發(fā)布時(shí)間：2018-06-14 10:49

  本文選題：消退素D1 + 燒傷痛�。� 參考：《鄭州大學(xué)》2016年碩士論文

【摘要】：背景消退素(resolvins,Rvs)是來源于ω-3多不飽和脂肪酸的內(nèi)源性促炎癥消退介質(zhì),包括D系和E系消退素,在多種炎性動物模型中具有抗炎和促炎癥消退作用。最近的研究發(fā)現(xiàn)Rvs還能通過調(diào)節(jié)各種瞬時(shí)受體電位通道的活性、促炎因子和抗炎介質(zhì)的表達(dá)及中樞敏化的形成等有效減輕炎性疼痛、術(shù)后疼痛和神經(jīng)病理性痛。脊髓膠質(zhì)細(xì)胞、小膠質(zhì)細(xì)胞內(nèi)的p-p38 MAPK和腦源性神經(jīng)營養(yǎng)因子(brain-derived neurotrophic factor,BDNF)及其受體原肌球蛋白相關(guān)激酶B(tropomyosin-related kinase B,Trk B)在神經(jīng)病理性痛的形成中發(fā)揮重要作用,而燒傷疼痛含有神經(jīng)病理性痛的成分。因此,它們可能也參與燒傷疼痛的形成。體內(nèi)外研究表明Rvs可以抑制小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞的活化,抑制小膠質(zhì)細(xì)胞內(nèi)p38 MAPK磷酸化,減少其分泌炎性介質(zhì)。因此,我們提出假說消退素D1(resolvin D1,Rv D1)可能通過抑制脊髓背角內(nèi)膠質(zhì)細(xì)胞活化、小膠質(zhì)細(xì)胞內(nèi)p38 MAPK磷酸化、下調(diào)BDNF/Trk B受體信號,從而減輕燒傷疼痛。目的本研究通過建立大鼠燒傷模型、腹腔注射Rv D1探索Rv D1在燒傷疼痛中的作用及其機(jī)制;通過鞘內(nèi)注射SB203580抑制p38 MAPK活化,觀察燒傷大鼠的痛行為學(xué)變化,檢測脊髓背角Iba-1、BDNF和Trk B的表達(dá)水平的變化,從而探討p38 MAPK活化在Rv D1對于燒傷疼痛作用機(jī)制中所發(fā)揮的作用;通過鞘內(nèi)注射Trk B-Fc抑制BDNF/Trk B信號,觀察燒傷大鼠的痛行為學(xué)變化,檢測脊髓背角Iba-1和p-p38 MAPK的表達(dá)水平的變化,從而探討B(tài)DNF/Trk B信號在Rv D1對于燒傷疼痛作用機(jī)制中所發(fā)揮的作用。方法1.140~150g清潔級雄性SD大鼠用隨機(jī)數(shù)字表法分為5組(n=6):假傷組(Sham+Veh1組),燒傷組(Burn+Veh1組),燒傷+Rv D1低劑量組(Burn+R(S)組),燒傷+Rv D1高劑量組(Burn+R(L)組),假傷+Rv D1高劑量組(Sham+R(L)組)。制備燒傷模型,于燒傷前30min及燒傷后1-7d分別腹腔注射100ng、300ng Rv D1或?qū)φ杖軇￢eh1。分別于燒傷前1d和燒傷后1、3、5、7、14d測定大鼠機(jī)械縮足反應(yīng)閾(MWT)。于燒傷后7d MWT測定結(jié)束后處死3只大鼠,用熒光免疫組化檢測脊髓小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞標(biāo)記物Iba-1和GFAP、p-p38MAPK、BDNF/Trk B的表達(dá)水平。用免疫熒光雙標(biāo)檢測p-p38 MAPK分別與Iba-1、GFAP和Neu N的共標(biāo)情況。2.140~150g清潔級雄性SD大鼠用隨機(jī)數(shù)字表法分為5組(n=6):Sham+Veh2組,Burn+Veh2組,Burn+SB203580組,Sham+SB203580組,Burn+R(L)組。制備燒傷模型,于造模后7d分別鞘內(nèi)注射SB203580或?qū)φ杖軇￢eh2,Burn+R(L)組腹腔注射Rv D1方法同前。分別于鞘內(nèi)注藥前、鞘內(nèi)注藥后15min、30min、1h和2h測定大鼠MWT。大鼠MWT測定結(jié)束后處死3只大鼠,用熒光免疫組化檢測脊髓小膠質(zhì)細(xì)胞標(biāo)記物Iba-1和BDNF/Trk B的表達(dá)水平。3.140~150g清潔級雄性SD大鼠用隨機(jī)數(shù)字表法分為5組(n=6):Sham+Veh3組,Burn+Veh3組,Burn+Trk B-Fc組,Sham+Trk B-Fc組,Burn+R(L)組。制備燒傷模型,于造模前1h和造模后1-7d用微量注射器通過鞘內(nèi)置管分別給予Trk B-Fc或?qū)φ杖軇￢eh3,Burn+R(L)組腹腔注射Rv D1方法同前。分別于燒傷前1d和燒傷后1、3、5、7、14d測定大鼠MWT。于燒傷后7d MWT測定結(jié)束后處死3只大鼠,用熒光免疫組化檢測脊髓小膠質(zhì)細(xì)胞標(biāo)記物Iba-1和p-p38 MAPK的表達(dá)水平。結(jié)果1.與Sham+Veh1組比較,Burn+Veh1組燒傷后各時(shí)點(diǎn)MWT降低(P0.05);與Burn+Veh1組比較,Burn+R(S)組和Burn+R(L)組燒傷后各時(shí)點(diǎn)MWT均升高(P0.05);與Burn+R(S)組比較,Burn+R(L)組燒傷后各時(shí)點(diǎn)MWT升高(P0.05)。免疫熒光顯示,與Sham+Veh1組比較,Burn+Veh1組脊髓背角Iba-1、GFAP、p-p38 MAPK、BDNF和Trk B受體表達(dá)水平均升高(P0.05);與Burn+Veh1組比較,Burn+R(L)組脊髓背角Iba-1、GFAP、p-p38 MAPK、BDNF和Trk B受體表達(dá)水平均降低(P0.05)。免疫熒光雙標(biāo)顯示,p-p38 MAPK只與Iba-1共標(biāo),而不與GFAP和Neu N共標(biāo)。2.與Sham+Veh2組相比,Burn+Veh2組MWT降低(P0.05);與Burn+Veh2組相比,Burn+SB203580組MWT升高(P0.05);與Burn+SB203580組注藥后各時(shí)點(diǎn)相比,Burn+R(L)組MWT差異無統(tǒng)計(jì)學(xué)意義(P0.05)。免疫熒光顯示,與Sham+Veh2組相比,Burn+Veh2組和Burn+SB203580組Iba-1、BDNF和Trk B受體表達(dá)水平均升高(P0.05);與Burn+Veh2組相比,Burn+SB203580組Iba-1、BDNF和Trk B受體表達(dá)水平均降低(P0.05)。