自噬在带状疱疹后神经痛中的作用及其机制探讨
发布时间：2018-08-08 18:17
【摘要】：目的:探讨不同自噬程度对带状疱疹后神经痛(postherpetic neuralgia,PHN)小鼠模型脊髓背角GABA能神经元的作用材料与方法:1.使用SPSS19.0的随机数字发生器将36只昆明小鼠随机分为生理盐水组(NS)、溶剂组(Solvent)与PHN组,每组12只。腹腔注射树脂毒素组(Resiniferotoxin,RTX)0.2μg/g的剂量构建PHN模型,Solvent组、NS组分别注射等体积的溶剂和生理盐水。每天定时用von-frey丝检测机械缩足阈值(Paw withdrawal mechanical thresholds,PMWT),热板法检测缩足时间阈值(Pain threshold in hot-plate test,HPPT)至疼痛稳定。然后分别处死各组小鼠,取脊髓L4~L6节段使用实时荧光定量PCR、免疫组织化学染色及western blot等方法检测LC3、beclin1的变化情况;透射电镜观察脊髓自噬小体。2.使用SPSS19.0的随机数字发生器将48只昆明小鼠随机分为PHN+Rapa组、PHN组、PHN+3-MA组及空白对照(Control,C)组,每组12只。C组不做任何处理,其余各组均在构建PHN模型成功后,连续14天分别腹腔注射1μg/(kg.d)的自噬诱导剂雷帕霉素(Rapamycin,Rapa)、生理盐水和2μg/(kg.d)的自噬抑制试剂3-甲基腺嘌呤(3-Methyladenine,3-MA)。提取脊髓L4~L6节段,使用western blot检测LC3、beclin-1和SQSTM1/P62的表达情况;ELSIA法检测脊髓匀浆液与动脉血中β-内啡肽和P物质浓度;统计各组小鼠PMWT、HPPT。3.使用SPSS19.0的随机数字发生器将48只昆明小鼠随机分为C组、PHN+Rapa组、PHN组及PHN+3-MA组,每组12只。C组不做任何处理,其余各组均在构建PHN模型成功后,连续14天分别腹腔注射1μg/(kg.d)的自噬诱导剂Rapa、生理盐水和2μg/(kg.d)的自噬抑制剂3-MA。检测PMWT、HPPT至稳定后处死各组小鼠,提取脊髓L4~6节段western blot法检测抑凋亡蛋白bcl-2和促凋亡Bax的表达;荧光Tunel法检测脊髓凋亡细胞数;免疫荧光标记脊髓背角GABA能神经元数。结果:1、使用0.2ug/g的RTX后PHN组PMWT显著降低、HPPT显著增加(p0.05),同时自噬相关蛋白LC3、beclin-1的m RNA和蛋白相对表达量显著性增加(p0.05);2、在腹腔注射雷帕霉素后,PHN+Rapa组的LC3II、beclin-1蛋白质水平的相对表达量显著增加,SQSTM1/P62则显著性降低(p0.05),PHN+3-MA组的LC3II、beclin-1蛋白质水平相对表达量显著降低、SQSTM1/P62则显著性增加(P0.05);与C组比较,PHN+Rapa、PHN及PHN+3-MA组血清与脊髓匀浆液中的β内啡肽与P物质浓度均显著性增加(P0.05)。与PHN组比较,PHN+Rapa血清与脊髓匀浆液中的β内啡肽显著性降低,而PHN+3-MA组则显著性增加(P0.05)。PHN+Rapa组的血清与脊髓匀浆液中P物质浓度较PHN组显著性增加,而PHN+3-MA组则显著性降低(P0.05);与PHN组比较,在PHN+Rapa组使用自噬诱导剂Rapa后PMWT显著降低、HPPT显著增加(p0.05),PHN+3-MA组使用自噬抑制剂3-MA后PMWT则显著增加、HPPT显著降低(p0.05);3、使用Rapa诱导PHN脊髓自噬后,PHN+Rapa组的bcl-2和Bax的蛋白相对表达程度均显著性增加,凋亡细胞数量显著增加,脊髓背角GABA能神经元数显著性减少(p0.05)。使用3-MA抑制自噬后,PHN+3-MA组的bcl-2表达显著增加,而Bax则显著性降低。Tunel法检测凋亡细胞数量显著减少。免疫荧光检测脊髓背角GABA能神经元数显著性增加(p0.05)。结论:1、腹腔注射0.2ug/g的RTX可以复制PHN小鼠模型;2、PHN小鼠模型脊髓自噬程度增加;3、脊髓自噬的过度激活促进了PHN的疼痛发生发展;4、PHN的自噬激活可能是导致脊髓GABA能神经元数量减少促进中枢敏化的重要因素之一。
[Abstract]:Objective: To investigate the effect of different autophagy on GABA neurons in the dorsal horn of postherpetic neuralgia (PHN) mouse model: 1. random digital generator of SPSS19.0 was used to randomly divide 36 Kunming mice into physiological saline group (NS), solvent group (Solvent) and PHN group, 12 rats in each group. The dosage of Resiniferotoxin (RTX) was 0.2 mu g/g to construct the PHN model, the Solvent group and the NS group were injected with the same volume of solvent and physiological saline respectively. Every day, the threshold of the mechanical contraction was measured by Von-Frey wire (Paw withdrawal mechanical thresholds, PMWT), and the hot plate method was used to detect the threshold of the contraction time. The pain was stable. Then the mice were killed in each group. The L4~L6 segment of the spinal cord was taken with real-time fluorescence quantitative PCR, immunohistochemical staining and Western blot were used to detect the changes of LC3 and Beclin1. The random digital generator of the autophagic corpuscle of the spinal cord.2. using SPSS19.0 was observed by transmission electron microscopy, and 48 Kunming mice were randomly divided into PHN+Rapa group, PHN. Group, PHN+3-MA group and blank control group (Control, C) group, each group of 12.C groups did not do