TDP-25对运动神经元延迟整流钾电流电生理特性的影响及双甲氧姜黄素的保护作用
发布时间：2018-09-18 20:02
【摘要】：肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis，ALS）是一个逐渐进展的、具有严重致死性的神经变性疾病，以进行性运动神经元丢失为特征，发病机制不明。TDP-43（TAR DNA bingding protein of43kDa）被认为是在肌萎缩侧索硬化（Amyotrophic lateral sclerosis，ALS）及额颞叶痴呆患者大脑中发现的泛素化包涵体的主要成分，主要功能为参与特定的前mRNA的剪接、转录、维持其稳定性及微小mRNA的生物合成。目前在TDP-43上发现了40种与散发性及家族性ALS有关的显性突变，为TDP-43的功能异常及神经变性病之间的直接联系提供了强有力的证据。TDP-25是在ALS患者的受累大脑区域中发现的分子量为25kDa的TDP-43的C末端片段。研究发现，TDP-25可以促进TDP-43包涵体形成，对运动神经元具有毒性作用，引起神经元变性，在疾病的发病过程中起重要作用，但其机制尚未明确。最初的观点认为：兴奋性毒性可以引起运动神经元损伤。然而，对ALS患者的神经传导的研究发现，轴索膜兴奋性的提高，及过兴奋性的程度与病人的生存期呈负相关。兴奋性的提高包括持续性钠电流的增加及钾电流的减小。然而对TDP-43模型是否存在高兴奋性尚不清楚。随着对ALS研究的不断深入及对K+通道的进一步了解，ALS与钾通道的关系日益受到人们的关注，延迟整流电流（delayed rectifierpotassium current，IKdr）是影响动作电位复极化过程的主要的电流,但有关ALS模型细胞水平上对钾电流的研究甚少。同时，双甲氧姜黄素（Dimethoxy Curcumin, DMC）是姜黄素的类似物，近年来因其具有抗氧化、抗炎、抗肿瘤、免疫调节等功效而受了广泛关注，本实验室研究也发现其对转染TDP-43的运动神经元样细胞线粒体有保护作用，可以增强其自噬清除毒性蛋白的能力，此外姜黄素还可以上调Nrf2和II相酶的水平，减少氧化应激产生的损害，进而保护细胞。 目的：检测在稳定转染TDP-25基因的运动神经元的延迟整流钾电流性质是否发生改变及动作电位相关指标的改变，以探讨TDP-25对运动神经元产生毒性作用的机制；进一步探讨DMC对电压门控钾通的道调节作用。 方法：应用稳定转染TDP-25基因和空质粒（Empty vector）的两种细胞，该细胞系由本实验室构建，，参考NSC34细胞株的培养方法进行培养，采用抗生素加压的方法筛选单克隆细胞，将筛选所得单克隆细胞系进行培养、传代。在全细胞膜片钳技术电压钳模式下记录上述两种细胞延迟整流钾电流并分析其性质改变。在电流钳模式下测定两种细胞的动作电位，分别给予两种细胞20pA、40pA、60pA和80pA4个阶梯递增的电流刺激动作电位发生，并记录其阈电位、潜伏期、幅度及动作电位半峰时程（action potential duration at50%repolarization，APD50）等指标以进行比较。将成功培养的稳定转染TDP-25和Empty的NSC34细胞系传代，接种于六孔板的小玻璃片上，12小时后给予换液并给予上述两种细胞浓度为10mΜ的DMC24小时。给予与对照组相同的刺激程序，观察DMC对延迟整流钾通道电流的影响。 结果：1、TDP-25组细胞的电流密度小于Empty组，在60mV时有统计学意义（TDP-25组10.65±3.24，Empty组13.26±6.51P0.05）。2、与Empty组相比，TDP-25组半数激活电压V1/2向超极化方向移动3.73mV左右(TDP-25组8.18±4.95mV，n=14, Empty组11.91±4.34mV，n=19,P0.05)，表明TDP-25组细胞的钾通道更容易在较负的电位下被激活。斜率K无统计学差异（TDP-25组11.46±2.41，Empty组12.82±1.89. P0.05）。3、TDP-25组(n=14)与Empty（n=11）组细胞的动作电位的阈电位值在各个刺激电流下无统计学差异；TDP-25组细胞的潜伏期值高于Empty组，在40pA时两组相比有统计学意义（分别为38.99±9.48ms和30.13±6.96ms,P0.05）；TDP-25组细胞的幅度低于Empty组，其中在20pA时有统计学差异（分别为42.96±7.29mV和47.10±8.69mV, P 0.05）。TDP-25组细胞的APD50显著长于Empty组，在40pA、60pA、80pA时有统计学差异（分别为80.41±25.51ms,64.03±23.35ms,55.16±18.97ms和53.18±7.65ms,44.13±5.58ms,36.56±9.24ms）。推断主要与TDP-25组细胞IKdr的激活动力学发生改变有关。4、在给予TDP-25组细胞10uM的DMC处理24小时后，电流密度大于未给药组细胞，表明DMC有增加延迟整流钾电流的趋势，但两组相比无统计学差异。5、给药后IKdr的稳态激活曲线发生了正向偏移，相应地，半数激活电压V1/2向去极化方向移动了8.93mV左右，未给药组V1/2=8.18±4.96mV，给药组V1/2=17.11±7.99mV（P＜0.05)，表明DMC改变了延迟整流钾电流的激活性质，具有促进钾通道开放的作用；激活曲线的斜率k由11.46±2.4增加为16.12±2.40（P＜0.01)，表明DMC使此钾通道对电压的敏感性增强。 结论：在本实验中，我们在运动神经元样细胞上成功检测出延迟整流钾电流，并发现TDP-25通过改变延迟整流钾电流的激活性质，抑制了延迟整流钾通道的活性，减小电流密度，延长动作电位的复极化时程，增大了细胞的放电频率，从而引起神经元兴奋性增高，对神经元产生兴奋毒性作用，据此推测TDP-25对钾电流的抑制产生的细胞兴奋性升高可能是其引起运动神经元变性的可能的机制之一。双甲氧姜黄素（DMC）具有促进延迟整流钾通道开放的作用，改变其激活特性，并可能因此增加延迟整流钾电流的幅度降低神经元兴奋性，从而对神经元起到保护作用。