3.與Sham+Veh3組相比,Burn+Veh3組燒傷后各時(shí)點(diǎn)MWT降低(P0.05);與Burn+Veh3組相比,Burn+Trk B-Fc組注藥后MWT升高(P0.05);與Burn+Trk B-Fc組相比,Burn+R(L)組MWT差異無統(tǒng)計(jì)學(xué)意義(P0.05)。免疫熒光顯示,與Sham+Veh3組相比,Burn+Veh3組和Burn+Trk B-Fc組Iba-1和p-p38MAPK表達(dá)水平均升高(P0.05);與Burn+Veh3組相比,Burn+Trk B-Fc組Iba-1和p-p38 MAPK表達(dá)水平均降低(P0.05)。結(jié)論消退素D1(resolvin D1,Rv D1)可通過抑制脊髓背角內(nèi)膠質(zhì)細(xì)胞活化、小膠質(zhì)細(xì)胞內(nèi)p38 MAPK磷酸化、下調(diào)BDNF/Trk B受體信號的表達(dá),從而減輕燒傷疼痛。
[Abstract]:Resolvins (Rvs) is an endogenous pro-inflammatory regression medium derived from Omega -3 polyunsaturated fatty acids, including the D and E system antiinflammatory agents, which have anti-inflammatory and proinflammatory response in a variety of inflammatory animal models. Recent studies have found that Rvs can also regulate the activity of various transient receptor potential channels, proinflammatory factors and anti-inflammatory factors. The expression of medium and the formation of central sensitization effectively alleviated inflammatory pain, postoperative pain and neuropathic pain. Spinal glial cells, p-p38 MAPK in microglia and brain-derived neurotrophic factor (BDNF) and their receptor promyocal egg white related kinase B (tropomyosin-related kinase B, Trk B) It plays an important role in the formation of neuropathic pain, and burn pain contains neuropathic pain components. Therefore, they may also participate in the formation of burn pain. In vivo and in vivo studies have shown that Rvs can inhibit the activation of microglia and astrocytes, inhibit the phosphorylation of p38 MAPK in microglia, and reduce the secretion of inflammatory mediates Therefore, we suggest that the hypothesis D1 (resolvin D1, Rv D1) may inhibit the activation of glial cells in the dorsal horn of the spinal cord, the phosphorylation of p38 MAPK in microglia, down regulation of the B receptor signal of BDNF/Trk, and thus reduce the pain of the BDNF/Trk, thus the purpose of this study was to establish a rat burn model by intraperitoneal injection of Rv D1 to explore the pain of Rv in the burn. To observe the changes in pain behavior in burned rats by intrathecal injection of SB203580 and to inhibit the activation of p38 MAPK, the changes in the expression of Iba-1, BDNF and Trk B in the dorsal horn of the spinal cord were detected, and the effect of p38 MAPK activation on the mechanism of the action of Rv D1 on the mechanism of burn pain was investigated. The changes in the pain behavior of the burned rats were observed and the changes of the expression level of Iba-1 and p-p38 MAPK in the dorsal horn of the spinal cord were detected, and the role of BDNF/Trk B signal in the mechanism of Rv D1 on the action of burn pain was explored. Method 1.140~150g clean grade male SD rats were divided into 5 groups (n=6) with random number table: false injury group (Sham+Veh1 group) and burn group ( Burn+Veh1 