【作者】 吕斌；

【导师】 董红梅；

【作者基本信息】 华中科技大学 ， 法医病理学， 2019， 硕士

【摘要】 【研究背景】弥漫性轴索损伤（diffuse axonal injury,DAI）是创伤性脑损伤（traumatic brain injury,TBI）中最常见及危害最大的一种病理类型,是指当头部因外力作用时,由于大脑各部位组织存在质量、密度差异而在脑组织内部产生剪切力、牵拉力导致的一种弥漫性的脑白质轴索损伤。DAI可分为原发性损伤和继发性损伤。原发性损伤是指外力作用于头部时脑组织内部产生剪切力、牵拉力引起原发性轴索断裂损伤,在损伤当时立即出血。而继发性损伤是指在伤后数小时至数十天内由创伤诱发的轴索局部损伤,经过一系列复杂的病理生理学过程,逐渐发展至轴索断裂,最终导致轴索坏死、崩解。继发性损伤是导致DAI预后不良的主要原因,其损伤机制尚不明确。在临床上,DAI尚无统一的诊断标准和有效的治疗手段。在法医学鉴定中,DAI的诊断也有很大困难。因此,深入研究DAI的发病机制,寻找可能的敏感的分子标记,对指导DAI的法医学鉴定有很大的潜在应用价值,同时对改进该病的临床诊治方法也具有重要意义。DAI的发生发展过程中包含了机械力信号和生化信号之间的转导。研究显示力信号与生化信号转导最可能的途径有2种,一种是力敏感受体途径,而另一种是力敏感的离子通道途径。机械敏感TRP离子通道（Mechanosensitive transient receptor potential channels）是一种常见的力敏感离子通道,也是细胞力学信号转导的重要途径之一。该类离子通道属于非选择性阳离子通道,在人体分布广泛,几乎表达于所有组织和细胞类型。目前已发现哺乳动物的TRP通道有六个亚族,即TRPA、TRPC、TRPM、TRPP、TRPV。其中TRPC1是TRPC亚族中的成员之一,在人体内大多数细胞中均有表达。研究发现TRPC1可以介导细胞膜拉伸时Ca2+、Na+等阳离子的内流。而快速、大量的Ca²⁺内流导致的Ca²⁺超载正是公认的DAI发病的中心环节。Ca²⁺超载可以引起轴索内不可逆的微丝、微管结构紊乱,进而引起轴索的肿胀、断裂。Ca2+超载还可以导致线粒体的损伤,导致线粒体膜电位下降,通透性转换孔开放,释放促凋亡因子,进而诱导细胞发生凋亡。综上所述,既参与了力信号与生化信号转导,又能介导Ca²⁺内流的机械敏感TRPC1离子通道很可能在DAI发展过程中起到了重要的调控作用。基于上述研究进展,本研究通过建立皮质神经元牵拉损伤模型体外模拟DAI损伤,初步探索机械敏感TRPC1通道在牵张力致神经元损伤中的调控作用。【研究目的】1、通过牵拉损伤皮质神经元建立DAI细胞模型,观察轴索损伤后的生化指标变化,检测LDH的表达水平和变化规律;2、初步探讨DAI损伤后的神经元中细胞骨架,线粒体膜电位,胞内活性氧生成量的变化情况;3、初步探讨TRPC1通道在DAI后神经元损伤中的调控作用。【研究方法】1、原代培养皮质神经元,采用神经元烯醇化酶NSE免疫荧光进行神经元纯度鉴定。选择纯度高,生长状态良好的神经元随机分组,参照Ellis方法建立DAI神经细胞牵拉损伤模型。前期研究已表明硅胶膜形变距离为10mm时可以对神经细胞造成牵拉损伤,引起轴索的形态学改变。本实验在在损伤后0h、4h、12h、24h及48h分别取培养基上清液,检测LDH含量变化对牵拉损伤造模情况进行再次验证。2、根据是否添加TRPC1抑制剂GsMTx-4将原代培养皮质神经元分为药物干预组和对照组。其中药物干预组在添加抑制剂后进行损伤造模,并根据损伤后不同时间点分为0h组、4h组、12h组、24h组及48h组,形变距离统一为10mm。对照组除未添加抑制剂外,其余处理与药物干预组相同3、通过微丝蛋白和微管蛋白免疫荧光双标染色,观察药物干预组和对照组损伤后不同时间段神经元细胞骨架变化情况。4、通过JC-1染色,观察药物干预组和对照组损伤后不同时间点神经元线粒体膜电位变化情况。5、通过DCFH-DA荧光探针检测,观察药物干预组和对照组不同时间点细胞内活性氧浓度变化情况。【研究结果】1、本实验使用形变量统一为10mm,形变时间1s对硅胶膜上的神经元进行牵拉后。损伤后0h可见部分神经元轴索扭曲呈“S”型,少量神经元轴索发生断裂。至48h,神经元死亡严重,出现细胞空泡变,轴索数量减少,形态不清,部分轴索增粗、扭曲、断裂、甚至发生崩解。经过牵拉损伤后上清液中的LDH含量有较明显的升高。而对照组的上清液LDH含量基本保持稳定。2、检测加力前后β-tubulin荧光强度,发现β-tubulin荧光强度在加力后呈不稳定的轻微上升趋势。但向DAI模型中加入TRPC1拮抗剂GsMTx-4,并未能显著逆转β-tubulin表达强度变化。以F-actin指示神经元轴索,对照组比药物干预组神经元轴索密度低,在12～48 h组尤为明显。对照组加力后轴索长度随着经历时间增加而变短,使用TRPC1抑制剂干预后轴索长度下降趋势有所缓解。3、可见在牵张力作用后,神经元线粒体膜电位出现了即时性下降（0h）,随着加力后时间的延长,神经元线粒体膜电位有一定程度的恢复,但并未恢复至加力前水平。向神经元加入TRPC1抑制剂GsMTx-4后进行检测发现加力后线粒体膜电位并未出现即时性下降,虽然随着加力后时间的延长有轻微的下降,但到加力后24小时的时候,线粒体膜电位逐渐恢复,而后48小时恢复到加力前的水平。4、发现对照组伤后0h时活性氧含量轻微升高,4h时出现降低,12h时恢复至损伤前水平,后逐渐降低。药物干预组的活性氧含量变化幅度较轻微。0h时药物干预组与对照组的差异较为明显。【研究结论】1、成功建立牵张力致神经元损伤细胞模型;2、牵张力对神经元的作用可导致迟发性轴索断裂,使用TRPC1抑制剂可以改善轴索迟发性断裂情况;3、牵张力对神经元的作用可导致线粒体膜电位降低,使用TRPC1抑制剂可以改善伤后线粒体膜电位降低的情况。更多还原

【Abstract】 BACKGROUND Diffuse axonal injury(DAI)is the most common and harmful pathological type of traumatic brain injury(TBI).It occurs when brain is attacked by external force.The brain tissue has a diffuse white matter axonal injury caused by shearing force and pulling force.DAI can be divided into primary and secondary injuries.Primary injury refers to the shearing force and pulling force inside the brain tissue caused by external force on the head.Secondary injury is induced by trauma within hours to tens of days after injury.The local injury of the axon,after a series of complicated pathophysiological processes,gradually develops into axonal rupture,which eventually leads to axonal necrosis and disintegration.Secondary injury is the main cause of poor prognosis of DAI,and the mechanism