囊泡靶向通路介导的自噬在脊髓损伤后锌毒性中的保护作用及机制研究

Brain-derived neurotrophic factor (BDNF) can significantly promote nerve regeneration after spinal cord injury (SCI), zinc could promote the expression of BDNF and its receptor TrKB after SCI, so the zinc has good potential clinical application as a means of treatment of SCI, but zinc has two sides, zinc overload and zinc toxicity after SCI become an urgent problem. Autophagy is activated and play a protective role after SCI, but the extent of its activation is not enough. Our previous studies have shown: as a channel that transport zinc out of intracellular, zinc transporter 1 (ZnT1) expression significantly increased after SCI, and there is a positive correlation between ZnT1 and autophagy in the acute phase, while zinc can induced autophagy, and cytoplasm to vacuole targeting pathway could improve the tolerance to high concentrations of zinc, all these suggested that cytoplasm to vacuole targeting pathway (Cvt) induced autophagy may play a protective role in zinc overload, zinc toxicity after SCI. This project intends to use the autophagy gene knockout mice and their SCI model; using RNA interference and transfection techniques to inhibit or promote autophagy in the cultured neurons; using cell SCI model to detect the protective roles of autophagy under a high level of zinc of in nerve cells in zinc overload and zinc toxicity and to find out the mechanism. All these will solve zinc overload and zinc toxicity after SCI.

锌由锌转运体1（ZnT1）介导可明显促进脊髓损伤(SCI)后脑源性神经营养因子(BDNF)分泌，促进SCI后神经修复，但锌过量会导致锌毒性，而机体对锌毒性的保护机制尚未阐明。我们发现在SCI后，损伤脊髓中锌大量聚集，ZnT1表达升高，自噬功能被明显激活，且自噬相关基因与ZnT1的表达在急性期正相关，提示锌能通过ZnT1诱导自噬，而囊泡靶向通路（Cvt）能提高细胞对高锌的耐受力，所以我们认为Cvt特异性自噬通路可能对SCI后神经细胞锌毒性具有保护作用。基于上述工作基础，本课题拟利用自噬APG9L基因敲除小鼠及其SCI模型，在高锌条件下检测脊髓锌的代谢分布，研究脊髓神经细胞存活与自噬通路变化的关系；利用RNA 干扰和基因转染技术干预体外培养脊髓神经元特异性自噬，阐明高锌条件下锌代谢分布与脊髓神经细胞的生物学功能变化情况，揭示Cvt特异性自噬通路在锌毒性中对神经细胞的保护作用及其机

脊髓损伤(spinal cord injury)是指由于外界直接或间接因素导致脊髓损伤，在损害的相应节段出现各种运动、感觉和括约肌功能障碍，肌张力异常及病理反射等的相应改变。脊髓损伤的程度和临床表现取决于原发性损伤的部位和性质。本研究团队分别研究锌处理对脊髓损伤功能毒性、治疗影响及其作用机制进行了深入研究。本课题利用脊髓打击损伤模型，随机分为高锌组，适量锌组，假手术组，研究锌干预对脊髓损伤动物行为学影响，利用Western blot技术检测锌干预对自噬及凋亡表达影响，初步探寻作用机制。利用尼氏染色和免疫荧光观察脊髓前角运动神经元形态，金属自显影技术检测锌离子在不同节段脊髓组织分布。电镜检测锌干预对脊髓损伤神经元的。结果：在正常小鼠脊髓背角存在丰富的游离锌离子，正常小鼠脊髓组织中自噬特异性蛋白表达较少，SCI小鼠脊髓组织自噬表达增加。高锌处理抑制脊髓损伤恢复，适量锌可能参与脊髓损伤修复。结论：锌处理能显著降低小鼠脊髓损伤。其机制可能与促进自噬、抑制凋亡活性，增加前角运动神经元的存活数量，促进自噬有关。其作用机制可能成为脊髓损伤治疗的新途径。