外周炎症性疼痛刺激诱导脊髓以上中枢胶质细胞激活及细胞因子的表达变化

发布时间：2018-09-04 15:40

【摘要】： 目的:观察完全弗氏佐剂(CFA)诱导的大鼠外周炎症性疼痛后不同时间点中枢神经系统内部分脑区:杏仁核、丘脑中线核群和板内核群、中脑导水管周围灰质(PAG)中星形胶质细胞、小胶质细胞标记物以及细胞因子(IL-1β,TNF-α)在基因水平和蛋白水平的表达变化。方法:(1)行为学检测:用辐射热刺激和机械刺激检测大鼠双侧后爪注射CFA后不同时间点的温度痛敏和机械痛敏的变化。(2)RT-PCR:21只SD大鼠随机分成7组,每组3只。第1组为正常对照组,第2-4组为注射CFA后4h、3d、14d组,第5-7组为注射生理盐水后4h、3d、14d组。取各组大鼠的杏仁核、丘脑中线核群和板内核群、PAG,提取总RNA,用半定量RT-PCR方法分析相同部位不同时间点各组星形胶质细胞激活标记物-GFAP、小胶质细胞激活标记物-CD14、炎症前细胞因子IL-1β、TNF-αmRNA表达的变化。以各泳道中GFAP、CD14、IL-1β、TNF-α与β-actin或GAPDH条带的光密度比值表示GFAP、CD14、IL-1β,TNF-αmRNA的相对表达量。使用Stata7.0统计学软件进行t检验后,行单因素方差分析两两比较。P 0.05为显著性界值。(3)Western-blot: 21只SD大鼠随机分成7组,每组3只。第1组为正常对照组,第2-4组为注射CFA后12h、3d、14d组,第5-7组为注射生理盐水后12h、3d、14d组。取各组大鼠的杏仁核、丘脑中线核群和板内核群、PAG,提取蛋白,用Western-blot方法分析相同部位不同时间点各组GFAP、IL-1β、TNF-α蛋白表达的变化。以各泳道中GFAP、IL-1β、TNF-α与β-actin条带的光密度比值表示GFAP、IL-1β、TNF-α蛋白的相对表达量。使用Stata7.0统计学软件进行t检验后,行单因素方差分析两两比较。P 0.05为显著性界值。(4)免疫组化:用荧光单标的方法观察3d组CFA以及生理盐水组大鼠杏仁核、丘脑中线核群和板内核群、PAG内星形胶质细胞标记物GFAP以及小胶质细胞标记物Iba表达的变化。结果:(1)注射前,大鼠对机械性刺激的缩腿反应阈值平均为11.00±1.32g,注射CFA后4小时出现痛觉过敏,反应阈值为7.33±0.42g(n = 6,p 0.05 VS注射前),注射后12小时达到最高峰1.00g(p 0.001 VS注射前),这种机械痛敏持续到21天时为6(gp 0.01 VS注射前)。而注射CFA前,大鼠对热刺激的缩腿潜伏期为10.32±0.74s,在注射后12小时敏感性达到最高峰,缩腿潜伏期为3.18±0.24s(n = 6,p 0.001 VS注射前)。炎性疼痛的热痛敏持续到14天基本恢复正常。(2)各脑区GFAP mRNA表达量在注射CFA后3d、14d与正常组相比有显著性增加,CD14、IL-1β、TNF-α在注射CFA后4h、3d、14d有显著性增加。(3)各脑区GFAP蛋白表达量在注射CFA后3d、14d增多,IL-1β、TNF-α则是在12h、3d、14d均有增加。(4)GFAP荧光单标显示,在PAG、丘脑、杏仁核内星形胶质细胞在3d组CFA组比生理盐水组显著增加。而Iba荧光单标显示,各脑区内小胶质细胞在3d组CFA与生理盐水组相比,数量上未有显著增加,但形态上增殖肥大。结论:小胶质细胞激活可能与炎性疼痛的起始相关,而星形胶质细胞可能与炎性疼痛的维持相关,细胞因子表达的增加可能对痛觉增敏的发生起着重要的作用。
[Abstract]:AIM: To observe the gene levels of astrocytes, microglial markers and cytokines (IL-1beta, TNF-alpha) in the central nervous system (CNS) at different time points after complete Freund's adjuvant (CFA) induced peripheral inflammatory pain in rats: amygdala, midline thalamic nucleus and intraplate nucleus, periaqueductal gray (PAG). Changes in protein level.
Methods: (1) Behavioral test: The changes of temperature and mechanical hyperalgesia were detected by radiation-induced thermal stimulation and mechanical stimulation at different time points after injection of CFA into bilateral hind paws of rats. (2) RT-PCR: 21 SD rats were randomly divided into 7 groups with 3 rats in each group. Total RNA was extracted from amygdala, median thalamic nucleus and intraplate nucleus, PAG, and semi-quantitative RT-PCR was used to analyze the expression of GFAP, CD14, IL-1beta and TNF-alpha mRNA in each swimming passage. The optical density ratio of AP, CD14, IL-1beta, TNF-alpha to beta-actin or GAPDH bands indicated the relative expression of GFAP, CD14, IL-1beta, and TNF-alpha mRNA. After t-test using Stata 7.0 statistical software, univariate analysis of variance was used to compare the two pairs. The control group, group 2-4, 12 h, 3 d, 14 d after injection of CFA, group 5-7, 12 h, 3 d, 14 d after injection of normal saline. The amygdala, midline thalamic nucleus and intraplate nucleus, PAG, protein were extracted and the expression of GFAP, IL-1beta and TNF-alpha were analyzed by Western-blot. The optical density ratios of beta, TNF-alpha and beta-actin bands indicated the relative expression of GFAP, IL-1beta and TNF-alpha proteins. Stata 7.0 statistical software was used to test the relative expression of GFAP, IL-1beta and TNF-alpha proteins. Results: (1) Before injection, the threshold of contraction reaction to mechanical stimulation was 11.00 (+ 1.32 g), and 4 hours after injection of CFA, the threshold of hyperalgesia was 7.33 (+ 0.42 g) (n = 6, P 0.05 VS) before injection. The peak of mechanical hyperalgesia was 1.00G (p 0.001 VS before injection) at 12 hours and 6 (p 0.01 VS before injection) at 21 days. The latency of thermal stimulation was 10.32 + 0.74 s before injection of CFA, and reached the peak at 12 hours after injection. The latency of leg contraction was 3.18 + 0.24 s (n = 6, P 0.001 VS) before injection. The expression of GFAP mRNA in all brain regions increased significantly at 3 and 14 days after injection of CFA, while CD14, IL-1beta and TNF-alpha increased significantly at 4, 3 and 14 days after injection of CFA. (3) The expression of GFAP protein in all brain regions increased at 3 and 14 days after injection of CFA, while IL-1beta, TNF-alpha increased at 12, 3 and 14 days after injection. 4) GFAP fluorescence single labeling showed that the number of astrocytes in PAG, thalamus and amygdala increased significantly in the three-day group than in the normal saline group. However, Iba fluorescence single labeling showed that the number of microglia in the three-day group was not significantly increased compared with that in the normal saline group, but the proliferation and hypertrophy of microglia were observed. The onset of pain is correlated with astrocytes, which may be associated with the maintenance of inflammatory pain. Increased expression of cytokines may play an important role in pain sensitization.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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