本文選題：Nav1.7 + 神經(jīng)病理痛　； 參考：《鄭州大學(xué)》2016年碩士論文

【摘要】：背景與目的神經(jīng)系統(tǒng)原發(fā)性損害或功能障礙所引起的異常痛覺、自發(fā)性疼痛、痛覺過敏的現(xiàn)象,稱為神經(jīng)病理痛。神經(jīng)病理痛患病率高,患者生存質(zhì)量差,治療效果不滿意。至今神經(jīng)病理痛的發(fā)生機制尚不完全清楚,機械損傷、神經(jīng)的代謝或營養(yǎng)性改變、病毒感染或放療的神經(jīng)毒性、缺血性神經(jīng)損害、神經(jīng)遞質(zhì)功能障礙均可導(dǎo)致神經(jīng)病理痛。目前,疼痛的治療方法主要是通過局麻藥、抗抑郁藥、中樞性抑制藥、抗癲癇藥等,療效短暫、副作用大,且易產(chǎn)生藥物依賴。近期的研究表明,電壓門控鈉離子通道Ⅸα亞基(Nav1.7,SCN9A編碼)可能與三種痛覺異常性疾病即肢端紅痛癥、陣發(fā)性劇痛癥、先天性無痛癥密切相關(guān)。其中Nav1.7功能錯義突變引起肢斑紅痛癥和陣發(fā)性劇痛癥,Nav1.7無義突變引起先天性無痛癥。電壓門控鈉離子通道Nav1.7主要表達在DRG和交感神經(jīng)節(jié)中。DRG中表達各種各樣的鈉、鉀離子通道。這些離子通道包括轉(zhuǎn)換、傳導(dǎo)神經(jīng)信號、調(diào)節(jié)突觸的運輸三大功能。神經(jīng)受傷后,受損或者未受損的神經(jīng)元均會出現(xiàn)異常的放電活動,這種異常放電是否是Nav1.7的異常表達所引起的?因此,我們設(shè)計以下實驗,并希望通過對Nav1.7表達的干預(yù)來治療神經(jīng)病理痛提供實驗數(shù)據(jù)。本研究通過建立SNL神經(jīng)病理痛模型和原代培養(yǎng)DRG神經(jīng)元細胞,腫瘤壞死因子-α(TNF-α)刺激神經(jīng)元細胞;構(gòu)建慢病毒,體內(nèi)、外實驗兩個方面感染慢病毒介導(dǎo)的siscn9a(lv-siscn9a),觀察行為學(xué)及鈉離子通道nav1.7的變化,闡明nav1.7在神經(jīng)病理性疼痛中的作用,為神經(jīng)病理性疼痛的轉(zhuǎn)基因治療提供重要的理論和實驗基礎(chǔ)。方法1.構(gòu)建慢病毒介導(dǎo)的siscn9a,培養(yǎng)pc12,感染慢病毒后并進行滴度測定。2.用tnf-α刺激體外培養(yǎng)出生21d的sd大鼠drg神經(jīng)元細胞,通過免疫組化及western-blot檢測nav1.7的表達情況。慢病毒介導(dǎo)的siscn9a分別感染有和無tnf-α刺激的神經(jīng)元細胞,western-blot檢測各個組別中nav1.7的表達情況,免疫熒光檢測慢病毒的感染效率。3.制作snl神經(jīng)病理痛模型,行為學(xué)檢測術(shù)前1d,術(shù)后3,7,10,14,21d大鼠機械痛閾值和熱縮足閾值的變化,免疫熒光雙標和western-blot檢測各組中術(shù)后7d時nav1.7的變化,統(tǒng)計nav1.7的灰度值。4.drg微量注射2μl的lv-siscn9a和lv-sc。行為學(xué)檢測機械痛閾值和熱縮足閾值,免疫熒光雙標和western-blot檢測各組中nav1.7的變化,并統(tǒng)計nav1.7的灰度值。免疫熒光檢測大鼠脊髓背角中cgrp的表達情況,并統(tǒng)計cgrp的熒光強度。結(jié)果1.構(gòu)建的慢病毒介導(dǎo)的sirna的基因測序為acagcatgatctgcttgttcc與目標基因scn9a的cds區(qū)的5084-5104堿基序列完全配對,慢病毒介導(dǎo)的sirna感染培養(yǎng)的pc12細胞選用的moi=10可以滿足我們的實驗需要。檢測慢病毒的滴度為6×108。2.體外原代培養(yǎng)的神經(jīng)細胞用100ng/mltnf-α刺激westernblot結(jié)果顯示與對照組相比,tnf-α組的nav1.7蛋白表達增加,`x±s分別為(0.78±0.07、0.34±0.04);p=0.013;免疫熒光顯示病毒攜帶的gfp在神經(jīng)細胞內(nèi)表達,lv-siscn9a感染神經(jīng)元細胞后感染效率達到80%左右。與lv-sc組相比,lv-siscn9a感染原代培養(yǎng)的神經(jīng)元后可以抑制tnf-α刺激引起的nav1.7的高表達`x±s分別為(0.26±0.05、0.73±0.08);p=0.02。3.制作snl神經(jīng)病理痛模型,與sham組相比,snl組大鼠術(shù)側(cè)的痛敏閾值(13.5±0.9到2.3±0.6)、熱縮足閾值(13.7±1.2到8.5±0.4)均降低,p=0.03;SNL模型建立成功;免疫熒光顯示SNL術(shù)后3d Nav1.7表達明顯增高(0.20±0.03到0.8±0.07);p=0.002。4.SNL模型DRG注射LV-siSCN9A與注射LV-SC組相比,SNL大鼠術(shù)側(cè)機械痛敏閾值(5.7±0.5到10.5±0.6)和熱痛敏閾值(7.9±0.3到13.5±0.7)均升高。Western blot顯示術(shù)后7d SNL+LV-siSCN9A組與SNL+LV-sc組相比Nav1.7的表達降低(0.5±0.04、0.8±0.05);p=0.03。免疫熒光可以實現(xiàn)GFP與Nav1.7的共標;結(jié)論LV-siSCN9A感染神經(jīng)元可有效下調(diào)TNF-α刺激和SNL神經(jīng)病理痛模型引起的Nav1.7的高表達,從而緩解神經(jīng)病理痛。