group, +Rv D1 low dose group (Burn+R (S) group), +Rv D1 high dose group (Burn+R (L) group), false wound +Rv D1 high dose group (Sham+R (Sham+R) group). The threshold of contraction reaction (MWT). After the end of 7D MWT, 3 rats were killed and the expression level of Iba-1, GFAP, p-p38MAPK, BDNF/Trk B of spinal microglia and astrocytes was detected by immunofluorescence. The male SD rats were divided into 5 groups (n=6): group Sham+Veh2, group Burn+Veh2, group Burn+SB203580, group Sham+SB203580, Burn+R (L). The model of burn was prepared by injection of SB203580 or controlled solvent Veh2 in the sheath. 3 rats were killed after MWT determination in MWT. rats and 3 rats were killed. The expression level of microglia markers Iba-1 and BDNF/Trk B in the male SD rats was detected by immunofluorescence. The random number table method was used to determine the level of.3.140~150g clean male SD rats into 5 groups (n=6): Sham+Veh3, Burn+Veh3, Burn+Trk. To prepare the burn model, 1-7d was given Trk B-Fc or Veh3, Burn+R (L) group by intraperitoneal injection of Rv D1 (Rv D1) before the 1H and the model of the model after the model of the model. 3 rats were killed and the rats were killed after the burn before and after the burn, respectively. The expression level of the microglia markers Iba-1 and p-p38 MAPK was measured. Results 1. compared with the Sham+Veh1 group, MWT decreased at each time point in Burn+Veh1 group (P0.05). Compared with the Burn+Veh1 group, both Burn+R (S) and Burn+R (L) groups increased at every point after burn. Immunofluorescence showed that the expression level of Iba-1, GFAP, p-p38 MAPK, BDNF and Trk B receptor in the spinal dorsal horn of group Burn+Veh1 increased (P0.05), compared with the Sham+Veh1 group, and the expression level of the spinal dorsal horn of the group was lower than that in the Burn+Veh1 group. Compared with the Sham+Veh2 group, the MWT decreased (P0.05) in the Burn+Veh2 group without the co labeling of GFAP and Neu N, and the MWT increased (P0.05) in the Burn+SB203580 group compared with the Burn+Veh2 group. The expression level of Iba-1, BDNF and Trk B receptors in the 0 groups increased (P0.05). Compared with the Burn+Veh2 group, the expression level of Iba-1, BDNF and Trk B receptors in the Burn+SB203580 group decreased (P0.05) was lower than that in the group. Compared with group Burn+R (L), there was no significant difference in MWT (P0.05). Immunofluorescence showed that the expression level of Iba-1 and p-p38MAPK increased in both Burn+Veh3 and Burn+Trk B-Fc groups (P0.05). By inhibiting the activation of glial cells in the dorsal horn of the spinal cord, the phosphorylation of p38 MAPK in the microglia and the down regulation of BDNF/Trk B receptor signaling can reduce the burn pain.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R644

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