any treatment. The rest of the other groups were injected 1 mu g/ (kg.d), the autophagic inducer, rapamycin (Rapamycin, Rapa), physiological saline and 2 mu g / (kg.d), the autophagy inhibitor 3- methyl adenine, after the construction of PHN model. The L4~L6 segment of the spinal cord was extracted and the expression of LC3, beclin-1 and SQSTM1/P62 were detected by Western blot. The ELSIA method was used to detect the concentration of beta endorphin and P in the spinal cord homogenate and arterial blood. The PMWT and HPPT.3. using SPSS19.0 random digital generator in each group were randomly divided into 48 Kunming mice. 12.C groups in each group did not do any treatment. After the construction of PHN model, the other groups were intraperitoneally injected with 1 micron g/ (kg.d) autophagic inducer Rapa, physiological saline and 2 micron g/ (kg.d) autophagic inhibitor 3-MA. to detect PMWT, and HPPT to the stability of each group. 2 and the expression of apoptotic Bax, the number of apoptotic cells in the spinal cord by fluorescence Tunel, and the number of GABA neurons in the dorsal horn of the spinal cord by immunofluorescence. Results: 1, the PMWT of PHN group decreased significantly after RTX of 0.2ug/g, HPPT increased significantly (P0.05), while the autophagy associated protein LC3, beclin-1 m and protein relative expression increased significantly; 2, in the abdominal cavity After injection of rapamycin, the relative expression of LC3II and beclin-1 protein levels in group PHN+Rapa increased significantly, while SQSTM1/P62 decreased significantly (P0.05), the relative expression of LC3II, beclin-1 protein in PHN+3-MA group decreased significantly, and SQSTM1/P62 increased significantly (P0.05). The concentration of beta endorphin and substance P in the serous was significantly increased (P0.05). Compared with the PHN group, the beta endorphin in the PHN+Rapa serum and the spinal cord homogenate decreased significantly, while the PHN+3-MA group significantly increased the concentration of P in the serum and spinal homogenates of the group P0.05.PHN+Rapa significantly increased, while the PHN+3-MA group decreased significantly (P). 0.05): compared with the PHN group, PMWT significantly decreased and HPPT increased significantly after the use of autophagic inducer Rapa in group PHN+Rapa (P0.05), and PMWT increased significantly in PHN+3-MA group with autophagic inhibitor 3-MA, and HPPT significantly decreased (P0.05). 3, the relative expression of protein and protein increased significantly after the induction of autophagy in the spinal cord. The number of dead cells increased significantly and the number of GABA neurons in the dorsal horn of the spinal cord decreased significantly (P0.05). Bcl-2 expression in the PHN+3-MA group increased significantly after the use of 3-MA to inhibit autophagy, while Bax significantly decreased the number of apoptotic cells by.Tunel method. The number of GABA energy neurons in the dorsal horn of the spinal cord was significantly increased (P0.05). Conclusion: 1, Intraperitoneal injection of 0.2ug/g RTX can copy the PHN mouse model; 2, the degree of autophagy increases in the spinal cord of PHN mice; 3, the excessive activation of autophagy in the spinal cord promotes the development of PHN pain; 4, the activation of autophagy may be one of the important factors that lead to the decrease in the number of GABA energy neurons in the spinal cord and to promote the central sensitization of the spinal cord neurons.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R752.12