[Abstract]:Background and objective primary damage or dysfunction of the nervous system caused by abnormal pain, spontaneous pain, hyperalgesia, called neuropathic pain, high prevalence of neuropathic pain, poor quality of life, and dissatisfied treatment. The mechanism of neuropathic pain is not completely clear, mechanical injury, and nerve metabolism is not yet fully understood. Or nutritional changes, the neurotoxicity of viral infection or radiotherapy, ischemic nerve damage, and neurotransmitter dysfunction can lead to neuropathic pain. At present, the treatment of pain is mainly through local anesthetics, antidepressants, central suppressants, antiepileptic drugs and so on. The curative effect is short, the side effects are large and the drug dependence is easy to produce. It is suggested that the voltage gated sodium channel IX alpha subunit (Nav1.7, SCN9A) may be closely related to three kinds of abnormal pain disorders, such as acromegalgia, paroxysmal pain, and congenital painless disease. The Nav1.7 function missense mutation causes red pain and paroxysmal pain in the limb, and the Nav1.7 nonsense mutation causes congenital painless disease. Voltage gating The sodium channel Nav1.7 mainly expresses a variety of sodium, potassium channels in the.DRG and the sympathetic ganglia in DRG and sympathetic ganglia. These channels include conversion, conduction of nerve signals, regulating three major functions of synapse transport. After nerve injury, the damaged or undamaged neurons will have abnormal discharge activity. It is caused by the abnormal expression of Nav1.7, so we designed the following experiments and hope to provide experimental data for the treatment of neuropathic pain by the intervention of Nav1.7 expression. In this study, the SNL neuropathic pain model and the primary cultured DRG neuron cells, the tumor necrosis factor - alpha (TNF- alpha) stimulation of the neuron cells, and the construction of the lentivirus, In vivo and in vitro, two aspects of siscn9a (lv-siscn9a) are infected by lentivirus, observe the changes in behavior and sodium channel Nav1.7, clarify the role of Nav1.7 in neuropathic pain, provide an important theoretical and experimental basis for the neuropathic pain transgene therapy. Method 1. the construction of lentivirus mediated siscn9a and the culture of PC12 After infection of lentivirus and titer,.2. was stimulated by tnf- alpha to stimulate DRG neuron cells of SD rats born 21d in vitro, and the expression of Nav1.7 was detected by immunohistochemistry and Western-blot. The siscn9a infected by lentivirus was respectively infected with neuron cells without tnf- alpha stimulation. Western-blot was used to detect the expression of Nav1.7 in each group. The immunofluorescent detection of the infection efficiency of the lentivirus.3. made the SNL neuropathic pain model, the behavioral test of 1D before operation, the change of the threshold of mechanical pain and the threshold of the thermal contraction in the 3,7,10,14,21d