【摘要】： 外周炎癥和組織損傷都能導致病理痛的產(chǎn)生。作為傷害性信號傳遞的中樞第一站，脊髓是傷害性信息傳遞和調(diào)控的重要部位。已有的報道表明，脊髓的中樞敏化是病理痛形成的主要機制之一；然而，人們對這種敏化的機制卻了解甚少。在炎性或神經(jīng)病理性疼痛狀態(tài)下，痛覺過敏產(chǎn)生的重要原因之一是由于脊髓背角神經(jīng)元表達的離子通道和受體受到調(diào)控，從而使其持續(xù)過度興奮。目前，有多種離子通道和受體被認為和疼痛的產(chǎn)生與維持相關(guān)。 酸敏感離子通道(ASICs)是一類由質(zhì)子(H~+)激活的陽離子通道，屬于ENaC／DEG家族，有四個編碼的基因：ASIC1、ASIC2、ASIC3、ASIC4。它們編碼六種亞基蛋白：ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。酸敏感離子通道的N-端和C-端均在胞內(nèi)，有兩個跨膜區(qū)，胞外環(huán)上富含半胱氨酸。最新的結(jié)構(gòu)生物學實驗發(fā)現(xiàn)，體內(nèi)的ASICs是由相同的或不同的亞基構(gòu)成的三聚體。除了ASIC3和ASIC1b只在外周神經(jīng)系統(tǒng)中特異表達外，ASICs其他亞基在外周和中樞神經(jīng)系統(tǒng)都有存在。目前對ASICs的大部分研究主要集中在外周神經(jīng)系統(tǒng)中，認為ASICs參與了痛覺、味覺以及機械感覺等的信息傳遞；而對中樞神經(jīng)系統(tǒng)中的ASICs功能了解較少，尤其是對病理條件下ASICs的變化如何了解不多。 目前，背根神經(jīng)節(jié)(DRG)的ASICs研究已有大量報道，并被認為與機械感受和痛覺相關(guān)。對于ASICs在中樞痛覺感受中的作用目前尚未見報道。已有報道提及ASICs在脊髓中有表達，另外在脊髓培養(yǎng)神經(jīng)元中也觀察到了酸誘導的電流。然而，對于脊髓背角神經(jīng)元中酸誘導電流的分子基礎、通道特性及其生理、病理功能卻很不清楚。 因此，在本課題中，我們首先證明了脊髓背角神經(jīng)元中主要表達Ca~(2+)通透性的ASIC1a通道。進一步，我們探討了ASIC1a通道參與脊髓水平痛覺傳遞和調(diào)制的作用及其機制。主要實驗結(jié)果如下： 1．脊髓背角神經(jīng)元中酸敏感離子通道特性研究 運用電生理的方法，我們首先發(fā)現(xiàn)，對于急性分離的脊髓背角神經(jīng)元，Psalmotoxin 1(PcTX1)，一種特異的ASIC1a同聚體通道抑制劑，能夠非常明顯的抑制酸性溶液(pH6.0)引發(fā)的內(nèi)向電流。采用RNA干擾的手段，我們特異的抑制培養(yǎng)的脊髓背角神經(jīng)元中ASIC1a的表達，隨后的電生理實驗發(fā)現(xiàn)，酸性溶液引發(fā)的內(nèi)向電流也明顯的降低。這提示我們ASIC1a同聚體通道是脊髓背角神經(jīng)元中主要的質(zhì)子感受器。進一步的鈣離子成像實驗表明，在脊髓背角神經(jīng)元中，ASIC1a同聚體通道介導了酸性溶液引起的Ca~(2+)內(nèi)流。 2．脊髓背角酸敏感離子通道與炎性痛覺超敏 運用完全弗氏佐劑(CFA)致炎模型，我們進一步研究了ASIC1a通道在炎癥痛中的作用。行為學實驗表明，大鼠腳底CFA致炎能夠?qū)е旅黠@的熱和機械超敏化，這種行為超敏化能夠持續(xù)超過七天。然而，鞘內(nèi)注射PcTX1能夠明暴地削弱這種外周炎癥引起的熱和機械超敏化。同時，鞘內(nèi)注射阿米洛利(anailoride)，一種非特異性的ASICs抑制劑，也產(chǎn)生同樣的效果。進一步，我們使用一條特異性的抑制ASIC1a蛋白表達的反義寡核苷酸發(fā)現(xiàn)，與急性抑制ASIC1a功能相似，在抑制脊髓背角ASIC1a蛋白表達之后，外周炎癥引起的熱和機械超敏化也被明顯削弱。與之相反的是，鞘內(nèi)注射PcTX1并不影響正常大鼠的傷害性信號傳遞。 為了進一步證明脊髓背角的ASIC1a通道參與炎性痛覺超敏，我們采用福爾馬林模型闡明脊髓背角和背根神經(jīng)節(jié)中的ASIC1a通道的功能異同。給大鼠腳背注射福爾馬林能夠使其產(chǎn)生持續(xù)約一小時的傷害性反應。根據(jù)持續(xù)時間，這種反應被分為兩相：第一相僅與背根神經(jīng)節(jié)細胞超興奮相關(guān)；第二相與脊髓背角神經(jīng)元超興奮關(guān)系密切。我們的實驗表明，鞘內(nèi)注射PcTX1僅抑制第二相持續(xù)反應，而不影響第一相反應。相反地，腳底注射PcTX1對傷害性傳遞沒有作用。