NGF调节M电流对大鼠TG神经元兴奋性的影响
发布时间：2018-07-25 17:25
【摘要】：M通道是电压及配体依赖性的慢激活、非失活、慢去活的电压门控性钾通道。KCNQ2~5这四个亚型构成的同源或异源四聚体是构成神经元M通道的分子基础，对维持神经元静息膜电位、调节神经元兴奋性和调节突触传递等神经生理活动有着重要的意义。KCNQ2~5亚型基因分别位于不同的染色体上，除了第五个亚型以外，其他亚型的突变均发现与人类遗传疾病相关，如良性家族性新生儿惊厥（benign familial neonatalconvulsions, BFNCs）和癫痫，先天性耳聋等。 近年来研究发现，神经元M电流参与了慢性疼痛和炎性疼痛的的信号转导。神经元和外周痛觉感受器M通道的抑制将会导致神经元去极化且兴奋性增高，从而引发疼痛。由多个通路介导的M电流抑制是疼痛产生的机制之一，增强KCNQ/M通道功能（如提高KCNQ/M通道转录水平和开放M通道等）可以明显减轻疼痛。KCNQ开放剂对多种神经病理性疼痛模型及炎性疼痛模型都具有很好的疼痛预防及治疗作用。 神经生长因子(Nerve growth factor, NGF)是最早被发现的神经营养素家族成员，它对中枢及周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用。这些生物学作用多是一种慢效应，然而近年来越来越多的证据表明，神经生长因子还表现出快速的调节作用，如神经递质的释放，突触的传递，离子通道功能和神经元兴奋性等，以往的研究认为NGF可以调节钠通道和钙通道以及钙激活的钾通道影响神经活动。 我们的前期研究发现NGF还可以抑制大鼠颈上交感神经节（superior cervical ganglion, SCG）和脊髓背根神经节（dosal root ganglion,DRG）神经元记录的M电流，提示M通道可能会成为NGF调节神经兴奋性的新途径。而对于三叉神经节（trigeminal ganglion, TG）神经元上是否能记录到M电流及NGF又有怎样地调节它？这对于研究TG神经元电活动相关的神经病理性疼痛——三叉神经痛有重要意义。本课题旨在TG神经元上验证M电流存在的基础上，观察NGF对其调节规律，并在三叉神经痛动物模型上观察NGF受体和M通道表达水平变化，为三叉神经痛治疗提供帮助。 目的：从TG经元上记录M电流并进行验证，在原代细胞和表达系统观察NGF对它的调节规律，并制备三叉神经疼痛动物模型，观察NGF受体和M通道表达变化，进而分析NGF通过这一途径参与神经病理性疼痛发挥作用的可能性。 方法： (1)质粒cDNA的扩增与提取： KCNQ2、3， TrkA基因克隆在pcDNA3.0质粒，GFP基因克隆在pEGFP-N1质粒，用TOP10感受态细菌转化、扩增之后用试剂盒提取，并用琼脂糖凝胶电泳和紫外分光光度计鉴定质粒的纯度与浓度。 (2)大鼠三叉神经节神经元分离及M电流的鉴别：急性分离乳鼠TG神经细胞，24小时后采用穿孔全细胞膜片钳记录方式，对去活尾电流进行鉴别，用KCNQ通道特异性开放剂RTG和特异性阻断剂XE991进行观察。利用KCNQ亚型对TEA的敏感度不同，分析TG神经元上所表达的亚型，再用western blot技术对典型的KCNQ2亚型进行验证。 (3)在表达细胞上，通过将KCNQ2、3，TrkA及GFP质粒共转染于HEK293B细胞，记录NGF激活TrkA受体实现对KCNQ2/3电流的调节作用。 (4)在原代培养的TG神经元细胞上观察神经生长因子（NGF）激活其受体后对M电流的作用，对不同浓度NGF进行研究，计算EC50，并与表达系统观察到的现象进行比较分析。 (5)用电流钳方式记录神经动作电位，通过观察不同浓度NGF和工具药物对TG神经元放电模式的影响，分析NGF对神经放电活动的调节作用是否与M电流的调节变化一致。 (6)通过眶下神经缩窄环（ION-CCI）手术制备三叉神经痛（trigeminalneuralgia, TN）模型，取三叉神经半月节，用免疫组化方法，观察TrkA受体及KCNQ2通道蛋白表达水平变化，分析NGF调节。 结果： (1)质粒提取与鉴定结果：克隆在pcDNA3.0(5.4Kbp)载体上的KCNQ2、 KCNQ3、 TrkA质粒电泳条带和绿色荧光蛋白pEGFP-N1(4.8Kbp)质粒的电泳条带均在5000bp左右。ND-1000紫外/可见光分光光度计测得质粒DNA的浓度值，其中KCNQ2、KCNQ3浓度均在400~600ng/μL之间， GFP和TrkA均在200~400ng/μL之间；OD260/OD280值均在1.8~1.9之间。 (2) TG神经元M通道的验证：以记录M电流的标准protocol：钳制电压在-20mV，跃迁至-50mV持续1s，再回到-20mV，分析-50mV段的慢去活尾电流（非失活的M电流）。M通道特异性激动剂RTG可以增大去活尾电流的水平，且这种激动作用可被M通道特异性阻断剂XE991完全抑制至更低水平。10μM RTG可以使M电流升高至173.98±21.43%，有显著性差异（P0.05，n=3）；3μM XE991可使M电流降低至19.97±7.58%，有极显著性差异（P0.01，n=3）。以不同浓度的TEA溶液（0.03~30mM）抑制M电流帮助判断M通道亚型，并用Logistic方程进行拟合，得到TEA对该尾电流的量效关系曲线，同时求得TEA的半数抑制浓度IC50为3.87±1.33mM，相关系数R2为0.99739。膜蛋白的Western blot结果显示，三叉神经半月节部位KCNQ2蛋白的免疫印迹条带明显，分布在100kD左右。 (3) NGF通过TrkA受体对表达的KCNQ2/3电流作用：记录的Protocol为钳制电压-80mV，去极化至-20mV并持续1s，再复极化至-60mV持续800ms。分析-20mV段的稳态激活KCNQ2/3电流大小。