rats, the changes of Nav1.7 in 7d after the operation of immunofluorescence and Western-blot, and the statistics of Nav1.7's gray value.4.drg microinjection of 2 micron L lv- Siscn9a and lv-sc. behaviours detected the threshold of mechanical pain and the threshold of thermal contraction, the changes of Nav1.7 in each group by double immunofluorescence and Western-blot, and the gray value of Nav1.7. The expression of CGRP in the spinal dorsal horn of rats was detected by immunofluorescence, and the fluorescence intensity of CGRP was counted. The gene mapping of siRNA, constructed by slow virus 1., was constructed. The sequence is the complete pairing of the 5084-5104 base sequence of the CDs region of acagcatgatctgcttgttcc and the target gene SCN9A. The moi=10 of PC12 cells cultured in the siRNA infected by the lentivirus can satisfy our experimental needs. The titer of the lentivirus is detected by the 6 x 108.2. in vitro primary cultured neurons with 100ng/mltnf- a stimulation of the Westernblot result. Compared with the control group, the expression of Nav1.7 protein in the tnf- alpha group increased, `x + s was (0.78 + 0.07,0.34 + 0.04), p=0.013, the immunofluorescence showed that the GFP in the virus was expressed in the nerve cells, and the infection efficiency of lv-siscn9a infected neurons was about 80%. Compared with the lv-sc group, the lv-siscn9a infected the primary cultured neurons. The high expression of `x + s caused by inhibition of tnf- alpha stimulation was (0.26 + 0.05,0.73 + 0.08); p=0.02.3. made SNL neuropathic pain model in p=0.02.3.. Compared with group sham, the threshold of pain sensitivity of group SNL group (13.5 + 0.9 to 2.3 + 0.6), the threshold of thermal contraction foot (13.7. 1.2 to 8.5 + 0.4) decreased, p=0.03; SNL model was established successfully; immunofluorescence showed SNL operation. The expression of 3D Nav1.7 was significantly increased (0.20 + 0.03 to 0.8 + 0.07), and DRG injection of LV-siSCN9A in p=0.002.4.SNL model, compared with the LV-SC group, increased the expression of the mechanical algesensitivity threshold (5.7 + 0.5 to 10.5 + 0.6) and the threshold of thermal pain sensitivity (7.9 + 0.3 to 13.5 + 0.7) in SNL rats (7.9 + 0.3 to 13.5 + 0.7), and increased the expression of 7D SNL+LV-siSCN9A group and SNL+LV-sc group after.Western blot display. The decrease (0.5 + 0.04,0.8 + 0.05); p=0.03. immunofluorescence can achieve the co labeling of GFP and Nav1.7; conclusion LV-siSCN9A infected neurons can effectively reduce the high expression of Nav1.7 caused by TNF- alpha stimulation and SNL neuropathic pain model, thus alleviating neuropathic pain.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R741

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