發(fā)布時(shí)間：2018-09-18 20:02

【摘要】：肌萎縮側(cè)索硬化（Amyotrophic Lateral Sclerosis，ALS）是一個(gè)逐漸進(jìn)展的、具有嚴(yán)重致死性的神經(jīng)變性疾病，以進(jìn)行性運(yùn)動(dòng)神經(jīng)元丟失為特征，發(fā)病機(jī)制不明。TDP-43（TAR DNA bingding protein of43kDa）被認(rèn)為是在肌萎縮側(cè)索硬化（Amyotrophic lateral sclerosis，ALS）及額顳葉癡呆患者大腦中發(fā)現(xiàn)的泛素化包涵體的主要成分，主要功能為參與特定的前mRNA的剪接、轉(zhuǎn)錄、維持其穩(wěn)定性及微小mRNA的生物合成。目前在TDP-43上發(fā)現(xiàn)了40種與散發(fā)性及家族性ALS有關(guān)的顯性突變，為TDP-43的功能異常及神經(jīng)變性病之間的直接聯(lián)系提供了強(qiáng)有力的證據(jù)。TDP-25是在ALS患者的受累大腦區(qū)域中發(fā)現(xiàn)的分子量為25kDa的TDP-43的C末端片段。研究發(fā)現(xiàn)，TDP-25可以促進(jìn)TDP-43包涵體形成，對(duì)運(yùn)動(dòng)神經(jīng)元具有毒性作用，引起神經(jīng)元變性，在疾病的發(fā)病過程中起重要作用，但其機(jī)制尚未明確。最初的觀點(diǎn)認(rèn)為：興奮性毒性可以引起運(yùn)動(dòng)神經(jīng)元損傷。然而，對(duì)ALS患者的神經(jīng)傳導(dǎo)的研究發(fā)現(xiàn)，軸索膜興奮性的提高，及過興奮性的程度與病人的生存期呈負(fù)相關(guān)。興奮性的提高包括持續(xù)性鈉電流的增加及鉀電流的減小。然而對(duì)TDP-43模型是否存在高興奮性尚不清楚。隨著對(duì)ALS研究的不斷深入及對(duì)K+通道的進(jìn)一步了解，ALS與鉀通道的關(guān)系日益受到人們的關(guān)注，延遲整流電流（delayed rectifierpotassium current，IKdr）是影響動(dòng)作電位復(fù)極化過程的主要的電流,但有關(guān)ALS模型細(xì)胞水平上對(duì)鉀電流的研究甚少。同時(shí)，雙甲氧姜黃素（Dimethoxy Curcumin, DMC）是姜黃素的類似物，近年來因其具有抗氧化、抗炎、抗腫瘤、免疫調(diào)節(jié)等功效而受了廣泛關(guān)注，本實(shí)驗(yàn)室研究也發(fā)現(xiàn)其對(duì)轉(zhuǎn)染TDP-43的運(yùn)動(dòng)神經(jīng)元樣細(xì)胞線粒體有保護(hù)作用，可以增強(qiáng)其自噬清除毒性蛋白的能力，此外姜黃素還可以上調(diào)Nrf2和II相酶的水平，減少氧化應(yīng)激產(chǎn)生的損害，進(jìn)而保護(hù)細(xì)胞。 目的：檢測(cè)在穩(wěn)定轉(zhuǎn)染TDP-25基因的運(yùn)動(dòng)神經(jīng)元的延遲整流鉀電流性質(zhì)是否發(fā)生改變及動(dòng)作電位相關(guān)指標(biāo)的改變，以探討TDP-25對(duì)運(yùn)動(dòng)神經(jīng)元產(chǎn)生毒性作用的機(jī)制；進(jìn)一步探討DMC對(duì)電壓門控鉀通的道調(diào)節(jié)作用。 方法：應(yīng)用穩(wěn)定轉(zhuǎn)染TDP-25基因和空質(zhì)粒（Empty vector）的兩種細(xì)胞，該細(xì)胞系由本實(shí)驗(yàn)室構(gòu)建，，參考NSC34細(xì)胞株的培養(yǎng)方法進(jìn)行培養(yǎng)，采用抗生素加壓的方法篩選單克隆細(xì)胞，將篩選所得單克隆細(xì)胞系進(jìn)行培養(yǎng)、傳代。在全細(xì)胞膜片鉗技術(shù)電壓鉗模式下記錄上述兩種細(xì)胞延遲整流鉀電流并分析其性質(zhì)改變。在電流鉗模式下測(cè)定兩種細(xì)胞的動(dòng)作電位，分別給予兩種細(xì)胞20pA、40pA、60pA和80pA4個(gè)階梯遞增的電流刺激動(dòng)作電位發(fā)生，并記錄其閾電位、潛伏期、幅度及動(dòng)作電位半峰時(shí)程（action potential duration at50%repolarization，APD50）等指標(biāo)以進(jìn)行比較。