本文编号：2172621
[Abstract]:Objective: To investigate the effect of different autophagy on GABA neurons in the dorsal horn of postherpetic neuralgia (PHN) mouse model: 1. random digital generator of SPSS19.0 was used to randomly divide 36 Kunming mice into physiological saline group (NS), solvent group (Solvent) and PHN group, 12 rats in each group. The dosage of Resiniferotoxin (RTX) was 0.2 mu g/g to construct the PHN model, the Solvent group and the NS group were injected with the same volume of solvent and physiological saline respectively. Every day, the threshold of the mechanical contraction was measured by Von-Frey wire (Paw withdrawal mechanical thresholds, PMWT), and the hot plate method was used to detect the threshold of the contraction time. The pain was stable. Then the mice were killed in each group. The L4~L6 segment of the spinal cord was taken with real-time fluorescence quantitative PCR, immunohistochemical staining and Western blot were used to detect the changes of LC3 and Beclin1. The random digital generator of the autophagic corpuscle of the spinal cord.2. using SPSS19.0 was observed by transmission electron microscopy, and 48 Kunming mice were randomly divided into PHN+Rapa group, PHN. Group, PHN+3-MA group and blank control group (Control, C) group, each group of 12.C groups did not do any treatment. The rest of the other groups were injected 1 mu g/ (kg.d), the autophagic inducer, rapamycin (Rapamycin, Rapa), physiological saline and 2 mu g / (kg.d), the autophagy inhibitor 3- methyl adenine, after the construction of PHN model. The L4~L6 segment of the spinal cord was extracted and the expression of LC3, beclin-1 and SQSTM1/P62 were detected by Western blot. The ELSIA method was used to detect the concentration of beta endorphin and P in the spinal cord homogenate and arterial blood. The PMWT and HPPT.3. using SPSS19.0 random digital generator in each group were randomly divided into 48 Kunming mice. 12.C groups in each group did not do any treatment. After the construction of PHN model, the other groups were intraperitoneally injected with 1 micron g/ (kg.d) autophagic inducer Rapa, physiological saline and 2 micron g/ (kg.d) autophagic inhibitor 3-MA. to detect PMWT, and HPPT to the stability of each group. 2 and the expression of apoptotic Bax, the number of apoptotic cells in the spinal cord by fluorescence Tunel, and the number of GABA neurons in the dorsal horn of the spinal cord by immunofluorescence. Results: 1, the PMWT of PHN group decreased significantly after RTX of 0.2ug/g, HPPT increased significantly (P0.05), while the autophagy associated protein LC3, beclin-1 m and protein relative expression increased significantly; 2, in the abdominal cavity After injection of rapamycin, the relative expression of LC3II