結(jié)合這兩個實驗，我們進一步證明脊髓背角ASIC1a通道參與傷害性信號傳遞。 3．脊髓背角酸敏感離子通道影響脊髓背角神經(jīng)元的興奮性和可塑性 脊髓背角ASIC1a通道是如何參與傷害性信號傳遞的呢?免疫組織化學實驗表明，在外周致炎后，脊髓背角神經(jīng)元中ASIC1a的蛋白表達量明顯增加。與之相反，ASIC1a在背根神經(jīng)節(jié)細胞的中樞端和膠質(zhì)細胞中的表達卻很少。進一步的在體電生理實驗表明，電刺激大鼠足底神經(jīng)激動C-纖維時，脊髓背角廣動力域(WDR)神經(jīng)元的興奮性改變，其表現(xiàn)形式為自發(fā)放電增強(“wind-up”現(xiàn)象)。而在脊髓急性給予PcTX1能構(gòu)有效的抑制“wind-up”現(xiàn)象。隨后，向WDR神經(jīng)元的外周感受野中心部位分別施加不同強度的刷(brushing)、壓(pressing)和夾(pinching)等機械性刺激；而在外周致炎的條件下，WDR神經(jīng)元對不同強度brushing、pressing和pinching等機械性刺激反應性明顯增強，說明有機械性痛敏產(chǎn)生。進一步的實驗表明，脊髓急性給予PcTX1，并不影響正常大鼠脊髓背角WDR神經(jīng)元對機械性刺激的反應。然而，脊髓急性給予PcTX1能夠明顯抑制致炎大鼠的WDR神經(jīng)元對傷害性機械刺激(包括pressing和pinching)的反應性，而對非傷害性機械刺激(brushing)引起的反應沒有作用。 4．炎性痛中脊髓背角酸敏感離子通道上調(diào)機制初探 在炎癥過程中，大量的神經(jīng)遞質(zhì)(Glu)和調(diào)質(zhì)[P物質(zhì)(SP)、BDNF]等從DRG細胞突觸終末釋放，影響脊髓背角的突觸傳遞。我們發(fā)現(xiàn)，慢性施加BDNF能夠濃度依賴性的增加培養(yǎng)的脊髓背角神經(jīng)元中ASIC1a表達。進一步，給大鼠鞘內(nèi)長期注射BDNF受體-TrkB的抑制劑K252a，能夠抑制外周炎癥引起的脊髓背角ASIC1a的增加和痛行為。而且，在K252a慢性處理的大鼠脊髓中注射PcTX1，不能進一步的抑制痛行為。這提示我們可能是BDNF介導了炎性痛中ASIC1a的上調(diào)。另外，穿孔膜片鉗實驗表明，PI3K信號通路，而不是PKA和CaMKⅡ信號通路，介導了BDNF引起的ASIC1a上調(diào)。 綜上所述，本文鑒定了Ca~(2+)通透性的ASIC1a同聚體通道是脊髓背角主要存在的ASIC；外周炎癥導致脊髓背角神經(jīng)元中ASIC1a的表達增加；過高表達的ASIC1a通道增加脊髓背角神經(jīng)元的興奮性和可塑性，并參與炎性痛覺敏化的過程。該研究結(jié)果揭示了生物體內(nèi)痛覺誘發(fā)和維持的一種新機制，，并且提示脊髓背角的ASIC1a通道可能成為研究鎮(zhèn)痛藥物的新靶點。
[Abstract]:Peripheral inflammation and tissue damage can cause pathological pain. As the first station of nociceptive signal transmission, the spinal cord is an important part of the transmission and regulation of nociceptive information. It has been reported that the central sensitization of the spinal cord is one of the main mechanisms of the formation of pathological pain; however, few people know the mechanism of this sensitization. In the state of inflammatory or neuropathic pain, one of the important causes of hyperalgesia is the regulation of ion channels and receptors expressed in the dorsal horn neurons of the spinal cord, which makes it excessively excited. At present, a variety of ion channels and receptors are considered to be associated with the production and maintenance of pain.