[Abstract]:Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by progressive loss of motor neurons. TDP-43 (TAR DNA binding protein of 43 kDa) is thought to be associated with amyotrophic lateral sclerosis (ALS). The main components of ubiquitinated inclusions in the brain of patients with frontotemporal dementia are involved in splicing, transcription, stability and micromRNA biosynthesis of specific pre-mRNAs. Forty dominant mutations related to sporadic and familial ALS have been found in TDP-43, which are dysfunction and neurodegeneration of TDP-43. TDP-25 is a C-terminal fragment of TDP-43 with a molecular weight of 25 kDa found in the affected brain regions of ALS patients. Studies have shown that TDP-25 promotes the formation of TDP-43 inclusion bodies, has a toxic effect on motor neurons, causes neuronal degeneration, and plays an important role in the pathogenesis of ALS. Initially, excitotoxicity was thought to cause motor neuron damage. However, studies of neural conduction in ALS patients found that increased excitability of the axonal membrane and the degree of hyperexcitability were negatively correlated with survival. Increased excitability included an increase in persistent sodium current and potassium. The relationship between ALS and potassium channel has attracted more and more attention. Delayed rectifier potassium current (IKdr) is the main factor affecting the repolarization process of action potential. At the same time, dimethoxycurcumin (DMC), a curcumin analogue, has attracted much attention in recent years because of its antioxidant, anti-inflammatory, anti-tumor, immune regulation and other effects. Our laboratory study also found that DMC can transfect the motor neurons of TDP-43. Mitochondria-like cells have protective effects, which can enhance the ability of autophagy to remove toxic proteins. Curcumin can also up-regulate the levels of Nrf2 and phase II enzymes, reduce the damage caused by oxidative stress, and protect cells.
AIM: To investigate whether the delayed rectifier potassium current (DCK) and action potential (AP) related parameters in the stably transfected motoneurons were altered in order to explore the mechanism of the toxic effect of TDP-25 on motoneurons, and to further explore the regulation of voltage-gated potassium channel by DMC.