of injury is still unclear.Clinically,there is no uniform diagnostic criteria and effective treatments for DAI.For forensic scientist,the diagnosis of DAI is also very difficult.Therefore,clarifying the pathogenesis of DAI,and searching for possible sensitive molecular markers,has great potential application value for guiding the forensic identification of DAI.Likewise,it is important for improving the clinical diagnosis and treatment of the disease.The development of DAI involves the transduction between mechanical force signals and biochemical signals.Studies have shown that there are two most likely pathways for force and biochemical signal transduction,one for the force-sensitive receptor pathway and the other for the force-sensitive ion channel pathway.Mechanosensitive transient receptor potential channels are a common force-sensitive ion channel.They are also important pathways for cell mechanical signal transduction.These ion channels are non-selective cation channels that are widely distributed and expressed in almost all tissues and cell types.It has been found that mammalian TRP channels have six subfamilies,namely TRPA,TRPC,TRPM,TRPP,TRPV.Among them,TRPC1 is a member of the TRPC subfamily and is expressed in most cells of the human body.It was found that TRPC1 can mediate the influx of Ca2+,Na+ and other cations when the cell membrane is stretched.The rapid and large Ca2+ overload caused by Ca2+ influx is the vital process of DAI.Ca2+ overload can cause irreversible microfilaments and microtubule structures in the axons,which can cause swelling and fracture of the axons.Ca2+ overload can also cause damage to mitochondria,leading to mitochondrial membrane potential decreasing,permeability transition pores opening and pro-apoptotic factors releasing.In summary,the mechanically sensitive TRPC1 ion channel,which is involved with both force transduction and Ca2+ influx,is likely to play an important role in the development of DAI.Based on the above research progress,this study established a cortical neuron pulling injury model to simulate DAI injury in vitro,and initially explored the regulation of mechanically sensitive TRPC1 channel in tension-induced neuronal injury.OBJECTIVES 1.Establish DAI cell model by pulling the injured cortical neurons,observe the changes of biochemical indexes,such as LDH;2.To investigate the changes of cytoskeleton,mitochondrial membrane potential and intracellular reactive oxygen species in neurons after DAI injury.3.To investigate the role of TRPC1 channel in neuronal injury after DAI.METHODS 1.Primary cultured cortical neurons were identified by immunofluorescence.Then neurons were randomly divided into groups,and the DAI nerve cell pulling injury model was established by using the Ellis method.Previous studies have shown that the silicone membrane deformation distance of 10 mm can cause tensile damage to nerve cells,causing morphological changes of axonal.In the experiment,the culture solution was taken at 0h,4h,12 h,24h and 48 h after injury,and the change of LDH was detected to verify the modeling of the traction damage.2.Primary cultured cortical neurons were divided into drug intervention group and control group according to whether or not TRPC1 inhibitor GsMTx-4 was added.The drug intervention group was