【摘要】：目的建立大鼠坐骨神經(jīng)離斷模型,產(chǎn)生瓦勒變性現(xiàn)象;靶向調(diào)控TOLL樣受體4(TLR4)信號通路,探討TLR4通路對大鼠坐骨神經(jīng)損傷早期瓦勒變性和神經(jīng)再生的影響。方法構(gòu)建大鼠周圍神經(jīng)離斷模型,觀察瓦勒變性。將40只Wistar大鼠隨機(jī)分為2組:假手術(shù)組(20只)和模型組(20只)。大鼠坐骨神經(jīng)離斷模型中干預(yù)TOLL樣受體4(TLR4)信號通路。將60只Wistar大鼠隨機(jī)分為3組:對照組(G1)(20只),TAK-242組(G2)(20只)和LPS組(G3)(20只)。模型組,對照組,TAK-242組和LPS組橫斷大鼠右側(cè)坐骨神經(jīng)后,行神經(jīng)外膜端端吻合;假手術(shù)組顯露坐骨神經(jīng)不橫斷,逐層縫合切口。TAK-242組術(shù)前1 h及術(shù)后7 d大鼠每天尾靜脈注射0.15 mg/kg TLR4拮抗劑TAK-242,LPS組大鼠在神經(jīng)斷端顯微注射LPS(2 g/L)1μL,對照組大鼠注射同等體積生理鹽水,模型組不作處理。于術(shù)后1.5h、24 h、3 d、4 d和7d取術(shù)側(cè)坐骨神經(jīng)。實(shí)時(shí)定量熒光PCR(qRT-PCR)檢測坐骨神經(jīng)中β-干擾素TIR結(jié)構(gòu)域銜接蛋白(TRIF)mRNA、白介素1β(IL-1β)m RNA和單核細(xì)胞趨化蛋白-1(MCP-1)mRNA水平;免疫熒光法分析坐骨神經(jīng)中CD68+細(xì)胞和iba1+細(xì)胞的表達(dá);勞克堅(jiān)牢藍(lán)(LFB)染色觀察坐骨神經(jīng)髓鞘變化;蘇木精-伊紅(HE)染色觀察坐骨神經(jīng)的病理變化;免疫組化法檢測TRIF蛋白和生長相關(guān)蛋白43蛋白(GAP-43)的表達(dá);坐骨神經(jīng)功能指數(shù)(SFI)分析大鼠下肢運(yùn)動(dòng)功能。結(jié)果建立大鼠坐骨神經(jīng)離斷模型:與假手術(shù)組相比,(1)qRT-PCR顯示,模型組的IL-1β和MCP-1 mRNA表達(dá)水平均顯著升高(P0.001,P0.001);(2)免疫熒光可見,模型組中CD68+細(xì)胞表達(dá)上調(diào)(P0.01);(4)HE染色可見,模型組軸突完全斷裂,大量炎性細(xì)胞浸潤;(3)LFB染色可見,與假手術(shù)組相比,模型組損傷遠(yuǎn)端發(fā)生脫髓鞘(P0.001);(5)坐骨神經(jīng)功能指數(shù)(SFI)顯示,模型組大鼠坐骨神經(jīng)功能完全喪失。大鼠離斷模型中干預(yù)TOLL樣受體4(TLR4)信號通路:(1)qRT-PCR顯示,與對照組相比,TAK-242組在TRIF、IL-1β和MCP-1 mRNA表達(dá)水平均顯著減低((P0.001,P0.001,P0.001),LPS組IL-1β和MCP-1mRNA的表達(dá)明顯升高(P0.01,P0.01);(2)免疫熒光可見,與對照組相比,iba1+細(xì)胞和CD68+細(xì)胞表達(dá)水平在TAK-242組均下調(diào)(P0.05,P0.05),在LPS組均上調(diào)(P0.05,P0.05);(4)HE染色可見,與對照組相比,炎性細(xì)胞及再生神經(jīng)纖維在TAK-242組較少,而在LPS組較多;(3)LFB染色可見,與對照組相比,神經(jīng)斷端髓鞘碎片清除率在TAK-242組降低(P0.05),而在LPS組升高(P0.05);(5)免疫組織化學(xué)可見,與對照組相比,TAK-242組中TRIF表達(dá)下調(diào)(P0.01);GAP-43表達(dá)下調(diào)(P0.05);(5)SFI顯示,與對照組相比,TAK-242組術(shù)后20,30和40 d同期比較明顯降低(P0.05),LPS組術(shù)后20 d明顯增高(P0.05)。結(jié)論TLR4信號通路可以調(diào)控免疫反應(yīng)影響大鼠周圍神經(jīng)損傷后早期髓鞘碎片清除,提示TLR4信號通路參與瓦勒變性。
[Abstract]:Objective to establish a rat model of sciatic nerve fragmentation and produce Vall degeneration, and to investigate the effect of TLR4 pathway on the early Vall degeneration and nerve regeneration in rats with sciatic nerve injury by regulating the signal pathway of TOLL like receptor 4 (TLR4). Methods the rat model of peripheral nerve dissection was established and Vall degeneration was observed. Forty Wistar rats were randomly divided into two groups: sham operation group (n = 20) and model group (n = 20). TOLL-like receptor 4 (TLR4) signaling pathway was interfered with in rat sciatic nerve transection model. Sixty Wistar rats were randomly divided into three groups: control group (G _ 1) (n = 20), TAK-242 group (G _ 2) (n = 20) and LPS group (G _ 3) (n = 20). The right sciatic nerve was transected in the model group, control group, TAK-242 group and LPS group, the sciatic nerve was transected by end-to-end anastomosis of the epineurium, and the sciatic nerve was not transected in the sham operation group. One hour before operation and seven days after operation, the rats in TAK-242 group were given LPS (2 g / L) 1 渭 L intravenously in TAK-242,LPS group, and the rats in control group were injected with the same volume of normal saline. The model group was not treated. The sciatic nerve was removed at 1. 