【摘要】：M通道是電壓及配體依賴性的慢激活、非失活、慢去活的電壓門控性鉀通道。KCNQ2~5這四個亞型構(gòu)成的同源或異源四聚體是構(gòu)成神經(jīng)元M通道的分子基礎(chǔ)，對維持神經(jīng)元靜息膜電位、調(diào)節(jié)神經(jīng)元興奮性和調(diào)節(jié)突觸傳遞等神經(jīng)生理活動有著重要的意義。KCNQ2~5亞型基因分別位于不同的染色體上，除了第五個亞型以外，其他亞型的突變均發(fā)現(xiàn)與人類遺傳疾病相關(guān)，如良性家族性新生兒驚厥（benign familial neonatalconvulsions, BFNCs）和癲癇，先天性耳聾等。 近年來研究發(fā)現(xiàn)，神經(jīng)元M電流參與了慢性疼痛和炎性疼痛的的信號轉(zhuǎn)導(dǎo)。神經(jīng)元和外周痛覺感受器M通道的抑制將會導(dǎo)致神經(jīng)元去極化且興奮性增高，從而引發(fā)疼痛。由多個通路介導(dǎo)的M電流抑制是疼痛產(chǎn)生的機(jī)制之一，增強(qiáng)KCNQ/M通道功能（如提高KCNQ/M通道轉(zhuǎn)錄水平和開放M通道等）可以明顯減輕疼痛。KCNQ開放劑對多種神經(jīng)病理性疼痛模型及炎性疼痛模型都具有很好的疼痛預(yù)防及治療作用。 神經(jīng)生長因子(Nerve growth factor, NGF)是最早被發(fā)現(xiàn)的神經(jīng)營養(yǎng)素家族成員，它對中樞及周圍神經(jīng)元的發(fā)育、分化、生長、再生和功能特性的表達(dá)均具有重要的調(diào)控作用。這些生物學(xué)作用多是一種慢效應(yīng)，然而近年來越來越多的證據(jù)表明，神經(jīng)生長因子還表現(xiàn)出快速的調(diào)節(jié)作用，如神經(jīng)遞質(zhì)的釋放，突觸的傳遞，離子通道功能和神經(jīng)元興奮性等，以往的研究認(rèn)為NGF可以調(diào)節(jié)鈉通道和鈣通道以及鈣激活的鉀通道影響神經(jīng)活動。 我們的前期研究發(fā)現(xiàn)NGF還可以抑制大鼠頸上交感神經(jīng)節(jié)（superior cervical ganglion, SCG）和脊髓背根神經(jīng)節(jié)（dosal root ganglion,DRG）神經(jīng)元記錄的M電流，提示M通道可能會成為NGF調(diào)節(jié)神經(jīng)興奮性的新途徑。而對于三叉神經(jīng)節(jié)（trigeminal ganglion, TG）神經(jīng)元上是否能記錄到M電流及NGF又有怎樣地調(diào)節(jié)它？這對于研究TG神經(jīng)元電活動相關(guān)的神經(jīng)病理性疼痛——三叉神經(jīng)痛有重要意義。本課題旨在TG神經(jīng)元上驗證M電流存在的基礎(chǔ)上，觀察NGF對其調(diào)節(jié)規(guī)律，并在三叉神經(jīng)痛動物模型上觀察NGF受體和M通道表達(dá)水平變化，為三叉神經(jīng)痛治療提供幫助。 目的：從TG經(jīng)元上記錄M電流并進(jìn)行驗證，在原代細(xì)胞和表達(dá)系統(tǒng)觀察NGF對它的調(diào)節(jié)規(guī)律，并制備三叉神經(jīng)疼痛動物模型，觀察NGF受體和M通道表達(dá)變化，進(jìn)而分析NGF通過這一途徑參與神經(jīng)病理性疼痛發(fā)揮作用的可能性。 方法： (1)質(zhì)粒cDNA的擴(kuò)增與提�。� KCNQ2、3， TrkA基因克隆在pcDNA3.0質(zhì)粒，GFP基因克隆在pEGFP-N1質(zhì)粒，用TOP10感受態(tài)細(xì)菌轉(zhuǎn)化、擴(kuò)增之后用試劑盒提取，并用瓊脂糖凝膠電泳和紫外分光光度計鑒定質(zhì)粒的純度與濃度。 (2)大鼠三叉神經(jīng)節(jié)神經(jīng)元分離及M電流的鑒別：急性分離乳鼠TG神經(jīng)細(xì)胞，24小時后采用穿孔全細(xì)胞膜片鉗記錄方式，對去活尾電流進(jìn)行鑒別，用KCNQ通道特異性開放劑RTG和特異性阻斷劑XE991進(jìn)行觀察。