NGF(20ng/mL)可以抑制药前水平到77.9±9.46%(P0.05, n=3)，再恢复至83.4±4.67%(P0.05, n=3)，但抑制后水平与恢复后水平相比无显著性差异(P0.05, n=3)。不同浓度NGF(20、40、80ng/mL)依次给药，三个浓度都表现出了较为明显的抑制作用，而且抑制后的电流很难恢复或恢复很小一部分。衡量稳定后电流大小，以80ng/mL NGF下激活电流的终水平作为最大抑制效应，20ng/mL NGF抑制达到30.6±4.00%的抑制效应，40ng/mL NGF为69.6±5.11%，与药前比均具有极显著性差异（P0.01,n=5）。用Hill方程拟合得到半数抑制浓度EC50为32.8ng/mL。 (4)原代培养的TG神经元上记录M电流，NGF抑制作用明显，NGF的浓度梯度设为0.02、0.1、1、10ng/mL，测量给药后出现的最低值作为NGF抑制最大值，将10ng/mL NGF的抑制作用设为100%（标准化），用Hill方程进行拟合量效曲线，求得EC50为0.0194±0.0047ng/mL。NGF在原代细胞上的反应性明显增强。一个很有意思的现象是，，当NGF浓度增大时抑制的电流恢复的比较明显，如果与药前比较，恢复稳定后测量，20ng/mL的NGF可以恢复到原来的114.6±3.35%(P0.05，n=3)。 (5) NGF对动作电位的影响，以电流钳方式记录，500ms电流诱发动作电位，以50pA递增的跃阶形式确定阈电流水平，再以1.5倍或3倍水平进行记录。结果表明，0.02ng/mL和0.1ng/mL对TG神经元动作电位个数的影响显著：从3.2±0.2分别增加到4.63±0.18和13.86±0.50(P0.01，n=4)。2ng/mL由对照的4.5±0.3个增加到7.8±0.5个，10ng/mL以上基本达到约10个Spike的最大值，静息膜电位水平随NGF随浓度增大而逐渐向去极化方向改变。 (6)大鼠三叉神经痛（trigeminal neuralgia, TN）行为学实验结果：成功建立了眶下神经紧缩结扎（ION-CCI）的三叉神经痛模型。模型组大鼠与手术前相比，鼠术侧（CCI-ipsi）第6天开始痛阈明显降低，第9~12天降至最低；非术侧（CCI-Ctrl）第9天痛阈明显降低，直到第12天均有显著差异；与假手术组比较，模型组动物术侧痛阈从第6天开始降低（P0.05），9~12天更明显(P0.01)。 (7)免疫组化结果：神经元胞体在三叉神经节内不同段分布不同，且无相对集中的密集处。TrkA受体和KCNQ2通道单位均在细胞膜上高表达，胞内较少。模型组术侧与假手组相比，TrkA受体的density(P0.01)、积分光密度值（IOD，P0.05)和阳性细胞率(P0.05)都显著升高；KCNQ2蛋白三个参数有升高趋势，但无统计学差异，但只有IOD值有统计学意义（P0.05）。模型对侧TrkA和KCNQ2与假手组相比三个参数均分别有所升高和降低，但是都只有IOD值有统计学意义（P0.05）。模型术侧与对侧相比，只有density值有显著性差异（P0.05）。 结论：三叉神经节（TG）神经元上确有M通道表达，而且KCNQ2/3亚型是构成M电流的主要成分，对TG神经元兴奋性起重要调节作用，而且NGF对M电流的调节作用很敏感。在三叉神经痛时，TrkA受体蛋白表达水平会上调，NGF可能会通过上调的受体进一步增加对M电流抑制性调控。
[Abstract]:The M channel is a slow activation of voltage and ligand dependent, non inactivation, and a slow deactivated voltage-gated potassium channel.KCNQ2~5, the four subtypes of homologous or heterologous four polymers are the molecular basis of the neuronal M channels, focusing on the maintenance of the resting membrane potential of the neurons, the regulation of neuronal excitability, and the regulation of synaptic transmission. The significant.KCNQ2~5 subtypes are located on different chromosomes. Except for fifth subtypes, the other subtypes are found to be associated with human genetic diseases, such as benign familial neonatal convulsions (benign familial neonatalconvulsions, BFNCs) and epilepsy, and congenital deafness.
