慢病毒介导的siSCN9A下调Nav1.7的表达在神经病理痛中的镇痛作用

发布时间：2018-04-25 02:14

本文选题：Nav1.7 + 神经病理痛　；参考：《郑州大学》2016年硕士论文

【摘要】：背景与目的神经系统原发性损害或功能障碍所引起的异常痛觉、自发性疼痛、痛觉过敏的现象,称为神经病理痛。神经病理痛患病率高,患者生存质量差,治疗效果不满意。至今神经病理痛的发生机制尚不完全清楚,机械损伤、神经的代谢或营养性改变、病毒感染或放疗的神经毒性、缺血性神经损害、神经递质功能障碍均可导致神经病理痛。目前,疼痛的治疗方法主要是通过局麻药、抗抑郁药、中枢性抑制药、抗癫痫药等,疗效短暂、副作用大,且易产生药物依赖。近期的研究表明,电压门控钠离子通道Ⅸα亚基(Nav1.7,SCN9A编码)可能与三种痛觉异常性疾病即肢端红痛症、阵发性剧痛症、先天性无痛症密切相关。其中Nav1.7功能错义突变引起肢斑红痛症和阵发性剧痛症,Nav1.7无义突变引起先天性无痛症。电压门控钠离子通道Nav1.7主要表达在DRG和交感神经节中。DRG中表达各种各样的钠、钾离子通道。这些离子通道包括转换、传导神经信号、调节突触的运输三大功能。神经受伤后,受损或者未受损的神经元均会出现异常的放电活动,这种异常放电是否是Nav1.7的异常表达所引起的?因此,我们设计以下实验,并希望通过对Nav1.7表达的干预来治疗神经病理痛提供实验数据。本研究通过建立SNL神经病理痛模型和原代培养DRG神经元细胞,肿瘤坏死因子-α(TNF-α)刺激神经元细胞;构建慢病毒,体内、外实验两个方面感染慢病毒介导的siscn9a(lv-siscn9a),观察行为学及钠离子通道nav1.7的变化,阐明nav1.7在神经病理性疼痛中的作用,为神经病理性疼痛的转基因治疗提供重要的理论和实验基础。方法1.构建慢病毒介导的siscn9a,培养pc12,感染慢病毒后并进行滴度测定。2.用tnf-α刺激体外培养出生21d的sd大鼠drg神经元细胞,通过免疫组化及western-blot检测nav1.7的表达情况。慢病毒介导的siscn9a分别感染有和无tnf-α刺激的神经元细胞,western-blot检测各个组别中nav1.7的表达情况,免疫荧光检测慢病毒的感染效率。3.制作snl神经病理痛模型,行为学检测术前1d,术后3,7,10,14,21d大鼠机械痛阈值和热缩足阈值的变化,免疫荧光双标和western-blot检测各组中术后7d时nav1.7的变化,统计nav1.7的灰度值。4.drg微量注射2μl的lv-siscn9a和lv-sc。行为学检测机械痛阈值和热缩足阈值,免疫荧光双标和western-blot检测各组中nav1.7的变化,并统计nav1.7的灰度值。免疫荧光检测大鼠脊髓背角中cgrp的表达情况,并统计cgrp的荧光强度。结果1.构建的慢病毒介导的sirna的基因测序为acagcatgatctgcttgttcc与目标基因scn9a的cds区的5084-5104碱基序列完全配对,慢病毒介导的sirna感染培养的pc12细胞选用的moi=10可以满足我们的实验需要。检测慢病毒的滴度为6×108。2.体外原代培养的神经细胞用100ng/mltnf-α刺激westernblot结果显示与对照组相比,tnf-α组的nav1.7蛋白表达增加,`x±s分别为(0.78±0.07、0.34±0.04);p=0.013;免疫荧光显示病毒携带的gfp在神经细胞内表达,lv-siscn9a感染神经元细胞后感染效率达到80%左右。与lv-sc组相比,lv-siscn9a感染原代培养的神经元后可以抑制tnf-α刺激引起的nav1.7的高表达`x±s分别为(0.26±0.05、0.73±0.08);p=0.02。3.制作snl神经病理痛模型,与sham组相比,snl组大鼠术侧的痛敏阈值(13.5±0.9到2.3±0.6)、热缩足阈值(13.7±1.2到8.5±0.4)均降低,p=0.03;SNL模型建立成功;免疫荧光显示SNL术后3d Nav1.7表达明显增高(0.20±0.03到0.8±0.07);p=0.002。4.SNL模型DRG注射LV-siSCN9A与注射LV-SC组相比,SNL大鼠术侧机械痛敏阈值(5.7±0.5到10.5±0.6)和热痛敏阈值(7.9±0.3到13.5±0.7)均升高。Western blot显示术后7d SNL+LV-siSCN9A组与SNL+LV-sc组相比Nav1.7的表达降低(0.5±0.04、0.8±0.05);p=0.03。免疫荧光可以实现GFP与Nav1.7的共标;结论LV-siSCN9A感染神经元可有效下调TNF-α刺激和SNL神经病理痛模型引起的Nav1.7的高表达,从而缓解神经病理痛。
[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.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R741

【相似文献】