and beclin-1 protein levels in group PHN+Rapa increased significantly, while SQSTM1/P62 decreased significantly (P0.05), the relative expression of LC3II, beclin-1 protein in PHN+3-MA group decreased significantly, and SQSTM1/P62 increased significantly (P0.05). The concentration of beta endorphin and substance P in the serous was significantly increased (P0.05). Compared with the PHN group, the beta endorphin in the PHN+Rapa serum and the spinal cord homogenate decreased significantly, while the PHN+3-MA group significantly increased the concentration of P in the serum and spinal homogenates of the group P0.05.PHN+Rapa significantly increased, while the PHN+3-MA group decreased significantly (P). 0.05): compared with the PHN group, PMWT significantly decreased and HPPT increased significantly after the use of autophagic inducer Rapa in group PHN+Rapa (P0.05), and PMWT increased significantly in PHN+3-MA group with autophagic inhibitor 3-MA, and HPPT significantly decreased (P0.05). 3, the relative expression of protein and protein increased significantly after the induction of autophagy in the spinal cord. The number of dead cells increased significantly and the number of GABA neurons in the dorsal horn of the spinal cord decreased significantly (P0.05). Bcl-2 expression in the PHN+3-MA group increased significantly after the use of 3-MA to inhibit autophagy, while Bax significantly decreased the number of apoptotic cells by.Tunel method. The number of GABA energy neurons in the dorsal horn of the spinal cord was significantly increased (P0.05). Conclusion: 1, Intraperitoneal injection of 0.2ug/g RTX can copy the PHN mouse model; 2, the degree of autophagy increases in the spinal cord of PHN mice; 3, the excessive activation of autophagy in the spinal cord promotes the development of PHN pain; 4, the activation of autophagy may be one of the important factors that lead to the decrease in the number of GABA energy neurons in the spinal cord and to promote the central sensitization of the spinal cord neurons.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R752.12
【参考文献】
相关期刊论文 前10条
1 赵明明;罗鹏;赵永博;尹丰;费舟;;自噬抑制剂3-MA对机械性神经元损伤后神经元凋亡的影响[J];中华神经外科疾病研究杂志;2016年03期
2 张桂友;王准;杨艳梅;栾静;郑莹;魏成博;吕丹;;自噬在大鼠炎性痛形成中的作用[J];中华麻醉学杂志;2016年04期
3 赵远波;洪杜北琦;陈英玉;;基于p62/SQSTM1-luciferase的细胞自噬水平检测方法的建立及鉴定[J];中国生物工程杂志;2016年01期
4 李燕尧;张爱民;蒋宗滨;吕时甲;何睿林;;脉冲射频对保留性神经损伤大鼠Nav1.8 mRNA表达的影响[J];中国疼痛医学杂志;2015年12期
5 Kai Gao;Yan-song Wang;Ya-jiang Yuan;Zhang-hui Wan;Tian-chen Yao;Hai-hong Li;Pei-fu Tang;Xi-fan Mei;;Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway[J];Neural Regeneration Research;2015年06期
6 贺俭;王汉兵;赵伟成;何万友;廖美娟;杨承祥;;脊髓自噬功能的激活在大鼠糖尿病神经病理性疼痛中的作用[J];中华医学杂志;2015年14期
7 范之丹;马柯;季晓燕;程志军;;电压门控钠通道Nav1.6在骨癌痛模型大鼠背根神经节中的表达[J];上海交通大学学报(医学版);2015年03期
8 冯涛;殷琴;翁泽林;张建成;王昆锋;袁世荧;程伟;;Rapamycin Ameliorates Neuropathic Pain by Activating Autophagy and Inhibiting Interleukin-1β in the Rat Spinal Cord[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2014年06期
9 薛照静;申乐;王之遥;黄宇光;;信号转导和转录激活子3抑制剂WP1066作为神经病理性疼痛治疗新靶点的可行性[J];协和医学杂志;2014年02期
10 叶永贤;林洪;沙漠;李招胜;伍磊;冯文龙;陈智彪;丁真奇;;神经病理性疼痛大鼠脊髓NF-κB、NR2B和iNOS的表达及意义[J];中国病理生理杂志;2014年04期
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