间充质干细胞干预对正己烷中毒大鼠受损神经功能恢复作用及机制

Chronic n-hexane poison is a kind of serious occupational diseases in our country and can cause peripheral polyneuropathy. So far, there is no effective method to restore the function of nerve tissue damaged heavily. MSCs can repair various of nerve system diseases which can divided into neurogliocyte and neuron. The results of the preliminary experiment shows that MSCs intervene can obviously improve the function of the limbs disturbance of model rats poisoned chronically by n-hexane. According to the results of the experiment and document analysis, the applicant comes up with the new theory innovatively. The theory is mainly about whether MSCs intervene can repair nerve tissue poisoned chronically by n-hexane and restore the defect functions. In the study the distribution of neurons and glial cells of MSCs differentiation in the damaged foci of nerve tissue is determined by transplant of MSCs labeled with BrdU using characteristics of MSCs automatically homing to the injury site. The recovery of neurological function is evaluated by nerve damage detection platform, electrophysiological and morphological observations. The intervention mechanism and optimization strategies of MSCs is clarified the Real Time PCR, Western blot and immunofluorescence technique. The research aims at providing new way to restore the function of never damaged by chronic n-hexane as well as to offer reference for further research about restoration of other chemicals-induced never damage through MSCs invetion.

慢性正己烷中毒是我国严重职业病之一，可诱发多发性周围神经病，目前尚无有效手段恢复其受损较重神经组织功能。间充质干细胞（MSCs）可分化为神经元和神经胶质细胞，对多种神经系统疾病病灶具有修复作用。预实验结果表明，MSCs干预对正己烷中毒大鼠的肢体功能障碍有明显改善作用。申请者根据文献分析和预实验结果，创新性地提出"MSCs干预修复慢性正己烷中毒受损神经组织，恢复其缺损功能"的全新理念。本研究移植BrdU标记MSCs给正己烷中毒大鼠，利用MSCs自动归巢于损伤部位特性，确定骨髓MSCs分化细胞在神经组织损伤灶分布；用神经损伤检测平台、电生理技术和形态学观察，评价神经功能恢复程度; 用Real Time PCR、Western blot和免疫荧光技术探讨MSCs干预机制和优化策略。本研究为慢性正己烷中毒受损神经功能恢复提供MSCs干预新方法，也为其它化学物所致损伤神经的干细胞修复提供参考依据。

慢性正己烷中毒可诱发多发性周围神经病，然而尚无有效手段恢复其受损较重神经组织功能。为研究间充质干细胞干预对正己烷中毒大鼠受损神经功能恢复作用及机制,我们首先建立大鼠骨髓间充质干细胞（BMSCs）提取、扩增和标记方法，制备正己烷神经毒作用代谢物2，5己二酮（HD）中毒性周围神经病大鼠模型和BMSCs移植大鼠模型以及进行体外细胞干预试验。1. 对神经行为及电生理影响结果显示：中毒大鼠出现后肢瘫痪、后脚掌上翻等异常神经行为表现，尾神经传导潜伏期(DL)延长,运动神经传导速度(MCV)降低。而BMSCs移植后，大鼠异常步态逐渐改善，DL显著缩短，MCV显著加快，提示MSCs移植对HD中毒大鼠受损神经功能恢复有明显疗效；2. 对轴突影响观察结果显示：中毒大鼠神经组织轴突变性，结构破坏，轴突萎缩成条索状或点状，NGF和GAP43表达水平以及TrkA、AKT、mTOR、CREB的磷酸化水平显著降低。然而MSC移植后，大鼠轴突内电子致密度提高，轴突多成片状形态趋于正常，这些蛋白表达水平或磷酸化水平显著升高。DRG细胞干预试验结果提示，BMSCs通过NGF依赖方式激活AKT/mTOR/CREB通路来促进HD中毒大鼠损伤轴突再生修复；3. 对髓鞘影响观察结果显示：中毒大鼠神经组织髓鞘形状不规则，髓鞘板层模糊、髓鞘变薄、脱失，MBP表达下调，Jagged1、Notch1、NICD、TNFα、RelB表达上调。然而MSC移植后，髓鞘板层结构趋于正常，髓鞘形状较规整，可见髓鞘的修复和新生，MBP、Hes1表达上调，TNFα、RelB表达下调。提示，MSCs对HD诱导脱髓鞘损伤有保护作用，其机制与TNFα/RelB介导的Notch通路抑制有关； 4. 凋亡检测结果表明，中毒大鼠神经组织凋亡率显著升高，TrkA、AKT和Bad磷酸化水平下调，Bad/Bcl-xl表达比值、细胞浆Cyt C表达水平及caspase 3活性上调。然而BMSCs移植后，通过激活NGF/AKT/Bad通路抑制HD诱导的神经组织凋亡；5.自噬检测结果表明，中毒大鼠神经组织自噬率显著升高，TrkA、AKT和mTOR的磷酸化水平下调。然而BMSCs移植后，通过激活NGF/AKT/mTOR通路抑制HD诱导的自噬增多。本研究为正己烷中毒受损神经功能恢复提供MSCs干预新方法，也为其它化学物所致损伤神经的干细胞修复提供参考依据。