【摘要】： 目的:觀察完全弗氏佐劑(CFA)誘導(dǎo)的大鼠外周炎癥性疼痛后不同時(shí)間點(diǎn)中樞神經(jīng)系統(tǒng)內(nèi)部分腦區(qū):杏仁核、丘腦中線核群和板內(nèi)核群、中腦導(dǎo)水管周圍灰質(zhì)(PAG)中星形膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞標(biāo)記物以及細(xì)胞因子(IL-1β,TNF-α)在基因水平和蛋白水平的表達(dá)變化。 方法:(1)行為學(xué)檢測(cè):用輻射熱刺激和機(jī)械刺激檢測(cè)大鼠雙側(cè)后爪注射CFA后不同時(shí)間點(diǎn)的溫度痛敏和機(jī)械痛敏的變化。(2)RT-PCR:21只SD大鼠隨機(jī)分成7組,每組3只。第1組為正常對(duì)照組,第2-4組為注射CFA后4h、3d、14d組,第5-7組為注射生理鹽水后4h、3d、14d組。取各組大鼠的杏仁核、丘腦中線核群和板內(nèi)核群、PAG,提取總RNA,用半定量RT-PCR方法分析相同部位不同時(shí)間點(diǎn)各組星形膠質(zhì)細(xì)胞激活標(biāo)記物-GFAP、小膠質(zhì)細(xì)胞激活標(biāo)記物-CD14、炎癥前細(xì)胞因子IL-1β、TNF-αmRNA表達(dá)的變化。以各泳道中GFAP、CD14、IL-1β、TNF-α與β-actin或GAPDH條帶的光密度比值表示GFAP、CD14、IL-1β,TNF-αmRNA的相對(duì)表達(dá)量。使用Stata7.0統(tǒng)計(jì)學(xué)軟件進(jìn)行t檢驗(yàn)后,行單因素方差分析兩兩比較。P 0.05為顯著性界值。(3)Western-blot: 21只SD大鼠隨機(jī)分成7組,每組3只。第1組為正常對(duì)照組,第2-4組為注射CFA后12h、3d、14d組,第5-7組為注射生理鹽水后12h、3d、14d組。取各組大鼠的杏仁核、丘腦中線核群和板內(nèi)核群、PAG,提取蛋白,用Western-blot方法分析相同部位不同時(shí)間點(diǎn)各組GFAP、IL-1β、TNF-α蛋白表達(dá)的變化。以各泳道中GFAP、IL-1β、TNF-α與β-actin條帶的光密度比值表示GFAP、IL-1β、TNF-α蛋白的相對(duì)表達(dá)量。使用Stata7.0統(tǒng)計(jì)學(xué)軟件進(jìn)行t檢驗(yàn)后,行單因素方差分析兩兩比較。P 0.05為顯著性界值。(4)免疫組化:用熒光單標(biāo)的方法觀察3d組CFA以及生理鹽水組大鼠杏仁核、丘腦中線核群和板內(nèi)核群、PAG內(nèi)星形膠質(zhì)細(xì)胞標(biāo)記物GFAP以及小膠質(zhì)細(xì)胞標(biāo)記物Iba表達(dá)的變化。結(jié)果:(1)注射前,大鼠對(duì)機(jī)械性刺激的縮腿反應(yīng)閾值平均為11.00±1.32g,注射CFA后4小時(shí)出現(xiàn)痛覺(jué)過(guò)敏,反應(yīng)閾值為7.33±0.42g(n = 6,p 0.05 VS注射前),注射后12小時(shí)達(dá)到最高峰1.00g(p 0.001 VS注射前),這種機(jī)械痛敏持續(xù)到21天時(shí)為6(gp 0.01 VS注射前)。而注射CFA前,大鼠對(duì)熱刺激的縮腿潛伏期為10.32±0.74s,在注射后12小時(shí)敏感性達(dá)到最高峰,縮腿潛伏期為3.18±0.24s(n = 6,p 0.001 VS注射前)。炎性疼痛的熱痛敏持續(xù)到14天基本恢復(fù)正常。(2)各腦區(qū)GFAP mRNA表達(dá)量在注射CFA后3d、14d與正常組相比有顯著性增加,CD14、IL-1β、TNF-α在注射CFA后4h、3d、14d有顯著性增加。(3)各腦區(qū)GFAP蛋白表達(dá)量在注射CFA后3d、14d增多,IL-1β、TNF-α則是在12h、3d、14d均有增加。(4)GFAP熒光單標(biāo)顯示,在PAG、丘腦、杏仁核內(nèi)星形膠質(zhì)細(xì)胞在3d組CFA組比生理鹽水組顯著增加。而Iba熒光單標(biāo)顯示,各腦區(qū)內(nèi)小膠質(zhì)細(xì)胞在3d組CFA與生理鹽水組相比,數(shù)量上未有顯著增加,但形態(tài)上增殖肥大。結(jié)論:小膠質(zhì)細(xì)胞激活可能與炎性疼痛的起始相關(guān),而星形膠質(zhì)細(xì)胞可能與炎性疼痛的維持相關(guān),細(xì)胞因子表達(dá)的增加可能對(duì)痛覺(jué)增敏的發(fā)生起著重要的作用。
[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.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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