NRG-1参与大鼠自体神经移植再生的机制研究
发布时间：2018-08-29 17:49
【摘要】：虽然显微外科技术已经有了很大的进步和发展,但是对于周围神经损伤和再生的病理生理机制我们仍然缺乏深入了解。周围神经缺损的治疗仍然是外科医生的一个重大难题。准确的掌握神经损伤修复的解剖学、病理生理学和外科重建技术是对神经损伤修复治疗的先决条件。对于大于5cm的神经缺失,直接的神经吻和会导致过大的张力,导致治疗的失败,需要进行自体神经移植或者应用套管技术。对于套管技术,虽然在生物医学工程方面取得了很大的创新和突破,但是由于免疫耐受的因素,仍然无法有效的应用于临床治疗。自体神经移植依然是治疗神经损伤的金标准,但是移植后修复的过程慢,坏死率高,是移植中常常遇到的问题。近年来,许多研究者发现NRG-1参与了神经损伤的修复,并且发挥着重要调节的作用。本研究中,为了探索NRG-1在神经移植后修复过程中的调节作用,我们采用SD大鼠自体神经移植模型,首先观察了NRG-1的表达变化及移植神经的修复过程,然后通过反义寡核苷酸技术,抑制NRG-1在自体神经移植模型中的表达,并采用SB203580特异性抑制P38αMAPK激活,并分别观察神经移植后髓鞘修复及神经功能恢复的过程,进一步分析NRG-1参与调控神经移植后髓鞘修复的作用及其信号转导机制。实验分为三部分:实验一NRG-1在大鼠自体神经移植再生过程中的变化目的:研究NRG-1在大鼠自体坐骨神经移植再生过程中的变化方法:采用清洁级健康雄性SD大鼠,40只,体重250-300g随机分为实验组和对照组,每组20只,按手术后3、7、14、21、28天分为5个时间点。实验组行坐骨神经倒转自体移植术,对照组单纯暴露坐骨神经后缝合处理,在不同时间点观察大鼠足迹的变化,计算坐骨神经指数(SFI),电生理检测运动神经传导速度(MNCV)变化,之后分别切取实验组移植段坐骨神经和对照组移植对应段的坐骨神经,透射电镜观察移植后的神经端髓鞘再生的变化,并通过RT-PCR技术检测II型和III型NRG-1 m RNA的表达变化,Western blot技术检测NRG-1蛋白表达变化。结果:术后各个时间点的实验组坐骨神经指数均低于对照组,且实验组坐骨神经指数随时间进行逐渐增加,差异有统计学意义(P0.01);在自体神经移植的3-28天实验组的坐骨神经运动神经传导速度小于对照组,并且有统计学差异(P均0.01);实验组有髓鞘神经纤维的面积(um2)在第7、14、21、28天与对照组比较有明显的统计学差异(P0.01),实验组轴突直径在第7、14、21天与对照组比较有明显的统计学差异(P0.01);RT-PCR结果分析显示,在移植的3-28天II型NRG-1 m RNA表达均较对照组增高,且差异有统计学意义(P分别0.01);Western blot结果显示在神经移植术后第3-28天,II型NRG-1和III型NRG-1蛋白表达也明显增高,且有显著性差异(P分别0.01)。结论:在神经移植后,NRG-1 m RNA及蛋白表达上调,II型NRG-1和III型NRG-1表达改变不同。NRG-1可能参与了神经移植后髓鞘再生的调节过程。实验二II型NRG-1在大鼠自体神经移植再生过程中的作用目的:研究II型NRG-1在大鼠自体坐骨神经移植再生过程中可能的作用。方法:采用清洁级健康雄性SD大鼠,54只,体重250-300g随机分为空白组(Blank)、缓冲液对照组(Model)和反义寡核苷酸抑制组(ASON),每组18只,按手术后3、7、14、21、28、35天分为6个时间点。缓冲液对照组和反义寡核苷酸抑制组行自体坐骨神经倒转移植术,在术后当日和第三天,Model组给予手术切口局部注射PBS缓冲液,ASON组给予注射II型NRG-1反义寡核苷酸;空白组单纯暴露坐骨神经后缝合处理。在不同时间点观察大鼠足迹的变化,计算坐骨神经指数,电生理检测运动神经传导速度。之后分别切取Model组及ASON组移植段坐骨神经和Blank组移植对应段的坐骨神经,透射电镜观察移植后的神经端髓鞘再生的变化,RT-PCR技术检测II型NRG-1 m RNA的变化,以及通过Western blot技术检测II型NRG-1蛋白变化,分析II型NRG-1在自体神经移植中可能的作用。结果:发现术后ASON组除第3天以外,其余各个时间点的ASON组坐骨神经指数均低于Model组的结果且差异有统计学意义(P0.01),在移植的14-35天,抑制II型NRG-1的表达,可减慢大鼠坐骨神经传导速度。说明缺少II型NRG-1减缓自体移植神经的再生。髓鞘的形态学分析提示II型NRG-1在自体神经移植3-28天,对髓鞘的崩解,雪旺细胞髓鞘化的过程中均发挥着一定的调节作用。RT-PCR和Western blot显示:II型NRG-1的m RNA和蛋白的表达均较Model组明显减低。结论:II型NRG-1在SD大鼠自体神经后神经再生的的过程中,在移植的3-28天,对髓鞘的崩解,雪旺细胞髓鞘化的过程中均发挥着一定的调节作用。缺少II型NRG-1可能影响移植后神经功能的恢复。实验三P38-MAPK参与NRG-1调控大鼠自体神经移植再生过程的机制研究目的:P38 MAPK在大鼠自体神经移植再生过程中对NRG-1/Erb B信号系统的转导作用方法:采用清洁级健康雄性SD大鼠,54只,体重250-300g随机分为空白组(Blank)、缓冲液对照组(Model)和P38αMAPK抑制组(P38αMAPK),每组18只,按手术后3、7、14、21、28、35天分为6个时间点。Model组和P38αMAPK抑制组行自体坐骨神经倒转移植术,在术后当日和第三天,Model组给予移植切口局部注射PBS缓冲液,P38αMAPK抑制组给予注射P38αMAPK抑制剂SB2035802;空白组单纯暴露坐骨神经后缝合不对其其他实验处理。在不同时间点观察大鼠足迹的变化,计算SFI,电生理检测MNCV,透射电镜观察移植后的神经端髓鞘再生的变化,RT-PCR技术检测髓鞘碱性蛋白(MBP)m RNA的变化,以及通过Western blot技术检测P-P38αMAPK蛋白变化。结果:SD大鼠的SFI值在移植的7、14、21、28、35五个时间点,P38αMAPK抑制组坐骨神经指数均低于Model组。MNCV值显示:在自体神经移植的14、21、28、35天P38αMAPK抑制组的传导速度小于Model组。在电镜的髓鞘观察和分析中有髓鞘神经纤维的面积(um2):P38αMAPK抑制组在第28、35天出现与Model组明显的统计学差异(P0.01),而前期无明显统计学差异;单位面积的有髓鞘神经纤维的数量:P38αMAPK抑制组与Model组无明显的统计学差异;轴突的直径:在第21、28、35天出现P38αMAPK抑制组与Model组明显的统计学差异(P0.01);对于G-ratio,在第28天和35天P38αMAPK抑制组均较Model组高且有明显的统计学差异(P0.01)。Western blot和RT-PCR显示:大鼠切口局部注射SB203580后,MBP的m RNA:P38αMAPK抑制组的表达较Model组减少;P-P38αMAPK:P38αMAPK抑制组均较Model组少。结论:在SD大鼠自体神经移植后,抑制P38αMAPK的激活,会出现大鼠神经功能恢复减慢,有髓鞘神经纤维的面积和轴突的直径会减小,G-ratio值增加。提示在自体神经移植模型中,P38αMAPK可能为NRG-1/Erb B信号转导途径的下游信号,并对神经功能的恢复和髓鞘的再生修复发挥着促进作用。
