机械敏感TRP离子通道在剪切力致神经元损伤中的调控作用及在弥漫性轴索损伤法医学鉴定中的应用探索

Diffuse axonal injury (DAI) is induced by shear force in brain injury, which results in a series of pathophysiological changes and eventually leads to the axonal disruption. It has been thought that Ca2+ overloding plays a central role in the pathophysiological process of DAI, during which Ca2+ overloading resulted from the mechanical signal transduction into biochemistry signal is included. Mechanosensitive channel is one of the most common pathways in mechnical signal transduction, in which transient receptor potential (TRP) channels are related with mechanotransduction and Ca2+ influx.The preliminary study indicated that the expression of several TRP mRNAs was increased by PCR chip.But the mechanism of Ca2+ overloding through TRP mechanosensitive channel in axons after external force is unclear, and its role in the pathogenesis of DAI is uncertain too. In the present study, the neurons under shear forces will be constructed to imitate DAI model in vitro. The experiment skills of electro-physiology,morphology and flueorescence etc will be used to observe the effects of TRP mechanosensitive channel on neurons under shear force.On the other hand, the TRP proteins, which were screened by PCR chip in DAI rat, were verified in neurons induced by shear force. The relationship between the expression of mechanosensitive channel TRP proteins and DAI in the brain tissue by autopsy will be observed too. This study would provide the research foundation for the development of diagnosis, treatment and forensic identification of DAI.

弥漫性轴索损伤（DAI）是死因鉴定的难点，其发生是由于外伤在脑组织内产生剪切力，引起病理生理变化，最终致轴索广泛断裂。Ca2+超载被认为是中心环节，期间包含着机械力与Ca2+超载之间力学-生化信号转导。机械敏感离子通道是力信号传导最常见途径之一，其中瞬时受体电位通道(TRP)与机械力传导和Ca2+通透均有关，前期研究通过PCR芯片筛选出多个TRP通道基因在DAI中表达增加，但受外力作用TRP通道如何引起神经元及轴浆内Ca2+超载以及在DAI中的作用尚不明确。本项目拟用剪切力加载神经元建立体外DAI模型，通过电生理、形态学及荧光检测等技术来探讨TRP离子通道在剪切力致神经元损伤中的作用及可能机制，另外在该模型观察筛选出的TRP通道蛋白的表达，并进一步在尸检脑组织研究这些通道蛋白与DAI病变的关系。本项目通过DAI发病力学-生化信号转导机制的探讨以期为该病法医鉴定及临床诊治的进展提供研究基础。

Ca2+超载被认为是弥漫性轴索损伤（DAI）发病的中心环节，期间包含着机械力与Ca2+超载之间力学-化学信号之间的转导。机械敏感离子通道是机械信号传导最常见的途径之一，其中的瞬时受体电位通道(TRP)与机械力传导和Ca2+通透有关。而有关研究表明，TRPC通道为TRP通道里最具有代表性的一种。本项目用牵张力加载培养的神经元模拟DAI模型，使用TPRC通道抑制剂SKF-96365探索TRPC通道在DAI模型造成神经元损伤中起的作用。研究显示微管蛋白β-tubulin在受力后呈现不稳定的上升趋势，受力48小时后变化显著（P<0.05）；微丝蛋白F-actin则没显著变化；轴索长度在受力后呈现显著性降低，给抑制剂后出现显著恢复；DAI后，神经元LDH分泌和神经元早期凋亡率没有显著改变（P>0.05）。线粒体膜电位加力后出现即时性下降，加力后一段时间也没有恢复至加力前水平，但加入抑制剂后，线粒体膜电位下降趋势不明显。钙离子浓度在加力后出现显著性升高（P<0.01），加抑制剂后再加力，钙离子浓度没有显著变化（P=0.088）。CaMK II磷酸化水平在加力后出现了即时性升高，但在48小时后恢复到加力前水平；而NFL与CaMK II重叠系数R则在加力后显著性下降（P=0.032），加抑制剂后，R下降趋势显著减慢（P=0.012）。上述研究结果显示DAI中，TRPC-Ca2+-CaMK II参与导致神经元NFL积累、轴索断裂，从而神经元二次损伤的机制，且此机制下导致神经元损伤结局并非细胞凋亡。