5 h, 24 h, 3 d, 4 d and 7 d after operation. The expression of IL-1 尾) m RNA (IL-1 尾) m RNA) and MCP-1 (MCP-1) mRNA in sciatic nerve was detected by real-time quantitative PCR (qRT-PCR), and the expression of CD68 cells and iba1 cells in sciatic nerve was analyzed by immunofluorescence. The changes of myelin sheath of sciatic nerve were observed by (LFB) staining, pathological changes of sciatic nerve were observed by (HE) staining of hematoxylin and eosin, expression of TRIF protein and growth-associated protein 43 protein (GAP-43) were detected by immunohistochemistry. Sciatic nerve function index (SFI) was used to analyze the motor function of lower extremities in rats. Results: (1) compared with sham-operated group, the expression of IL-1 尾 and MCP-1 mRNA in model group was significantly increased (P0.001, P0.001); (2), and the expression of CD68 cells was up-regulated (P0.01); (4) HE staining in model group. (3) LFB staining showed that the distal end of injury occurred demyelinating (P0.001); (5) sciatic nerve function index (SFI) in the model group, and the sciatic nerve function was completely lost in the model group. Intervention of TOLL like receptor 4 (TLR4) signal pathway in rat model: (1) qRT-PCR showed that the expression levels of TRIF,IL-1 尾 and MCP-1 mRNA in the TAK-242 group were significantly lower than those in the control group (P0.001 + P0.001), LPS group, the expression of IL-1 尾 and MCP-1mRNA increased significantly (P0.01); (2). Compared with the control group, the expression of iba1 cells and CD68 cells were down-regulated in the TAK-242 group (P0.05P 0.05), and up-regulated in the LPS group (P0.05P 0.05); (4) HE staining. Compared with the control group, the inflammatory cells and regenerated nerve fibers were less in the TAK-242 group and more in the LPS group. (3) LFB staining was observed. Compared with the control group, the clearance rate of myelin shards in the TAK-242 group was lower than that in the control group (P0.05), while in the LPS group it was increased (P0.05); (5) immunohistochemistry. Compared with the control group, the expression of TRIF was down-regulated (P0.01) in the TAK-242 group, and the expression of GAP-43 was down-regulated (P0.05); (5) SFI showed. Compared with the control group, the TAK-242 group was significantly lower than the control group at the same time of 20 days and 40 days after operation (P0.05), LPS group 20 days after operation significantly increased (P0.05). Conclusion TLR4 signaling pathway can regulate the immune response and affect the early clearance of myelin sheath fragments after peripheral nerve injury in rats, suggesting that TLR4 signaling pathway is involved in Vall degeneration.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R688

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