TOLL样受体4信号对大鼠周围神经损伤后瓦勒变性及神经再生的影响
发布时间：2018-10-23 19:16
【摘要】：目的建立大鼠坐骨神经离断模型,产生瓦勒变性现象;靶向调控TOLL样受体4(TLR4)信号通路,探讨TLR4通路对大鼠坐骨神经损伤早期瓦勒变性和神经再生的影响。方法构建大鼠周围神经离断模型,观察瓦勒变性。将40只Wistar大鼠随机分为2组:假手术组(20只)和模型组(20只)。大鼠坐骨神经离断模型中干预TOLL样受体4(TLR4)信号通路。将60只Wistar大鼠随机分为3组:对照组(G1)(20只),TAK-242组(G2)(20只)和LPS组(G3)(20只)。模型组,对照组,TAK-242组和LPS组横断大鼠右侧坐骨神经后,行神经外膜端端吻合;假手术组显露坐骨神经不横断,逐层缝合切口。TAK-242组术前1 h及术后7 d大鼠每天尾静脉注射0.15 mg/kg TLR4拮抗剂TAK-242,LPS组大鼠在神经断端显微注射LPS(2 g/L)1μL,对照组大鼠注射同等体积生理盐水,模型组不作处理。于术后1.5h、24 h、3 d、4 d和7d取术侧坐骨神经。实时定量荧光PCR(qRT-PCR)检测坐骨神经中β-干扰素TIR结构域衔接蛋白(TRIF)mRNA、白介素1β(IL-1β)m RNA和单核细胞趋化蛋白-1(MCP-1)mRNA水平;免疫荧光法分析坐骨神经中CD68+细胞和iba1+细胞的表达;劳克坚牢蓝(LFB)染色观察坐骨神经髓鞘变化;苏木精-伊红(HE)染色观察坐骨神经的病理变化;免疫组化法检测TRIF蛋白和生长相关蛋白43蛋白(GAP-43)的表达;坐骨神经功能指数(SFI)分析大鼠下肢运动功能。结果建立大鼠坐骨神经离断模型:与假手术组相比,(1)qRT-PCR显示,模型组的IL-1β和MCP-1 mRNA表达水平均显著升高(P0.001,P0.001);(2)免疫荧光可见,模型组中CD68+细胞表达上调(P0.01);(4)HE染色可见,模型组轴突完全断裂,大量炎性细胞浸润;(3)LFB染色可见,与假手术组相比,模型组损伤远端发生脱髓鞘(P0.001);(5)坐骨神经功能指数(SFI)显示,模型组大鼠坐骨神经功能完全丧失。大鼠离断模型中干预TOLL样受体4(TLR4)信号通路:(1)qRT-PCR显示,与对照组相比,TAK-242组在TRIF、IL-1β和MCP-1 mRNA表达水平均显著减低((P0.001,P0.001,P0.001),LPS组IL-1β和MCP-1mRNA的表达明显升高(P0.01,P0.01);(2)免疫荧光可见,与对照组相比,iba1+细胞和CD68+细胞表达水平在TAK-242组均下调(P0.05,P0.05),在LPS组均上调(P0.05,P0.05);(4)HE染色可见,与对照组相比,炎性细胞及再生神经纤维在TAK-242组较少,而在LPS组较多;(3)LFB染色可见,与对照组相比,神经断端髓鞘碎片清除率在TAK-242组降低(P0.05),而在LPS组升高(P0.05);(5)免疫组织化学可见,与对照组相比,TAK-242组中TRIF表达下调(P0.01);GAP-43表达下调(P0.05);(5)SFI显示,与对照组相比,TAK-242组术后20,30和40 d同期比较明显降低(P0.05),LPS组术后20 d明显增高(P0.05)。结论TLR4信号通路可以调控免疫反应影响大鼠周围神经损伤后早期髓鞘碎片清除,提示TLR4信号通路参与瓦勒变性。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R688
本文编号：2290228
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R688
【参考文献】
相关期刊论文 前4条
1 李志行;吕敬雷;陈萍;赵仁亮;;缺血后处理局灶性脑缺血再灌注模型大鼠相关通路以及白细胞介素8的表达[J];中国组织工程研究;2015年49期
2 刘洁;花放;;TLR4介导的信号转导通路在新生儿缺血缺氧性脑病中的作用[J];解剖科学进展;2015年06期
3 郑为娜;王珊;生晓娜;孙文蕾;张国华;;TLR4与神经变性疾病和多发性硬化[J];中国神经免疫学和神经病学杂志;2015年04期
4 王大永;徐翔;孙立倩;姜立先;郑宇;;亚低温治疗对脑出血大鼠TLR4/NF-κB介导的神经炎症反应的影响[J];神经解剖学杂志;2015年03期
，本文编号：2290228
本文链接：https://www.wllwen.com/yixuelunwen/waikelunwen/2290228.html
最近更新
教材专著