In recent years, it has been found that neuronal M current is involved in signal transduction of chronic pain and inflammatory pain. Inhibition of M channels in neurons and external Zhou Tongjue receptors will lead to neuron depolarization and increased excitement, causing pain. The M current inhibition mediated by multiple pathways is one of the mechanisms of pain generation, enhancing the KCNQ/M channel work Energy (such as increasing the transcriptional level of the KCNQ/M channel and opening the M channel) can obviously relieve the pain of the.KCNQ open agent, which has a good effect on the pain prevention and treatment of various neuropathic pain models and inflammatory pain models.
Nerve growth factor (NGF) is a member of the earliest known family of neurotrophin. It plays an important role in regulating the development, differentiation, growth, regeneration and functional properties of central and peripheral neurons. These biological functions are mostly slow effects. However, more and more evidence has been shown in recent years. Growth factors also show a rapid regulatory effect, such as the release of neurotransmitters, synaptic transmission, ion channel function and neuronal excitability. Previous studies suggest that NGF can regulate sodium channels and calcium channels and calcium activated potassium channels to affect neural activity.
Our previous study found that NGF could also inhibit the M current recorded by the superior cervical ganglion (SCG) and the spinal dorsal root ganglion (dosal root ganglion, DRG) neurons, suggesting that the M channel may become a new path for NGF to regulate nerve excitability. How can the M current and NGF be adjusted to regulate it? This is of great significance for studying the neuropathic pain associated with the electrical activity of TG neurons - trigeminal neuralgia. This subject aims to observe the regulation of NGF on the existence of M current on the basis of the existence of the M current, and to observe the N in the animal model of trigeminal neuralgia. The change of GF receptor and M channel expression level is helpful for the treatment of trigeminal neuralgia.
Objective: to record the current and verify the M current from the TG, observe the regulation of NGF in the primary cell and expression system, and prepare the animal model of trigeminal neuralgia, observe the changes in the expression of NGF receptor and M channel, and then analyze the possibility that NGF can play a role in neuropathic pain through this pathway.
Method:
(1) amplification and extraction of plasmid cDNA: KCNQ2,3, TrkA gene cloned in pcDNA3.0 plasmid, GFP gene cloned in pEGFP-N1 plasmid, transformed by TOP10 receptive bacteria, and then extracted with kits, and the purity and concentration of plasmids were identified by agarose gel electrophoresis and ultraviolet spectrophotometer.
