-7d-5p过表达或siNGF、NGF中和抗体、TrkA/Akt/mTOR抑制剂干预，均明显抑制MSC-exo对坐骨神经轴突的修复作用。这些结果提示，MSC-exo介导Let-7调控NGF/Akt/mTOR通路，促进HD暴露大鼠坐骨神经轴突修复；同时，MSC-exo上调HD暴露大鼠脊髓神经PTEN、p-Akt、p-GSK-3β及GAP-43表达。而miR-21 inhibitor、siPTEN、MK2206和TDZD-8干预，显著抑制MSC-exo对脊髓神经轴突修复作用。这些结果提示，MSC-exo介导miR-21调控PTEN/Akt/GSK-3β通路，促进HD暴露大鼠脊髓神经轴突修复。总之，通过本研究，表明MSC-exo对HD暴露大鼠神经组织损伤具有明显修复作用。同时，阐明了MSC-exo对HD暴露大鼠坐骨和脊髓神经轴突修复的部分机制。本研究成果，将为慢性正己烷中毒受损神经轴突修复，提供MSC-exo干预新策略；也为其它化学物暴露所致神经组织轴突损伤的MSC-exo修复，提供参考依据。