[Abstract]:Although microsurgical techniques have made great progress and development, the pathophysiological mechanisms of peripheral nerve injury and regeneration are still poorly understood. The treatment of peripheral nerve defects remains a major challenge for surgeons. Accurate understanding of the anatomy, pathophysiology and surgical reconstruction of nerve injury repair is essential. Technology is a prerequisite for the repair and treatment of nerve injuries. For a nerve defect greater than 5 cm, a direct nerve kiss can lead to excessive tension, leading to failure of treatment, autologous nerve grafting or cannulation technology is needed. For cannulation technology, although great innovations and breakthroughs have been made in biomedical engineering, however, it is necessary to use cannulation technology. Autologous nerve transplantation is still the gold standard for the treatment of nerve injury, but the slow process of repair and high rate of necrosis are common problems in transplantation. In recent years, many researchers have found that NRG-1 participates in the repair of nerve injury and plays an important role. In this study, in order to explore the regulatory role of NRG-1 in the process of nerve graft repair, we used the SD rat autologous nerve transplantation model. First, we observed the expression of NRG-1 and the repair process of the grafted nerve. Then we inhibited the expression of NRG-1 in the autologous nerve transplantation model by antisense oligonucleotide technology. SB203580 was used to specifically inhibit the activation of P38 alpha MAPK, and to observe the process of myelin repair and nerve function recovery after nerve transplantation, and further analyze the role of NRG-1 in regulating myelin repair after nerve transplantation and its signal transduction mechanism. AIM: To study the changes of NRG-1 in the process of sciatic nerve autograft regeneration in rats: 40 healthy male SD rats of clean grade, weighing 250-300g, were randomly divided into experimental group and control group, 20 rats in each group, divided into five time points according to 3, 7, 14, 21 and 28 days after operation. After the sciatic nerve was sutured, the footprints of rats were observed at different time points, the sciatic nerve index (SFI) was calculated, and the motor nerve conduction velocity (MNCV) was measured by electrophysiology. Then the sciatic nerve of the experimental group and the corresponding sciatic nerve of the control group were harvested and the myelin sheath regeneration at the nerve end was observed by transmission electron microscope. Results: The sciatic nerve index of the experimental group was lower than that of the control group at each time point after operation, and the sciatic nerve index of the experimental group increased gradually with time, the difference was statistically significant (P 0.01). The motor nerve conduction velocity of sciatic nerve in the experimental group was lower than that in the control group at 3-28 days after transplantation, and the difference was statistically significant (P 0.01); the area of myelinated nerve fibers (um2) in the experimental group was significantly different from that in the control group at 7, 14, 21 and 28 days (P 0.01); the diameter of axons in the experimental group was significantly different from that in the control group at 7, 14 and 21 days. The results of RT-PCR showed that the expression of type II NRG-1 m RNA was significantly higher than that of the control group at 3-28 days after transplantation (P 0.01, respectively); Western blot showed that the expression of type II NRG-1 and type III NRG-1 protein was also significantly higher at 3-28 days after nerve transplantation (P 0.01, respectively). CONCLUSION: After nerve transplantation, the expression of NRG-1 m RNA and protein is up-regulated, and the expression of NRG-1 type II and NRG-1 type III is different. NRG-1 may be involved in the regulation of myelin regeneration after nerve