自噬在帶狀皰疹后神經(jīng)痛中的作用及其機制探討
發(fā)布時間：2018-08-08 18:17
【摘要】：目的:探討不同自噬程度對帶狀皰疹后神經(jīng)痛(postherpetic neuralgia,PHN)小鼠模型脊髓背角GABA能神經(jīng)元的作用材料與方法:1.使用SPSS19.0的隨機數(shù)字發(fā)生器將36只昆明小鼠隨機分為生理鹽水組(NS)、溶劑組(Solvent)與PHN組,每組12只。腹腔注射樹脂毒素組(Resiniferotoxin,RTX)0.2μg/g的劑量構(gòu)建PHN模型,Solvent組、NS組分別注射等體積的溶劑和生理鹽水。每天定時用von-frey絲檢測機械縮足閾值(Paw withdrawal mechanical thresholds,PMWT),熱板法檢測縮足時間閾值(Pain threshold in hot-plate test,HPPT)至疼痛穩(wěn)定。然后分別處死各組小鼠,取脊髓L4~L6節(jié)段使用實時熒光定量PCR、免疫組織化學(xué)染色及western blot等方法檢測LC3、beclin1的變化情況;透射電鏡觀察脊髓自噬小體。2.使用SPSS19.0的隨機數(shù)字發(fā)生器將48只昆明小鼠隨機分為PHN+Rapa組、PHN組、PHN+3-MA組及空白對照(Control,C)組,每組12只。C組不做任何處理,其余各組均在構(gòu)建PHN模型成功后,連續(xù)14天分別腹腔注射1μg/(kg.d)的自噬誘導(dǎo)劑雷帕霉素(Rapamycin,Rapa)、生理鹽水和2μg/(kg.d)的自噬抑制試劑3-甲基腺嘌呤(3-Methyladenine,3-MA)。提取脊髓L4~L6節(jié)段,使用western blot檢測LC3、beclin-1和SQSTM1/P62的表達情況;ELSIA法檢測脊髓勻漿液與動脈血中β-內(nèi)啡肽和P物質(zhì)濃度;統(tǒng)計各組小鼠PMWT、HPPT。3.使用SPSS19.0的隨機數(shù)字發(fā)生器將48只昆明小鼠隨機分為C組、PHN+Rapa組、PHN組及PHN+3-MA組,每組12只。C組不做任何處理,其余各組均在構(gòu)建PHN模型成功后,連續(xù)14天分別腹腔注射1μg/(kg.d)的自噬誘導(dǎo)劑Rapa、生理鹽水和2μg/(kg.d)的自噬抑制劑3-MA。檢測PMWT、HPPT至穩(wěn)定后處死各組小鼠,提取脊髓L4~6節(jié)段western blot法檢測抑凋亡蛋白bcl-2和促凋亡Bax的表達;熒光Tunel法檢測脊髓凋亡細胞數(shù);免疫熒光標(biāo)記脊髓背角GABA能神經(jīng)元數(shù)。結(jié)果:1、使用0.2ug/g的RTX后PHN組PMWT顯著降低、HPPT顯著增加(p0.05),同時自噬相關(guān)蛋白LC3、beclin-1的m RNA和蛋白相對表達量顯著性增加(p0.05);2、在腹腔注射雷帕霉素后,PHN+Rapa組的LC3II、beclin-1蛋白質(zhì)水平的相對表達量顯著增加,SQSTM1/P62則顯著性降低(p0.05),PHN+3-MA組的LC3II、beclin-1蛋白質(zhì)水平相對表達量顯著降低、SQSTM1/P62則顯著性增加(P0.05);與C組比較,PHN+Rapa、PHN及PHN+3-MA組血清與脊髓勻漿液中的β內(nèi)啡肽與P物質(zhì)濃度均顯著性增加(P0.05)。與PHN組比較,PHN+Rapa血清與脊髓勻漿液中的β內(nèi)啡肽顯著性降低,而PHN+3-MA組則顯著性增加(P0.05)。PHN+Rapa組的血清與脊髓勻漿液中P物質(zhì)濃度較PHN組顯著性增加,而PHN+3-MA組則顯著性降低(P0.05);與PHN組比較,在PHN+Rapa組使用自噬誘導(dǎo)劑Rapa后PMWT顯著降低、HPPT顯著增加(p0.05),PHN+3-MA組使用自噬抑制劑3-MA后PMWT則顯著增加、HPPT顯著降低(p0.05);3、使用Rapa誘導(dǎo)PHN脊髓自噬后,PHN+Rapa組的bcl-2和Bax的蛋白相對表達程度均顯著性增加,凋亡細胞數(shù)量顯著增加,脊髓背角GABA能神經(jīng)元數(shù)顯著性減少(p0.05)。使用3-MA抑制自噬后,PHN+3-MA組的bcl-2表達顯著增加,而Bax則顯著性降低。Tunel法檢測凋亡細胞數(shù)量顯著減少。免疫熒光檢測脊髓背角GABA能神經(jīng)元數(shù)顯著性增加(p0.05)。結(jié)論:1、腹腔注射0.2ug/g的RTX可以復(fù)制PHN小鼠模型;2、PHN小鼠模型脊髓自噬程度增加;3、脊髓自噬的過度激活促進了PHN的疼痛發(fā)生發(fā)展;4、PHN的自噬激活可能是導(dǎo)致脊髓GABA能神經(jīng)元數(shù)量減少促進中樞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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R752.12
本文編號：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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R752.12
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