本文選題：背根神經(jīng)節(jié) + GABA回路��； 參考：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：軀體感覺神經(jīng)元是機(jī)體感覺系統(tǒng)的重要組成部分,其胞體存在于背根神經(jīng)節(jié)(dorsal root ganglion,DRG),DRG神經(jīng)元外周軸突末梢可感受各種各樣的外周刺激例如溫度刺激、化學(xué)刺激和機(jī)械刺激等,并將其轉(zhuǎn)化為神經(jīng)沖動向中樞傳遞,從而形成溫度覺、痛覺和觸覺等感覺。我們前期的研究發(fā)現(xiàn)DRG神經(jīng)元不僅具有痛覺信號形成和傳遞作用,而且還兼具痛覺信號調(diào)節(jié)作用。DRG局部存在功能性的GABA回路系統(tǒng),DRG組織和培養(yǎng)細(xì)胞在多種興奮性刺激作用下可有GABA釋放,DRG局部應(yīng)用GABA對緩激肽誘發(fā)的急性炎性疼痛有顯著的抑制作用。這些均提示DRG部位(特別是該部位的GABA系統(tǒng))可能作為疼痛治療的潛在靶點。慢性神經(jīng)病理性疼痛是臨床常見疾病,其發(fā)病機(jī)制尚不清楚,目前臨床針對慢性神經(jīng)病理性疼痛尚缺乏有效的治療手段。本論文中我們將重點關(guān)注DRG局部GABA系統(tǒng)在慢性神經(jīng)病理性疼痛的發(fā)生和治療中的作用。首先,為了研究DRG局部GABA系統(tǒng)在疼痛發(fā)生過程中的作用,我們建立了一個全新的“sniffer patch”電生理實驗方法,證明大鼠DRG神經(jīng)元在興奮情況下可直接釋放GABA,這為DRG局部存在功能性的GABA系統(tǒng)提供了更直接的證據(jù)。其次,我們通過制備大鼠坐骨神經(jīng)慢性擠壓損傷(CCI)神經(jīng)病理性疼痛模型,觀察疼痛進(jìn)展期DRG神經(jīng)元GABA電流密度的改變,以研究DRG局部GABA系統(tǒng)在神經(jīng)病理性疼痛發(fā)生中的作用。最后,我們通過滲透泵靶向DRG給藥系統(tǒng),觀察GABA對神經(jīng)性病理性疼痛反應(yīng)的影響,分析DRG局部GABA系統(tǒng)作為慢性神經(jīng)病理性疼痛治療靶點的可能性。第一部分通過“Sniffer Patch”方法證明DRG神經(jīng)元的GABA釋放功能目的:建立一種全新的電生理實驗方法“sniffer patch”,證明DRG神經(jīng)元具有直接釋放GABA的功能。方法:打孔“sniffer patch”電生理實驗方法,半定量PCR技術(shù)VGAT-ChR2-YFP小鼠鑒定,冰凍切片-免疫熒光技術(shù),統(tǒng)計方法為t檢驗。結(jié)果:①藥物刺激DRG神經(jīng)元“Sniffer Patch”結(jié)果:將表達(dá)有GABAA受體的HEK293B細(xì)胞與大鼠急性分離DRG細(xì)胞共培養(yǎng),與小直徑DRG緊密相鄰的轉(zhuǎn)染了 GABAA受體的HEK293B細(xì)胞在200μLM GABA作用下產(chǎn)生很大的GABA-Cl-電流。而當(dāng)給予TRPV1受體的激動劑辣椒素激活小直經(jīng)DRG神經(jīng)元,用sniffer patch的方法在相鄰HEK293B細(xì)胞記錄到類似的GABA樣電流。而辣椒素不能誘發(fā)單獨培養(yǎng)的轉(zhuǎn)染了 GABAA受體的HEK293B細(xì)胞,以及與DRG共培養(yǎng)的空白HEK293B細(xì)胞產(chǎn)生任何電流。②機(jī)械刺激DRG神經(jīng)元的“Sniffer Patch”結(jié)果:GABAA受體轉(zhuǎn)染HEK293B細(xì)胞與大鼠DRG細(xì)胞共培養(yǎng),給予表達(dá)有GAB AA受體的HEK293B細(xì)胞緊密相鄰的小直徑DRG機(jī)械刺激,未能誘發(fā)HEK293B細(xì)胞產(chǎn)生GABA樣電流。③光激活VGAT-ChR2-YFP 小鼠 DRG 神經(jīng)元的“Sniffer Patch”結(jié)果:分離VGAT-ChR2-YFP小鼠DRG細(xì)胞,如上述方法進(jìn)行共培養(yǎng),在473nm激光刺激下,在與轉(zhuǎn)基因小鼠VGAT-ChR2-YFP的DRG緊密相鄰的表達(dá)有GABAA受體的HEK293B細(xì)胞記錄到一個小的內(nèi)向電流,比在單獨培養(yǎng)的表達(dá)有GABAA受體的HEK293B細(xì)胞,以及與野生型小鼠的DRG緊密相鄰的表達(dá)有GABAA受體的HEK293B細(xì)胞記錄的電流大,且具有統(tǒng)計學(xué)意義。結(jié)論:大鼠DRG神經(jīng)元在capsaicin刺激下可直接釋放GABA,轉(zhuǎn)基因小鼠VGAT-ChR2-YFP的DRG神經(jīng)元在473 nm激光刺激下也有類似GABA釋放現(xiàn)象,從而證實了 DRG神經(jīng)元具有直接釋放GABA的功能。為DRG局部存在功能性的GABA系統(tǒng)提供了更直接的證據(jù)。第二部分DRG部位GABA系統(tǒng)對神經(jīng)病理性疼痛的影響目的:研究CCI手術(shù)后不同直徑大小DRG神經(jīng)元的GABA電流密度的改變。采用滲透泵靶向DRG給藥的給藥方法,觀察DRG局部GABA系統(tǒng)對病理性疼痛的治療作用。方法:制備神經(jīng)病理性模型CCI;應(yīng)用Von Frey纖維刺痛針和熱刺痛儀,測定大鼠機(jī)械痛、熱痛閾值;建立滲透泵靶向DRG給藥系統(tǒng);全細(xì)胞膜片鉗技術(shù);統(tǒng)計方法為兩個獨立樣本的非參數(shù)檢驗Mann-Whitney Test。結(jié)果:①CCI模型建立:CCI組出現(xiàn)明顯的痛覺過敏癥狀,其機(jī)械痛和熱痛閾值在術(shù)后第一天明顯下降,并持續(xù)至第十四天,與對照組比存在顯著性差異(P0.05)。②電生理結(jié)果:用電生理技術(shù)分別記錄大鼠CCI手術(shù)后第五天和第十四天的術(shù)側(cè)和對側(cè)大鼠大、中、小直徑L4-6DRG神經(jīng)元的GABA-Cl-電流。大直徑和小直徑L4-6DRG神經(jīng)元CCI術(shù)后第五天、第十四天術(shù)側(cè)DRG神經(jīng)元的電流密度和對GABA的反應(yīng)數(shù)目與對側(cè)DRG神經(jīng)元無明顯區(qū)別。中等直徑L4-6DRG神經(jīng)元在CCI術(shù)后第五天大鼠術(shù)側(cè)的DRG神經(jīng)元GABA-C1-電流密度大于對側(cè)(P0.05),而第十四天術(shù)側(cè)與對側(cè)無顯著區(qū)別。第五天和第十四天對側(cè)的DRG神經(jīng)元對GABA有反應(yīng)的神經(jīng)元數(shù)目遠(yuǎn)少于術(shù)側(cè)。③行為學(xué)實驗結(jié)果:通過滲透泵靶向DRG給藥系統(tǒng)對CCI模型大鼠部位持續(xù)給予GABA(200μM)個周,對照組給予生理鹽水;在術(shù)前及術(shù)后第1,5,7,10,14天進(jìn)行機(jī)械痛和熱痛實驗的測定。結(jié)果顯示GABA的應(yīng)用可使術(shù)后第一和第五天熱痛閾值明顯增大(P0.05),而GABA的應(yīng)用對CCI大鼠機(jī)械痛閾無明顯影響。結(jié)論:以上實驗證明在神經(jīng)病理性疼痛發(fā)生中伴有一定DRG局部GABA系統(tǒng)敏感性變化,特別是中直徑DRG神經(jīng)元GABA敏感性增高更為明顯;在神經(jīng)病理性疼痛發(fā)生過程中激活DRG局部GABA系統(tǒng)可一定程度上緩解疼痛癥狀,尤其是對熱痛過敏癥狀的緩解作用更為突出。