Acid sensitive ion channel (ASICs) is a class of cationic channels activated by protons (H~+), belonging to the ENaC / DEG family. There are four encoded genes: ASIC1, ASIC2, ASIC3, and ASIC4., which encode six subunits: ASIC1a, ASIC1b, ASIC2a, ASIC2b, and two transmembrane regions. The outer ring is rich in cysteine. The latest structural biology experiments have found that the ASICs in the body is a trimer composed of the same or different subunits. In addition to the specific expression of ASIC3 and ASIC1b in the peripheral nervous system, the other subunits of ASICs are stored in the peripheral and central nervous systems. Most of the research on ASICs is mainly concentrated. In the peripheral nervous system, ASICs is considered to be involved in the transmission of information of pain, taste and mechanical sensation, but less on the ASICs function in the central nervous system, especially on how the changes of ASICs in the pathological conditions are not well understood.
Currently, the ASICs study of the dorsal root ganglion (DRG) has been widely reported and is considered to be associated with mechanical perception and pain. The role of ASICs in central pain perception has not yet been reported. It has been reported that ASICs has been expressed in the spinal cord, and acid induced current is also observed in spinal cultured neurons. The molecular basis, channel characteristics, physiological and pathological functions of acid induced currents in neurons of the dorsal horn of the spinal cord are unclear.
Therefore, in this subject, we first demonstrated the ASIC1a channel that mainly expressed Ca~ (2+) permeability in the spinal dorsal horn neurons. Further, we explored the role and mechanism of the ASIC1a channel in the spinal level of spinal level pain transmission and modulation. The main experimental results are as follows:
Characteristics of acid sensing ion channels in spinal dorsal horn neurons in 1.
Using electrophysiological methods, we first found that, for the acute isolated spinal dorsal horn neurons, Psalmotoxin 1 (PcTX1), a specific ASIC1a homopolymer inhibitor, can significantly inhibit the inward current induced by acidic solution (pH6.0). Using RNA interference, we specifically inhibit the cultured spinal dorsal horn neurons. The expression of ASIC1a, followed by electrophysiological experiments, found that the inward current caused by the acid solution was also significantly reduced. This suggests that our ASIC1a homopolymer channel is the main proton receptor in the spinal dorsal horn neurons. Further calcium imaging experiments showed that the ASIC1a homopolymer channel mediated acid dissolution in the dorsal horn of the spinal cord of the spinal cord. Ca~ (2+) internal flow caused by liquid.
2. acid sensitive ion channels in spinal dorsal horn and inflammatory hyperalgesia
Using the complete Freund adjuvant (CFA) induced inflammatory model, we further studied the role of the ASIC1a channel in inflammatory pain. Behavioral experiments showed that the CFA induced inflammation in the sole of the rat can lead to obvious thermal and mechanical hyper sensitization, and this behavioral hypersensitization can last over seven days. However, intrathecal PcTX1 can clearly weaken the peripheral inflammation. Thermal and mechanical hyper sensitization. Meanwhile, intrathecal amiloride (anailoride), a nonspecific ASICs inhibitor, also produced the same effect. Further, we use a specific antisense oligodeoligonucleotide to inhibit the expression of ASIC1a protein, similar to the acute inhibition of ASIC1a function, in inhibiting the ASIC1a protein table in the dorsal horn of the spinal cord. After that, the heat and mechanical hyper sensitization caused by peripheral inflammation were also significantly weakened. In contrast, intrathecal injection of PcTX1 did not affect the nociceptive signal transmission in normal rats.