transplantation. Methods: Fifty-four healthy male SD rats weighing 250-300 g were randomly divided into blank group (Blank), buffer control group (Model) and antisense oligonucleotide inhibition group (ASON), 18 rats in each group, divided into 6 time points according to 3, 7, 14, 21, 28 and 35 days after operation. On the day of operation and the third day after operation, PBS buffer was injected into the incision in Model group and NRG-1 antisense oligonucleotide was injected into ASON group. In blank group, the sciatic nerve was simply exposed and sutured. Nerve conduction velocity. Sciatic nerves of model group and ASON group were harvested and corresponding segments of sciatic nerves of Blank group were transplanted. The changes of myelin sheath regeneration were observed by transmission electron microscopy. The changes of type II NRG-1 m RNA were detected by RT-PCR, and the changes of type II NRG-1 protein were detected by Western blot. Results: The sciatic nerve index of ASON group was lower than that of Model group at all other time points except the 3rd day after transplantation, and the difference was statistically significant (P 0.01). Inhibition of NRG-1 expression in type II could slow down the sciatic nerve conduction velocity of rats at 14-35 days after transplantation. G-1 slowed the regeneration of autologous nerve graft. Morphological analysis of myelin sheath suggested that type II NRG-1 played a certain role in the process of myelin breakdown and Schwann cell myelination at 3-28 days after autologous nerve transplantation. RT-PCR and Western blot showed that the expression of M RNA and protein of type II NRG-1 was significantly lower than that of model group. In the process of nerve regeneration after autologous nerve transplantation in SD rats, P38-MAPK played a role in the process of myelin sheath disintegration and Schwann cell myelination. Lack of type II NRG-1 may affect the recovery of nerve function after autologous nerve transplantation. Experiment 3 P38-MAPK participated in the mechanism of NRG-1 regulating nerve regeneration after autologous nerve transplantation in rats. Objective:To study the transduction of P38 MAPK on NRG-1/Erb B signal system in the process of nerve autograft regeneration in rats.Fifty-four clean-grade healthy male SD rats weighing 250-300 g were randomly divided into blank group(Blank),buffer control group(Model) and P38 alpha MAPK inhibitory group(P38 alpha MAPK) with 18 rats in each group at 3,7,14,21,28,35 days after operation. The model group and P38 alpha MAPK inhibitor group were treated with retrograde sciatic nerve transplantation. PBS buffer was injected into the graft incision on the first day and the third day after transplantation. The P38 alpha MAPK inhibitor SB2035802 was injected into the P38 alpha MAPK inhibitor group. The changes of rat footprints were observed at different time points, SFI was calculated, MNCV was detected by electrophysiology, myelin regeneration was observed by transmission electron microscopy, myelin basic protein (MBP) m RNA was detected by RT-PCR, and P-P38 alpha MAPK protein was detected by Western blot. At the time point, the sciatic nerve index of P38 alpha MAPK inhibitor group was lower than that of Model group. MNCV value showed that the conduction velocity of P38 alpha MAPK inhibitor group was lower than that of Model group at 14, 21, 28 and 35 days after autologous nerve transplantation. There was no significant difference in the number of myelinated nerve fibers per unit area between the P38 alpha MAPK inhibitor group and the Model