(2) isolation of trigeminal ganglion neurons and identification of M current: acute isolation of TG neurons in milk rats. After 24 hours, a perforated whole cell patch clamp recording method was used to identify the active tail current. The KCNQ channel specific open agent RTG and the specific blocker XE991 were observed. The sensitivity of KCNQ subtype to TEA was different. The subtypes expressed on TG neurons were verified by Western blot technology for typical KCNQ2 subtypes.
(3) on the expression cells, KCNQ2,3, TrkA and GFP plasmids were co transfected into HEK293B cells, and NGF activated TrkA receptor was recorded to regulate KCNQ2/3 current.
(4) to observe the effect of nerve growth factor (NGF) on M current after activation of its receptor on the primary cultured TG neurons, study the different concentrations of NGF, calculate EC50, and compare and analyze the phenomena observed by the expression system.
(5) the nerve action potential was recorded by current clamp, and the effects of different concentrations of NGF and tool drugs on the discharge patterns of TG neurons were observed, and the regulation of NGF on the activity of nerve discharge was consistent with the regulation of the M current.
(6) the trigeminal neuralgia (TrigeminalNeuralgia, TN) model was prepared by the operation of the suborbital nerve coarctation ring (ION-CCI). The semilunar node of the trigeminal nerve was taken. The expression of TrkA receptor and KCNQ2 channel protein expression was observed by immunohistochemical method, and the NGF regulation was analyzed.
Result:
(1) Plasmid Extraction and identification results: KCNQ2, KCNQ3, TrkA plasmid electrophoresis strips and green fluorescent protein pEGFP-N1 (4.8Kbp) plasmids on pcDNA3.0 (5.4Kbp) vector were cloned to determine the concentration of plasmid DNA in 5000bp.ND-1000 ultraviolet / visible light spectrophotometer. GFP and TrkA are between 200~400ng/ and L, and OD260/OD280 values are between 1.8~1.9.
(2) validation of the M channel of TG neurons: to record the standard protocol of the M current: clamp voltage in -20mV, jump to -50mV continuous 1s, return to -20mV, analyze the slow active tail current (non deactivated M current) of the -50mV segment,.M channel specific agonist can increase the level of the deactivated tail current, and this action can be blocked by specific resistance of the channel. The breaking agent XE991 was completely suppressed to a lower level of.10 mu M RTG to increase the M current to 173.98 + 21.43%, with significant difference (P0.05, n=3); 3 mu M XE991 can reduce M current to 19.97 + 7.58%, and there is a significant difference (P0.01, n=3). In line fitting, the dose effect relationship curve of the tail current was obtained by TEA, and the median inhibitory concentration of TEA was 3.87 + 1.33mM, and the correlation coefficient R2 was Western blot of 0.99739. membrane protein. The result showed that the immunoblotting strip of the KCNQ2 protein in the trigeminal part of the trigeminal nerve was obvious and distributed around 100kD.
(3) NGF acts on the expressed KCNQ2/3 current through the TrkA receptor: the recorded Protocol is the clamp voltage -80mV, depolarizing to -20mV and continuous 1s, then repolarization to -60mV continuous 800ms. analysis -20mV segment, the steady-state activation KCNQ2/3 current magnitude can inhibit the pre drug level to 77.9 + 9.46%, and then recover to 83.4 + 4.67%. N=3), but there is no significant difference between the level after inhibition and after the recovery (P0.05, n=3). Different concentrations of NGF (20,40,80ng/mL) are given in turn, the three concentrations have shown a more obvious inhibitory effect, and the suppressed current is difficult to recover or restore a very small part. Measure the size of the current after the stabilization and activate the current under 80ng/mL NGF. The final level as the maximum inhibitory effect, 20ng/mL NGF inhibition reached 30.6 + 4% inhibitory effect, 40ng/mL NGF was 69.6 + 5.11%, and the pre drug ratio was significantly different (P0.01, n=5). The median inhibitory concentration EC50 was 32.8ng/mL. with Hill equation.
(4) the M current was recorded on the primary cultured TG neurons, the inhibitory effect of NGF was obvious, the concentration gradient of NGF was set to 0.02,0.1,1,10ng/mL. The lowest value of the NGF was measured as the maximum of NGF inhibition, and the inhibition effect of 10ng/mL NGF was set to the standard, and the Hill equation was used to fit the volume effect curve, and the EC50 was 0.0194 + 0.0047ng/mL.NGF. The reactivity of the primary cells is obviously enhanced. One interesting phenomenon is that the current recovery of the suppressed NGF is more obvious when the concentration is increased, and the NGF of 20ng/mL can be recovered to the original 114.6 + 3.35% (P0.05, n=3) if compared with the before and after the restoration of stability.