[Abstract]:Somatosensory neurons are important components of the sensory system of the body, and their bodies exist in the dorsal root ganglion (dorsal root ganglion, DRG). The peripheral axons of DRG neurons can feel a variety of peripheral stimuli such as temperature stimulation, chemical stimulation and mechanical stimulation, and transform them into nerve impulses to the center, thus forming a nerve impulse. Our previous studies have found that DRG neurons not only have the formation and transmission of pain signals, but also have a functional GABA loop system in which.DRG is localized in the regulation of pain signals, and the DRG tissue and culture cells can be released by GABA under a variety of excitatory stimuli, and DRG local application of GABA. The acute inflammatory pain induced by bradykinin has a significant inhibitory effect. These all suggest that the DRG site (especially the GABA system in this area) may be a potential target for pain treatment. Chronic neuropathic pain is a common clinical disease, its pathogenesis is not yet clear, and there is still a lack of effective treatment for chronic neuropathic pain at present. In this paper, we will focus on the role of DRG local GABA system in the occurrence and treatment of chronic neuropathic pain. First, in order to study the role of DRG local GABA system in the process of pain, we have established a new "sniffer patch" electrophysiological experiment to prove that the rat DRG neurons are in the process. GABA can be released directly under the excitation condition, which provides more direct evidence for the functional GABA system in the local DRG. Secondly, we observe the GABA current density of DRG neurons in the DRG neurons of the pain progression by preparing the neuropathic pain model of the chronic crush injury (CCI) of the rat sciatic nerve, to study the neuropathy in the DRG local GABA system in the neuropathy. The role of rational pain in the occurrence of pain. Finally, we observe the effect of GABA on neuropathic pain by targeting the osmotic pump targeting DRG drug delivery system, and analyze the possibility of DRG local GABA system as a target for chronic neuropathic pain treatment. The first part uses the "Sniffer Patch" method to prove the GABA release function of DRG neurons. Objective: to establish a new method of electrophysiological experiment "sniffer patch", which proves that DRG neurons have the function of releasing GABA directly. Method: electrophysiological experimental method of "sniffer patch", semi quantitative PCR technique, VGAT-ChR2-YFP mouse identification, frozen section immunofluorescence technique, and statistical method for t test. Results: (1) drug stimulates DRG. The result of neuron "Sniffer Patch": co culture the HEK293B cells expressing the GABAA receptor with the acute isolated DRG cells of rats. The HEK293B cells transfected with the GABAA receptor, which is closely adjacent to the small diameter DRG, produce a large GABA-Cl- current under the action of 200 micron GABA. Neurons, sniffer patch method was used to record similar GABA like current in adjacent HEK293B cells. Capsaicin could not induce HEK293B cells transfected with GABAA receptor alone, and the blank HEK293B cells co cultured with DRG produce any current. 