利用KCNQ亞型對TEA的敏感度不同，分析TG神經(jīng)元上所表達(dá)的亞型，再用western blot技術(shù)對典型的KCNQ2亞型進(jìn)行驗證。 (3)在表達(dá)細(xì)胞上，通過將KCNQ2、3，TrkA及GFP質(zhì)粒共轉(zhuǎn)染于HEK293B細(xì)胞，記錄NGF激活TrkA受體實現(xiàn)對KCNQ2/3電流的調(diào)節(jié)作用。 (4)在原代培養(yǎng)的TG神經(jīng)元細(xì)胞上觀察神經(jīng)生長因子（NGF）激活其受體后對M電流的作用，對不同濃度NGF進(jìn)行研究，計算EC50，并與表達(dá)系統(tǒng)觀察到的現(xiàn)象進(jìn)行比較分析。 (5)用電流鉗方式記錄神經(jīng)動作電位，通過觀察不同濃度NGF和工具藥物對TG神經(jīng)元放電模式的影響，分析NGF對神經(jīng)放電活動的調(diào)節(jié)作用是否與M電流的調(diào)節(jié)變化一致。 (6)通過眶下神經(jīng)縮窄環(huán)（ION-CCI）手術(shù)制備三叉神經(jīng)痛（trigeminalneuralgia, TN）模型，取三叉神經(jīng)半月節(jié)，用免疫組化方法，觀察TrkA受體及KCNQ2通道蛋白表達(dá)水平變化，分析NGF調(diào)節(jié)。 結(jié)果： (1)質(zhì)粒提取與鑒定結(jié)果：克隆在pcDNA3.0(5.4Kbp)載體上的KCNQ2、 KCNQ3、 TrkA質(zhì)粒電泳條帶和綠色熒光蛋白pEGFP-N1(4.8Kbp)質(zhì)粒的電泳條帶均在5000bp左右。ND-1000紫外/可見光分光光度計測得質(zhì)粒DNA的濃度值，其中KCNQ2、KCNQ3濃度均在400~600ng/μL之間， GFP和TrkA均在200~400ng/μL之間；OD260/OD280值均在1.8~1.9之間。 (2) TG神經(jīng)元M通道的驗證：以記錄M電流的標(biāo)準(zhǔn)protocol：鉗制電壓在-20mV，躍遷至-50mV持續(xù)1s，再回到-20mV，分析-50mV段的慢去活尾電流（非失活的M電流）。M通道特異性激動劑RTG可以增大去活尾電流的水平，且這種激動作用可被M通道特異性阻斷劑XE991完全抑制至更低水平。10μM RTG可以使M電流升高至173.98±21.43%，有顯著性差異（P0.05，n=3）；3μM XE991可使M電流降低至19.97±7.58%，有極顯著性差異（P0.01，n=3）。以不同濃度的TEA溶液（0.03~30mM）抑制M電流幫助判斷M通道亞型，并用Logistic方程進(jìn)行擬合，得到TEA對該尾電流的量效關(guān)系曲線，同時求得TEA的半數(shù)抑制濃度IC50為3.87±1.33mM，相關(guān)系數(shù)R2為0.99739。膜蛋白的Western blot結(jié)果顯示，三叉神經(jīng)半月節(jié)部位KCNQ2蛋白的免疫印跡條帶明顯，分布在100kD左右。 (3) NGF通過TrkA受體對表達(dá)的KCNQ2/3電流作用：記錄的Protocol為鉗制電壓-80mV，去極化至-20mV并持續(xù)1s，再復(fù)極化至-60mV持續(xù)800ms。分析-20mV段的穩(wěn)態(tài)激活KCNQ2/3電流大小。NGF(20ng/mL)可以抑制藥前水平到77.9±9.46%(P0.05, n=3)，再恢復(fù)至83.4±4.67%(P0.05, n=3)，但抑制后水平與恢復(fù)后水平相比無顯著性差異(P0.05, n=3)。不同濃度NGF(20、40、80ng/mL)依次給藥，三個濃度都表現(xiàn)出了較為明顯的抑制作用，而且抑制后的電流很難恢復(fù)或恢復(fù)很小一部分。