(5) the effect of NGF on action potential was recorded by current clamp, 500ms current induced action potential, the threshold current level was determined by the step of 50pA increasing step, and then recorded at 1.5 times or 3 times. The results showed that the effect of 0.02ng/mL and 0.1ng/mL on the number of action potential of TG neurons was significantly increased from 3.2 + 0.2 to 4.63 + 0.18 and 13, respectively. .86 + 0.50 (P0.01, n=4).2ng/mL increased from 4.5 + 0.3 of the control to 7.8 + 0.5, and above 10ng/mL the maximum of about 10 Spike was basically reached. The resting membrane potential level gradually changed to depolarization direction as NGF increased with the concentration of NGF.
(6) the experimental results of trigeminal neuralgia (trigeminal neuralgia, TN) in rats: a trigeminal neuralgia model of the supraorbital nerve tightening and ligation (ION-CCI) was successfully established. The model group was compared with before the operation, and the pain threshold decreased significantly at the beginning of the operation (CCI-ipsi) on the sixth day of operation (CCI-ipsi), the 9~12 day decreased to the lowest, and the ninth day pain threshold of the non operative side (CCI-Ctrl) decreased obviously. There was a significant difference between the twelfth days. Compared with the sham operated group, the pain threshold of the model group decreased from sixth days (P0.05) to 9~12 days (P0.01).
(7) immunohistochemical results: the distribution of neurons in the different segments of the trigeminal ganglia was different, and the dense.TrkA receptor and KCNQ2 channel were highly expressed on the cell membrane without relative concentration. The model group was compared with the artificial hand group, the density (P0.01) of the TrkA receptor, the integral light density value (IOD, P0.05) and the positive cell rate (P0.05). The three parameters of KCNQ2 protein increased, but there was no statistical difference, but only the IOD value was statistically significant (P0.05). The three parameters of the model contralateral TrkA and KCNQ2 were all higher and lower respectively compared with the artificial hand group, but only the IOD value was statistically significant (P0.05). The model side was only density value compared to the opposite side. There were significant differences (P0.05).
Conclusion: the trigeminal ganglion (TG) neurons have M channel expression, and the KCNQ2/3 subtype is the main component of the M current, which plays an important role in regulating the excitatory of TG neurons, and NGF is sensitive to the regulation of M current. In trigeminal neuralgia, the level of the TrkA receptor protein surface is up to up, NGF may go through the up - regulated receptor. Step by step increases the control of M current inhibition.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R338
本文编号：2144516
[Abstract]:The M channel is a slow activation of voltage and ligand dependent, non inactivation, and a slow deactivated voltage-gated potassium channel.KCNQ2~5, the four subtypes of homologous or heterologous four polymers are the molecular basis of the neuronal M channels, focusing on the maintenance of the resting membrane potential of the neurons, the regulation of neuronal excitability, and the regulation of synaptic transmission. The significant.KCNQ2~5 subtypes are located on different chromosomes. Except for fifth subtypes, the other subtypes are found to be associated with human genetic diseases, such as benign familial neonatal convulsions (benign familial neonatalconvulsions, BFNCs) and epilepsy, and congenital deafness.
In recent years, it has been found that neuronal M current is involved in signal transduction of chronic pain and inflammatory pain. Inhibition of M channels in neurons and external Zhou Tongjue receptors will lead to neuron depolarization and increased excitement, causing pain. The M current inhibition mediated by multiple pathways is one of the mechanisms of pain generation, enhancing the KCNQ/M channel work Energy (such as increasing the transcriptional level of the KCNQ/M channel and opening the M channel) can obviously relieve the pain of the.KCNQ open agent, which has a good effect on the pain prevention and treatment of various neuropathic pain models and inflammatory pain models.
Nerve growth factor (NGF) is a member of the earliest known family of neurotrophin. It plays an important role in regulating the development, differentiation, growth, regeneration and functional properties of central and peripheral neurons. These biological functions are mostly slow effects. However, more and more evidence has been shown in recent years. Growth factors also show a rapid regulatory effect, such as the release of neurotransmitters, synaptic transmission, ion channel function and neuronal excitability. Previous studies suggest that NGF can regulate sodium channels and calcium channels and calcium activated potassium channels to affect neural activity.