METHODS: TDP-25 gene and Empty vector were stably transfected into two kinds of cells. The cell line was constructed in our laboratory and cultured with reference to the culture method of NSC34 cell line. Monoclonal cells were screened by antibiotic pressurization method. The selected monoclonal cell lines were cultured and subcultured. The whole cell patch clamp technique was used to electrocute the whole cell. Delayed rectifier potassium currents were recorded and their properties were analyzed under voltage clamp mode. Action potentials of the two cells were measured under current clamp mode. The action potentials of 20 pA, 40 pA, 60 pA and 80 pA were given to the two cells respectively. The threshold potential, latency, amplitude and half peak time of action potential were recorded. The NSC34 cell lines transfected with TDP-25 and Empty were subcultured and inoculated on the small glass slices of six-hole plate. After 12 hours, the cells were transfused and given DMC of 10 mfor 24 hours. The same stimulation was given to the control group. The effect of DMC on the current of delayed rectifier potassium channel was observed.
Results: 1. The cell current density of TDP-25 group was lower than that of Empty group, and there was statistical significance at 60 mV (10.65+3.24 in TDP-25 group and 13.26+6.51P 0.05 in Empty group). 2. Compared with Empty group, the half activation voltage of TDP-25 group moved 3.73 mV (8.18+4.95 mV, n=14 in TDP-25 group, 11.91+4.34mV in Empty group, n=19, P 0.05), indicating that TDP-25 group moved toward hyperpolarization by 3.73 mV (8.18+4.95 mV, n=14, 11.91+4.34mV, n=19, P 0.05). There was no significant difference in the slope K between TDP-25 group and Empty group (11.46+2.41, 12.82+1.89.P 0.05). 3. There was no significant difference in the threshold potential of action potential between TDP-25 group (n=14) and Empty group (n=11); the cell latency of TDP-25 group was higher than that of Empty group (40 p). There was significant difference between the two groups at 20 pA (42.96 [7.29] mV and 47.10 [8.69] mV, P 0.05). APD50 of the TDP-25 group was significantly longer than that of the Empty group at 40 pA, 60 pA, and 80 pA, respectively. It was concluded that the change of activation kinetics of IKdr in TDP-25 cells was mainly related to the change of the activation kinetics of IKdr. However, there was no significant difference between the two groups. The steady-state activation curve of IKdr shifted positively after administration. Accordingly, half of the activation voltage V1/2 shifted to depolarization direction by about 8.93 mV. In the untreated group, V1/2 = 8.18 + 4.96 mV, and in the untreated group, V1/2 = 17.11 + 7.99 mV (P < 0.05), indicating that DMC changed the activation property of delayed rectifier potassium current and promoted it. The slope of activation curve K increased from 11.46 (+ 2.4) to 16.12 (+ 2.40) (P < 0.01), indicating that DMC enhanced the sensitivity of potassium channel to voltage.
CONCLUSION: In this experiment, delayed rectifier potassium currents were successfully detected in motor neuron-like cells. TDP-25 inhibited the activity of delayed rectifier potassium channels, decreased the current density, prolonged the repolarization duration of action potential, and increased the cell discharge frequency by altering the activation properties of delayed rectifier potassium currents. It may be one of the possible mechanisms of motor neuron degeneration. Dimethoxycurcumin (DMC) can promote delayed rectifier potassium channel opening, change its activation characteristics, and may also be involved in the mechanism. It can therefore increase the amplitude of delayed rectifier potassium current and decrease neuronal excitability, thus playing a protective role on neurons.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.8
本文编号：2248975
[Abstract]:Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by progressive loss of motor neurons. TDP-43 (TAR DNA binding protein of 43 kDa) is thought to be associated with amyotrophic lateral sclerosis (ALS). The main components of ubiquitinated inclusions in the brain of patients with frontotemporal dementia are involved in splicing, transcription, stability and micromRNA biosynthesis of specific pre-mRNAs. Forty dominant mutations related to sporadic and familial ALS have been found in TDP-43, which are dysfunction and neurodegeneration of TDP-43. TDP-25 is a C-terminal fragment of TDP-43 with a molecular weight of 25 kDa found in the affected brain regions of ALS patients. Studies have shown that TDP-25 promotes the formation of TDP-43 inclusion bodies, has a toxic effect on motor neurons, causes neuronal degeneration, and plays an important role in the pathogenesis of ALS. Initially, excitotoxicity was thought to cause motor neuron damage. However, studies of neural conduction in ALS patients found that increased excitability of the axonal membrane and the degree of hyperexcitability were negatively correlated with survival. Increased excitability included an increase in persistent sodium current and potassium. The relationship between ALS and potassium channel has attracted more and more attention. Delayed rectifier potassium current (IKdr) is the main factor affecting the repolarization process of action potential. At the same time, dimethoxycurcumin (DMC), a curcumin analogue, has attracted much attention in recent years because of its antioxidant, anti-inflammatory, anti-tumor, immune regulation and other effects. Our laboratory study also found that DMC can transfect the motor neurons of TDP-43. Mitochondria-like cells have protective effects, which can enhance the ability of autophagy to remove toxic proteins. Curcumin can also up-regulate the levels of Nrf2 and phase II enzymes, reduce the damage caused by oxidative stress, and protect cells.