2. The "Sniffer Patch" result of the DRG nerve element stimulation: GABAA receptor turns. HEK293B cells were co cultured with rat DRG cells, and the HEK293B cells expressing the GAB AA receptor were closely adjacent to the small diameter DRG mechanical stimulation and failed to induce the HEK293B cells to produce the GABA like current. Co culture, under 473nm laser stimulation, a small inward current was recorded by HEK293B cells expressing GABAA receptor with the GABAA receptor closely adjacent to the DRG of the transgenic mouse DRG, and the HEK293B cells expressing GABAA receptors in the isolated culture, and HEK293B with the GABAA receptor in close proximity to the DRG of the wild type mice. The cell recording current is large and has statistical significance. Conclusion: the rat DRG neurons can release GABA directly under the stimulation of capsaicin. The DRG neurons of the transgenic mice VGAT-ChR2-YFP also have the similar GABA release under the 473 nm laser stimulation, which confirms that the DRG neurons have the function of releasing GABA directly. It has the function of the DRG part. The GABA system provides more direct evidence. Second part of the effect of the DRG site GABA system on neuropathic pain is to study the changes in the GABA current density of DRG neurons of different diameters in CCI after CCI operation. The therapeutic effect of the local GABA system on the pathological pain of DRG is observed by using the osmotic pump target to the DRG administration. Method: the neuropathic model CCI was prepared; the mechanical pain and the heat pain threshold of the rats were measured with the Von Frey fiber prick needle and the thermo pain instrument; the osmotic pump targeted DRG administration system was established; the whole cell patch clamp technique was established; the statistical method was the non parametric test of the Mann-Whitney Test. results of two independent samples: (1) the CCI model was established: the CCI group appeared obvious pain. The threshold of mechanical pain and heat pain decreased significantly on the first day after the operation and lasted to fourteenth days, and there was a significant difference compared with the control group (P0.05). 2. Electrophysiological results: electrophysiological techniques were used to record the GABA-Cl- of the large, medium and small diameter L4-6DRG neurons in the fifth and fourteenth days of the rats after the operation and the fifth and fourteenth days after the operation. The current density of DRG neurons and the number of responses to GABA were not significantly different from that of the contralateral DRG neurons on the fourteenth day after the operation of the large diameter and small diameter L4-6DRG neurons on the fourteenth day. The GABA-C1- current density of the DRG neurons on the side of the middle diameter L4-6DRG neuron fifth days after CCI was greater than that of the contralateral DRG (P0.05), and Fourteenth There was no significant difference between the operation side and the contralateral side. The number of neurons that reacted to the contralateral DRG neurons on the fifth and fourteenth days was far less than that of the operation. (3) the results of the behavioral experiment: the CCI model rats were continuously given GABA (200 u M) weeks by the osmotic pump target to the CCI model rats, and the control group was given physiological saline; before and after the operation, 1,5,7 was given. The results of the test of mechanical pain and heat pain in 10,14 days showed that the application of GABA could significantly increase the heat pain threshold of the first and fifth days after operation (P0.05), and the application of GABA had no obvious effect on the mechanical pain threshold of CCI rats. In particular, the increased sensitivity of GABA in the middle diameter DRG neurons is more obvious. In the process of neuropathic pain, activation of DRG local GABA system can relieve pain symptoms to a certain extent, especially for the relief of heat pain allergic symptoms.
【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】：2015
【分類號】：R402

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