【作者】 曹剑；

【导师】 尹维田；

【作者基本信息】 吉林大学 ， 外科学， 2011， 博士

【摘要】 目的：观察巴西苏木素在体内、外实验中对Balb/c小鼠免疫机能的影响,探讨Balb/c小鼠坐骨神经损伤后巴西苏木素抑制免疫反应及促进周围神经再生的作用机制。方法：体外细胞实验选用Balb/c小鼠的脾细胞,并在不同浓度巴西苏木素及苏木乙醇提取物(SME)干预下观察淋巴细胞转化能力；体内实验应用Balb/c小鼠坐骨神经损伤模型,通过免疫组化、Real-time PCR、Western-blot以及TUNEL技术观察应用巴西苏木素后GAP-43、NF-kB的变化,坐骨神经脱髓鞘程度,有髓神经纤维再生数目以及神经功能恢复的情况。结果：通过体外细胞实验观察到巴西苏木素可以显著抑制Balb/c小鼠T/B淋巴细胞转化能力；通过体内实验观察到巴西苏木素可以降低损伤局部及实验动物体内的细胞免疫强度,抑制NF-kB的过度表达,促进GAP-43的表达,降低损伤及修复再生过程中坐骨神经的脱髓鞘程度和神经元的凋亡数目,同时增加了有髓神经纤维再生数目,并促进周围神经损伤后的功能恢复。结论：体外细胞实验说明巴西苏木素能明显抑制Balb/c小鼠的免疫机能；体内实验说明周围神经损伤后体内产生一系列免疫变化,而巴西苏木素可以降低神经损伤引发的免疫反应的强度,减少免疫反应引起的继发性神经损害,从而促进周围神经损伤后的再生及功能恢复。更多还原

【Abstract】 Objective:To observe the effect of Brazilin on immune function, and to explore its suppression of autoimmune response to the promotion of nerve regeneration in Balb/ c mice.Methods and Results:In vitro and in vivo experiments in different concentrations of the intervention of the Brazilin observed by Balb/c mouse lymphocyte function changes; in vivo experiments with different concentrations of Brazilin observed after a sciatic nerve injury in mice include local infiltration of lymphocyte, changes in immune function, nerve demyelination the extent of myelinated nerve fibers and nerve function recovery situation, and the data for image analysis and statistical analysis.1 The study on Balb/c mice immune function inhibition of Brazilin Methods: The sciatic nerve injury models in mice a total of 60 patients were randomly divided into Brazilin high dose group, middle dose group, low dose group and saline control group. Brazilin respectively 10mg/kg/d,5mg/kg/d,2.5mg/kg/d, intraperitoneal injection of saline solution, the control group were injected with normal saline. Continuous administration to death, isolation and culture of mouse spleen lymphocytes by MTT method spleen T lymphocytes and B lymphocyte transformation capability. At the same time isolated normal Balb/c mice spleen lymphocytes in different concentrations of ethanol in Brazilin and hematoxylin extract (SME) intervention, the application MTT method spleen T lymphocytes and B lymphocyte transformation capability. The results show that:Brazilin intervention, whether in vitro or in vivo in mice, can significantly on the ability of lymphocyte transformation inhibition and dose-dependent effect.2.The effect of Brazilin after peripheral nerve injury repair and regeneration-related protein expression in experimentalMethods:320 cases of sciatic nerve injury in Balb/c mouse model, according to the first part of the experimental design and conduct drug intervention groups.By immunohistochemical assay. Real-time PCR, and western blot, the GAP-43. NF-kB expression in sciatic nerve spinal cord segment in response to brazilin were detected. The results show that:IHC,Western-blot and Real-time PCR results showed that after sciati nerve injury, gap-43 in the spinal cord segment was activated and increased in expression; the high, middle dose of brazilin groups had significantly higher gap-43 expression than both the low dose group and the blank control, and continuously in 2 weeks. It can draw the conclusion that after sciatic nerve injury, brazilin had a sustained stimulative effect on gap-43 activation of the anterior horn cells in the spinal cord.IHC,Western-blot and Real-time PCR results also showed that after sciatic nerve injury. NF-kB in the spinal cord segment was activated and increased in expression; the high, middle dose of brazilein groups had significantly lower NF-κB expression than both the low dose group and the blank control. Its underlying mechanism may be through the inhibition of immune reaction, and regulates the activation of NF-kB of anterior horn cells in the spinal cord, thus promote the neural regeneration.3 Brazilin inhibite the immune response after injury mechanisms to promote nerve regeneration experimentsMethods:160 cases of sciatic nerve injury in Balb/c mouse model, grouped according to the first part of the experimental design and for drug intervention. General condition of mice. LFB staining the number of myelinated nerve fibers and myelin changes by HE staining the general form of skeletal muscle fibers, muscle fiber cross-sectional area and fiber number, etc.. on the sciatic functional index and nerve conduction measurement speed and amplitude of nerve functional recovery after injury, applied TUNEL staining neurons apoptosis. The results show that:the application of Brazilin did not increase mortality in mice, Brazilin can reduce the immune response after peripheral nerve injury caused by nerve damage and lower secondary nerve repair and regeneration process of demyelination level. Fast blue staining showed that the experimental group, the number of myelinated nerve fibers are more uniform, less degeneration of myelin. the control group, reduction in the number of myelinated nerve fibers, myelin sheath degeneration more common. Can be observed by HE staining, the muscle cells in experimental groups arranged closely, the larger cross-sectional area of muscle fibers, connective tissue hyperplasia less; the control group, the small cross-sectional area of muscle fiber, muscle fiber gap was widening, connective tissue proliferation more common. Sciatic functional index and the determination of nerve conduction velocity and amplitude of the experimental group showed recovery of neurological function than the control group. High dose group which apoptotic cell counts at all time points significantly lower than the low dose group and control group with significant differences.Conclusion:1 Brazilin inhibited Balb/c mice immune function, lymphocyte transformation by inhibiting the ability to reduce cellular and humoral immune response intensity, and within a certain range showed dose dependent manner.2 Brazilin by nerve growth associated protein GAP-43 growth associated protein expression promoted peripheral nerve injury repair and regeneration. Through the nuclear transcription factor NF-kB expression of inhibitory immune over-expression of inflammatory response, help promote the repair of peripheral nerve injury and regeneration.3 Brazilin intervention can reduce the peripheral nerve injury-induced secondary immune response caused by nerve damage, which reduces the process of nerve repair and regeneration of the extent of demyelination, to promote nerve repair after injury and regeneration.更多还原