Our previous study found that NGF could also inhibit the M current recorded by the superior cervical ganglion (SCG) and the spinal dorsal root ganglion (dosal root ganglion, DRG) neurons, suggesting that the M channel may become a new path for NGF to regulate nerve excitability. How can the M current and NGF be adjusted to regulate it? This is of great significance for studying the neuropathic pain associated with the electrical activity of TG neurons - trigeminal neuralgia. This subject aims to observe the regulation of NGF on the existence of M current on the basis of the existence of the M current, and to observe the N in the animal model of trigeminal neuralgia. The change of GF receptor and M channel expression level is helpful for the treatment of trigeminal neuralgia.
Objective: to record the current and verify the M current from the TG, observe the regulation of NGF in the primary cell and expression system, and prepare the animal model of trigeminal neuralgia, observe the changes in the expression of NGF receptor and M channel, and then analyze the possibility that NGF can play a role in neuropathic pain through this pathway.
Method:
(1) amplification and extraction of plasmid cDNA: KCNQ2,3, TrkA gene cloned in pcDNA3.0 plasmid, GFP gene cloned in pEGFP-N1 plasmid, transformed by TOP10 receptive bacteria, and then extracted with kits, and the purity and concentration of plasmids were identified by agarose gel electrophoresis and ultraviolet spectrophotometer.
(2) isolation of trigeminal ganglion neurons and identification of M current: acute isolation of TG neurons in milk rats. After 24 hours, a perforated whole cell patch clamp recording method was used to identify the active tail current. The KCNQ channel specific open agent RTG and the specific blocker XE991 were observed. The sensitivity of KCNQ subtype to TEA was different. The subtypes expressed on TG neurons were verified by Western blot technology for typical KCNQ2 subtypes.
(3) on the expression cells, KCNQ2,3, TrkA and GFP plasmids were co transfected into HEK293B cells, and NGF activated TrkA receptor was recorded to regulate KCNQ2/3 current.
(4) to observe the effect of nerve growth factor (NGF) on M current after activation of its receptor on the primary cultured TG neurons, study the different concentrations of NGF, calculate EC50, and compare and analyze the phenomena observed by the expression system.
(5) the nerve action potential was recorded by current clamp, and the effects of different concentrations of NGF and tool drugs on the discharge patterns of TG neurons were observed, and the regulation of NGF on the activity of nerve discharge was consistent with the regulation of the M current.
(6) the trigeminal neuralgia (TrigeminalNeuralgia, TN) model was prepared by the operation of the suborbital nerve coarctation ring (ION-CCI). The semilunar node of the trigeminal nerve was taken. The expression of TrkA receptor and KCNQ2 channel protein expression was observed by immunohistochemical method, and the NGF regulation was analyzed.
Result:
(1) Plasmid Extraction and identification results: KCNQ2, KCNQ3, TrkA plasmid electrophoresis strips and green fluorescent protein pEGFP-N1 (4.8Kbp) plasmids on pcDNA3.0 (5.4Kbp) vector were cloned to determine the concentration of plasmid DNA in 5000bp.ND-1000 ultraviolet / visible light spectrophotometer. GFP and TrkA are between 200~400ng/ and L, and OD260/OD280 values are between 1.8~1.9.
(2) validation of the M channel of TG neurons: to record the standard protocol of the M current: clamp voltage in -20mV, jump to -50mV continuous 1s, return to -20mV, analyze the slow active tail current (non deactivated M current) of the -50mV segment,.M channel specific agonist can increase the level of the deactivated tail current, and this action can be blocked by specific resistance of the channel. The breaking agent XE991 was completely suppressed to a lower level of.10 mu M RTG to increase the M current to 173.98 + 21.43%, with significant difference (P0.05, n=3); 3 mu M XE991 can reduce M current to 19.97 + 7.58%, and there is a significant difference (P0.01, n=3). In line fitting, the dose effect relationship curve of the tail current was obtained by TEA, and the median inhibitory concentration of TEA was 3.87 + 1.33mM, and the correlation coefficient R2 was Western blot of 0.99739. membrane protein. The result showed that the immunoblotting strip of the KCNQ2 protein in the trigeminal part of the trigeminal nerve was obvious and distributed around 100kD.