subjected to injury modeling after adding inhibitors,and was divided into 0h,4h,12 h,24h and 48 h according to different time points after injury.The deformation distance was unified to 10 mm.The control group was the same as the drug intervention group except that no inhibitor was added.3.Immunofluorescence of microfilament protein and tubulin was used to observe the changes of neuronal cytoskeleton in drug intervention group and control group.4.JC-1 was used to detect the changes of mitochondrial membrane potential in the drug intervention group and the control group.5.DCFH-DA fluorescent probe was used to detect the changes of intracellular reactive oxygen species in the drug intervention group and the control group.RESULTS 1.In this experiment,the deformation distance was unified to 10 mm,and the deformation time was 1s.At 0h after injury,the axonal distortion of some neurons was "S" type,and a small number of neurons axonal fracture.By 48 h,neuronal death was severe,cell vacuolation occurred,the number of axons decreased,the shape was unclear,and some axonal thickening,distortion,fracture,and even disintegration occurred.After the traction injury,the LDH content in the supernatant increased significantly.The LDH content in the supernatant of the control group remained basically stable.2.It was found that the fluorescence intensity of β-tubulin showed an unstable upward trend after stretching.However,the addition of the TRPC1 antagonist GsMTx-4 to the DAI model did not significantly reverse the change in β-tubulin expression intensity.The axons of neurons were indicated by F-actin.The axial density of neurons in the control group was lower than that in the drug-treated group,especially in the 12-48 h group.After stretching,the length of the axon in control group became shorter as the elapsed time increased.The trend of the axonal length weakened after intervention with the TRPC1 inhibitor.3.It can be seen that after stretching,the mitochondrial membrane potential of the neurons decreased immediately(0h).With the prolongation of the time after the force,the mitochondrial membrane potential of the neurons recovered to some extent,but did not return to previous level.After adding GsMTx-4 to the neurons,it was found that there was no immediate decrease in mitochondrial membrane potential after the addition,although there was a slight decrease immediately after stretching,but 24 hours later,the mitochondrial membrane potential gradually recovered,and returned to previous level 48 hours later.4.It was found that the active oxygen content increased slightly at 0h after injury,and decreased at 4h,and returned to the pre-injury level at 12 h,then gradually decreased.The change of reactive oxygen species in the drug intervention group was mild.At 0h,the difference between the drug intervention group and the control group was more obvious.CONCLUSIONS 1.The neuronal stretch-induced injury model was established successfully.2.The effect of tension on neurons can lead to delayed axonal rupture,the use of TRPC1 inhibitors can improve the axonal delayed fracture;3.The effect of stretching on neurons can lead to a decrease in mitochondrial membrane potential.The use of TRPC1 inhibitor can improve the mitochondrial membrane potential decrease after injury.更多还原