AIM: To investigate whether the delayed rectifier potassium current (DCK) and action potential (AP) related parameters in the stably transfected motoneurons were altered in order to explore the mechanism of the toxic effect of TDP-25 on motoneurons, and to further explore the regulation of voltage-gated potassium channel by DMC.
METHODS: TDP-25 gene and Empty vector were stably transfected into two kinds of cells. The cell line was constructed in our laboratory and cultured with reference to the culture method of NSC34 cell line. Monoclonal cells were screened by antibiotic pressurization method. The selected monoclonal cell lines were cultured and subcultured. The whole cell patch clamp technique was used to electrocute the whole cell. Delayed rectifier potassium currents were recorded and their properties were analyzed under voltage clamp mode. Action potentials of the two cells were measured under current clamp mode. The action potentials of 20 pA, 40 pA, 60 pA and 80 pA were given to the two cells respectively. The threshold potential, latency, amplitude and half peak time of action potential were recorded. The NSC34 cell lines transfected with TDP-25 and Empty were subcultured and inoculated on the small glass slices of six-hole plate. After 12 hours, the cells were transfused and given DMC of 10 mfor 24 hours. The same stimulation was given to the control group. The effect of DMC on the current of delayed rectifier potassium channel was observed.
Results: 1. The cell current density of TDP-25 group was lower than that of Empty group, and there was statistical significance at 60 mV (10.65+3.24 in TDP-25 group and 13.26+6.51P 0.05 in Empty group). 2. Compared with Empty group, the half activation voltage of TDP-25 group moved 3.73 mV (8.18+4.95 mV, n=14 in TDP-25 group, 11.91+4.34mV in Empty group, n=19, P 0.05), indicating that TDP-25 group moved toward hyperpolarization by 3.73 mV (8.18+4.95 mV, n=14, 11.91+4.34mV, n=19, P 0.05). There was no significant difference in the slope K between TDP-25 group and Empty group (11.46+2.41, 12.82+1.89.P 0.05). 3. There was no significant difference in the threshold potential of action potential between TDP-25 group (n=14) and Empty group (n=11); the cell latency of TDP-25 group was higher than that of Empty group (40 p). There was significant difference between the two groups at 20 pA (42.96 [7.29] mV and 47.10 [8.69] mV, P 0.05). APD50 of the TDP-25 group was significantly longer than that of the Empty group at 40 pA, 60 pA, and 80 pA, respectively. It was concluded that the change of activation kinetics of IKdr in TDP-25 cells was mainly related to the change of the activation kinetics of IKdr. However, there was no significant difference between the two groups. The steady-state activation curve of IKdr shifted positively after administration. Accordingly, half of the activation voltage V1/2 shifted to depolarization direction by about 8.93 mV. In the untreated group, V1/2 = 8.18 + 4.96 mV, and in the untreated group, V1/2 = 17.11 + 7.99 mV (P < 0.05), indicating that DMC changed the activation property of delayed rectifier potassium current and promoted it. The slope of activation curve K increased from 11.46 (+ 2.4) to 16.12 (+ 2.40) (P < 0.01), indicating that DMC enhanced the sensitivity of potassium channel to voltage.
CONCLUSION: In this experiment, delayed rectifier potassium currents were successfully detected in motor neuron-like cells. TDP-25 inhibited the activity of delayed rectifier potassium channels, decreased the current density, prolonged the repolarization duration of action potential, and increased the cell discharge frequency by altering the activation properties of delayed rectifier potassium currents. It may be one of the possible mechanisms of motor neuron degeneration. Dimethoxycurcumin (DMC) can promote delayed rectifier potassium channel opening, change its activation characteristics, and may also be involved in the mechanism. It can therefore increase the amplitude of delayed rectifier potassium current and decrease neuronal excitability, thus playing a protective role on neurons.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.8
【引证文献】
相关硕士学位论文 前1条
1 李辉;肌萎缩侧索硬化和运动神经元内在高兴奋性的关系[D];河北医科大学;2016年
本文编号：2248975
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