(3) NGF acts on the expressed KCNQ2/3 current through the TrkA receptor: the recorded Protocol is the clamp voltage -80mV, depolarizing to -20mV and continuous 1s, then repolarization to -60mV continuous 800ms. analysis -20mV segment, the steady-state activation KCNQ2/3 current magnitude can inhibit the pre drug level to 77.9 + 9.46%, and then recover to 83.4 + 4.67%. N=3), but there is no significant difference between the level after inhibition and after the recovery (P0.05, n=3). Different concentrations of NGF (20,40,80ng/mL) are given in turn, the three concentrations have shown a more obvious inhibitory effect, and the suppressed current is difficult to recover or restore a very small part. Measure the size of the current after the stabilization and activate the current under 80ng/mL NGF. The final level as the maximum inhibitory effect, 20ng/mL NGF inhibition reached 30.6 + 4% inhibitory effect, 40ng/mL NGF was 69.6 + 5.11%, and the pre drug ratio was significantly different (P0.01, n=5). The median inhibitory concentration EC50 was 32.8ng/mL. with Hill equation.
(4) the M current was recorded on the primary cultured TG neurons, the inhibitory effect of NGF was obvious, the concentration gradient of NGF was set to 0.02,0.1,1,10ng/mL. The lowest value of the NGF was measured as the maximum of NGF inhibition, and the inhibition effect of 10ng/mL NGF was set to the standard, and the Hill equation was used to fit the volume effect curve, and the EC50 was 0.0194 + 0.0047ng/mL.NGF. The reactivity of the primary cells is obviously enhanced. One interesting phenomenon is that the current recovery of the suppressed NGF is more obvious when the concentration is increased, and the NGF of 20ng/mL can be recovered to the original 114.6 + 3.35% (P0.05, n=3) if compared with the before and after the restoration of stability.
(5) the effect of NGF on action potential was recorded by current clamp, 500ms current induced action potential, the threshold current level was determined by the step of 50pA increasing step, and then recorded at 1.5 times or 3 times. The results showed that the effect of 0.02ng/mL and 0.1ng/mL on the number of action potential of TG neurons was significantly increased from 3.2 + 0.2 to 4.63 + 0.18 and 13, respectively. .86 + 0.50 (P0.01, n=4).2ng/mL increased from 4.5 + 0.3 of the control to 7.8 + 0.5, and above 10ng/mL the maximum of about 10 Spike was basically reached. The resting membrane potential level gradually changed to depolarization direction as NGF increased with the concentration of NGF.
(6) the experimental results of trigeminal neuralgia (trigeminal neuralgia, TN) in rats: a trigeminal neuralgia model of the supraorbital nerve tightening and ligation (ION-CCI) was successfully established. The model group was compared with before the operation, and the pain threshold decreased significantly at the beginning of the operation (CCI-ipsi) on the sixth day of operation (CCI-ipsi), the 9~12 day decreased to the lowest, and the ninth day pain threshold of the non operative side (CCI-Ctrl) decreased obviously. There was a significant difference between the twelfth days. Compared with the sham operated group, the pain threshold of the model group decreased from sixth days (P0.05) to 9~12 days (P0.01).
(7) immunohistochemical results: the distribution of neurons in the different segments of the trigeminal ganglia was different, and the dense.TrkA receptor and KCNQ2 channel were highly expressed on the cell membrane without relative concentration. The model group was compared with the artificial hand group, the density (P0.01) of the TrkA receptor, the integral light density value (IOD, P0.05) and the positive cell rate (P0.05). The three parameters of KCNQ2 protein increased, but there was no statistical difference, but only the IOD value was statistically significant (P0.05). The three parameters of the model contralateral TrkA and KCNQ2 were all higher and lower respectively compared with the artificial hand group, but only the IOD value was statistically significant (P0.05). The model side was only density value compared to the opposite side. There were significant differences (P0.05).
Conclusion: the trigeminal ganglion (TG) neurons have M channel expression, and the KCNQ2/3 subtype is the main component of the M current, which plays an important role in regulating the excitatory of TG neurons, and NGF is sensitive to the regulation of M current. In trigeminal neuralgia, the level of the TrkA receptor protein surface is up to up, NGF may go through the up - regulated receptor. Step by step increases the control of M current inhibition.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R338
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