將成功培養(yǎng)的穩(wěn)定轉(zhuǎn)染TDP-25和Empty的NSC34細(xì)胞系傳代，接種于六孔板的小玻璃片上，12小時(shí)后給予換液并給予上述兩種細(xì)胞濃度為10mΜ的DMC24小時(shí)。給予與對(duì)照組相同的刺激程序，觀察DMC對(duì)延遲整流鉀通道電流的影響。 結(jié)果：1、TDP-25組細(xì)胞的電流密度小于Empty組，在60mV時(shí)有統(tǒng)計(jì)學(xué)意義（TDP-25組10.65±3.24，Empty組13.26±6.51P0.05）。2、與Empty組相比，TDP-25組半數(shù)激活電壓V1/2向超極化方向移動(dòng)3.73mV左右(TDP-25組8.18±4.95mV，n=14, Empty組11.91±4.34mV，n=19,P0.05)，表明TDP-25組細(xì)胞的鉀通道更容易在較負(fù)的電位下被激活。斜率K無統(tǒng)計(jì)學(xué)差異（TDP-25組11.46±2.41，Empty組12.82±1.89. P0.05）。3、TDP-25組(n=14)與Empty（n=11）組細(xì)胞的動(dòng)作電位的閾電位值在各個(gè)刺激電流下無統(tǒng)計(jì)學(xué)差異；TDP-25組細(xì)胞的潛伏期值高于Empty組，在40pA時(shí)兩組相比有統(tǒng)計(jì)學(xué)意義（分別為38.99±9.48ms和30.13±6.96ms,P0.05）；TDP-25組細(xì)胞的幅度低于Empty組，其中在20pA時(shí)有統(tǒng)計(jì)學(xué)差異（分別為42.96±7.29mV和47.10±8.69mV, P 0.05）。TDP-25組細(xì)胞的APD50顯著長(zhǎng)于Empty組，在40pA、60pA、80pA時(shí)有統(tǒng)計(jì)學(xué)差異（分別為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）。推斷主要與TDP-25組細(xì)胞IKdr的激活動(dòng)力學(xué)發(fā)生改變有關(guān)。4、在給予TDP-25組細(xì)胞10uM的DMC處理24小時(shí)后，電流密度大于未給藥組細(xì)胞，表明DMC有增加延遲整流鉀電流的趨勢(shì)，但兩組相比無統(tǒng)計(jì)學(xué)差異。5、給藥后IKdr的穩(wěn)態(tài)激活曲線發(fā)生了正向偏移，相應(yīng)地，半數(shù)激活電壓V1/2向去極化方向移動(dòng)了8.93mV左右，未給藥組V1/2=8.18±4.96mV，給藥組V1/2=17.11±7.99mV（P＜0.05)，表明DMC改變了延遲整流鉀電流的激活性質(zhì)，具有促進(jìn)鉀通道開放的作用；激活曲線的斜率k由11.46±2.4增加為16.12±2.40（P＜0.01)，表明DMC使此鉀通道對(duì)電壓的敏感性增強(qiáng)。 結(jié)論：在本實(shí)驗(yàn)中，我們?cè)谶\(yùn)動(dòng)神經(jīng)元樣細(xì)胞上成功檢測(cè)出延遲整流鉀電流，并發(fā)現(xiàn)TDP-25通過改變延遲整流鉀電流的激活性質(zhì)，抑制了延遲整流鉀通道的活性，減小電流密度，延長(zhǎng)動(dòng)作電位的復(fù)極化時(shí)程，增大了細(xì)胞的放電頻率，從而引起神經(jīng)元興奮性增高，對(duì)神經(jīng)元產(chǎn)生興奮毒性作用，據(jù)此推測(cè)TDP-25對(duì)鉀電流的抑制產(chǎn)生的細(xì)胞興奮性升高可能是其引起運(yùn)動(dòng)神經(jīng)元變性的可能的機(jī)制之一。雙甲氧姜黃素（DMC）具有促進(jìn)延遲整流鉀通道開放的作用，改變其激活特性，并可能因此增加延遲整流鉀電流的幅度降低神經(jīng)元興奮性，從而對(duì)神經(jīng)元起到保護(hù)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R744.8

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 李輝;肌萎縮側(cè)索硬化和運(yùn)動(dòng)神經(jīng)元內(nèi)在高興奮性的關(guān)系[D];河北醫(yī)科大學(xué);2016年

本文編號(hào)：2248975