To further demonstrate the involvement of the ASIC1a channel in the dorsal horn of the spinal cord in inflammatory pain hypersensitivity, we used the Faure Marin model to elucidate the functional differences in the ASIC1a channel in the dorsal horn of the spinal cord and the dorsal root ganglion. The injections of the dorsum of the rat to the dorsum of the rat were able to produce a persistent nociceptive response for about one hour. The first phase was related only to the superexcitability of the dorsal root ganglion cells; the second phase was closely related to the superexcitability of the dorsal horn neurons of the spinal cord. Our experiment showed that intrathecal injection of PcTX1 only inhibited the second phase, but did not affect the first phase reaction. On the contrary, the infoot injection of PcTX1 had no effect on nociceptive transmission. We further demonstrate that the ASIC1a channel in the dorsal horn of the spinal cord is involved in noxious signaling.
3. the acid sensitive ion channel of spinal dorsal horn affects the excitability and plasticity of neurons in spinal dorsal horn.
How does the spinal dorsal horn ASIC1a channel participate in nociceptive signal transduction? Immunohistochemical experiments show that the protein expression of ASIC1a in the dorsal horn neurons of the spinal cord increases obviously after the peripheral inflammation. On the contrary, the expression of ASIC1a in the central and glial cells of the dorsal root ganglion cells is few. The experimental results showed that the excitatory changes of the spinal dorsal horn (WDR) neurons were stimulated by electrical stimulation of the C- fibers in the plantar nerve of the rat, and the manifestation was the spontaneous discharge enhancement ("wind-up"). The acute administration of PcTX1 in the spinal cord could effectively inhibit the "wind-up" image. Then, the central part of the peripheral receptive field of the WDR neurons was divided. Do not apply mechanical stimuli such as brushing, pressing and pinching, and the mechanical irritation of WDR neurons to brushing, pressing, pinching and other mechanical stimuli in the peripheral inflammation, indicating that mechanical pain is produced. Further experiments suggest that PcTX1 is given acute spinal cord in the spinal cord. The response to mechanical stimulation of WDR neurons in the dorsal horn of the spinal cord of normal rats was not affected. However, acute spinal cord administration of the spinal cord in the spinal cord could significantly inhibit the reactivity of WDR neurons in the inflammatory rats to nociceptive mechanical stimulation (including pressing and pinching), but had no effect on the response to non nociceptive mechanical stimulation (brushing).
Mechanism of upregulated acid sensing ion channels in spinal dorsal horn in 4. inflammatory pain
During the process of inflammation, a large number of neurotransmitters (Glu) and substance [P (SP) and BDNF] are released from DRG cell synaptic terminals, affecting synaptic transmission in the dorsal horn of the spinal cord. We found that the chronic exertion of BDNF can increase the ASIC1a expression in the cultured spinal dorsal horn neurons in a concentration dependent manner. Further, the long-term injection of BDNF receptor -TrkB in the rat sheath K252a, a inhibitor, can inhibit the increase and pain behavior of the spinal dorsal horn ASIC1a caused by peripheral inflammation. Moreover, the injection of PcTX1 in the spinal cord of K252a chronic treated rats can not further inhibit the pain behavior. This suggests that we may mediate the up regulation of ASIC1a in inflammatory pain. Furthermore, the perforated patch clamp experiment indicates that the PI3K signal pathway is indicated by the perforated patch clamp test. Rather than PKA and CaMK II signaling pathways, BDNF induced ASIC1a upregulation.
In summary, the Ca~ (2+) permeability ASIC1a homopolymer channel is the main ASIC in the dorsal horn of the spinal cord; peripheral inflammation leads to an increase in the expression of ASIC1a in the dorsal horn neurons of the spinal cord; the exorbitant ASIC1a channel increases the excitability and plasticity of the spinal dorsal horn neurons and participates in the process of inflammatory pain sensitization. The results reveal a new mechanism of pain induction and maintenance in vivo, and suggest that ASC1a channel in spinal dorsal horn may be a new target for the study of analgesics.
【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R363

【引證文獻】

相關(guān)碩士學位論文 前1條

1 陸瑤;外周酸敏感離子通道在蜜蜂毒誘致的自發(fā)痛、痛敏和炎癥中的作用[D];遼寧醫(yī)學院;2012年

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