衡量穩(wěn)定后電流大小，以80ng/mL NGF下激活電流的終水平作為最大抑制效應(yīng)，20ng/mL NGF抑制達(dá)到30.6±4.00%的抑制效應(yīng)，40ng/mL NGF為69.6±5.11%，與藥前比均具有極顯著性差異（P0.01,n=5）。用Hill方程擬合得到半數(shù)抑制濃度EC50為32.8ng/mL。 (4)原代培養(yǎng)的TG神經(jīng)元上記錄M電流，NGF抑制作用明顯，NGF的濃度梯度設(shè)為0.02、0.1、1、10ng/mL，測量給藥后出現(xiàn)的最低值作為NGF抑制最大值，將10ng/mL NGF的抑制作用設(shè)為100%（標(biāo)準(zhǔn)化），用Hill方程進(jìn)行擬合量效曲線，求得EC50為0.0194±0.0047ng/mL。NGF在原代細(xì)胞上的反應(yīng)性明顯增強(qiáng)。一個很有意思的現(xiàn)象是，，當(dāng)NGF濃度增大時抑制的電流恢復(fù)的比較明顯，如果與藥前比較，恢復(fù)穩(wěn)定后測量，20ng/mL的NGF可以恢復(fù)到原來的114.6±3.35%(P0.05，n=3)。 (5) NGF對動作電位的影響，以電流鉗方式記錄，500ms電流誘發(fā)動作電位，以50pA遞增的躍階形式確定閾電流水平，再以1.5倍或3倍水平進(jìn)行記錄。結(jié)果表明，0.02ng/mL和0.1ng/mL對TG神經(jīng)元動作電位個數(shù)的影響顯著：從3.2±0.2分別增加到4.63±0.18和13.86±0.50(P0.01，n=4)。2ng/mL由對照的4.5±0.3個增加到7.8±0.5個，10ng/mL以上基本達(dá)到約10個Spike的最大值，靜息膜電位水平隨NGF隨濃度增大而逐漸向去極化方向改變。 (6)大鼠三叉神經(jīng)痛（trigeminal neuralgia, TN）行為學(xué)實驗結(jié)果：成功建立了眶下神經(jīng)緊縮結(jié)扎（ION-CCI）的三叉神經(jīng)痛模型。模型組大鼠與手術(shù)前相比，鼠術(shù)側(cè)（CCI-ipsi）第6天開始痛閾明顯降低，第9~12天降至最低；非術(shù)側(cè)（CCI-Ctrl）第9天痛閾明顯降低，直到第12天均有顯著差異；與假手術(shù)組比較，模型組動物術(shù)側(cè)痛閾從第6天開始降低（P0.05），9~12天更明顯(P0.01)。 (7)免疫組化結(jié)果：神經(jīng)元胞體在三叉神經(jīng)節(jié)內(nèi)不同段分布不同，且無相對集中的密集處。TrkA受體和KCNQ2通道單位均在細(xì)胞膜上高表達(dá)，胞內(nèi)較少。模型組術(shù)側(cè)與假手組相比，TrkA受體的density(P0.01)、積分光密度值（IOD，P0.05)和陽性細(xì)胞率(P0.05)都顯著升高；KCNQ2蛋白三個參數(shù)有升高趨勢，但無統(tǒng)計學(xué)差異，但只有IOD值有統(tǒng)計學(xué)意義（P0.05）。模型對側(cè)TrkA和KCNQ2與假手組相比三個參數(shù)均分別有所升高和降低，但是都只有IOD值有統(tǒng)計學(xué)意義（P0.05）。模型術(shù)側(cè)與對側(cè)相比，只有density值有顯著性差異（P0.05）。 結(jié)論：三叉神經(jīng)節(jié)（TG）神經(jīng)元上確有M通道表達(dá)，而且KCNQ2/3亞型是構(gòu)成M電流的主要成分，對TG神經(jīng)元興奮性起重要調(diào)節(jié)作用，而且NGF對M電流的調(diào)節(jié)作用很敏感。在三叉神經(jīng)痛時，TrkA受體蛋白表達(dá)水平會上調(diào)，NGF可能會通過上調(diào)的受體進(jìn)一步增加對M電流抑制性調(diào)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R338

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