group; the diameter of axon: there was significant difference between the P38 alpha MAPK inhibitor group and the Model group on the 21st, 28 and 35 days (P 0.01); for the G-ratio, there was significant difference in the number of P38 alpha MAPK on the 28th and 35th days (P 0.01). Western blot and RT-PCR showed that after local injection of SB203580, the expression of M RNA: P38 alpha MAPK of MB P in the inhibition group was lower than that in the model group, and that of P-P38 alpha MAPK: P38 alpha MAPK in the inhibition group was lower than that in the model group. It is suggested that P38 alpha MAPK may be the downstream signal of NRG-1/Erb B signal transduction pathway in the autologous nerve transplantation model, and may play an important role in the recovery of nerve function and the regeneration and repair of myelin sheath.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622.3
本文编号：2211956
[Abstract]:Although microsurgical techniques have made great progress and development, the pathophysiological mechanisms of peripheral nerve injury and regeneration are still poorly understood. The treatment of peripheral nerve defects remains a major challenge for surgeons. Accurate understanding of the anatomy, pathophysiology and surgical reconstruction of nerve injury repair is essential. Technology is a prerequisite for the repair and treatment of nerve injuries. For a nerve defect greater than 5 cm, a direct nerve kiss can lead to excessive tension, leading to failure of treatment, autologous nerve grafting or cannulation technology is needed. For cannulation technology, although great innovations and breakthroughs have been made in biomedical engineering, however, it is necessary to use cannulation technology. Autologous nerve transplantation is still the gold standard for the treatment of nerve injury, but the slow process of repair and high rate of necrosis are common problems in transplantation. In recent years, many researchers have found that NRG-1 participates in the repair of nerve injury and plays an important role. In this study, in order to explore the regulatory role of NRG-1 in the process of nerve graft repair, we used the SD rat autologous nerve transplantation model. First, we observed the expression of NRG-1 and the repair process of the grafted nerve. Then we inhibited the expression of NRG-1 in the autologous nerve transplantation model by antisense oligonucleotide technology. SB203580 was used to specifically inhibit the activation of P38 alpha MAPK, and to observe the process of myelin repair and nerve function recovery after nerve transplantation, and further analyze the role of NRG-1 in regulating myelin repair after nerve transplantation and its signal transduction mechanism. AIM: To study the changes of NRG-1 in the process of sciatic nerve autograft regeneration in rats: 40 healthy male SD rats of clean grade, weighing 250-300g, were randomly divided into experimental group and control group, 20 rats in each group, divided into five time points according to 3, 7, 14, 21 and 28 days after operation. After the sciatic nerve was sutured, the footprints of rats were observed at different time points, the sciatic nerve index (SFI) was calculated, and the motor nerve conduction velocity (MNCV) was measured by electrophysiology. Then the sciatic nerve of the experimental group and the corresponding sciatic nerve of the control group were harvested and the myelin sheath regeneration at the nerve end was observed by transmission electron microscope. Results: The sciatic nerve index of the experimental group was lower than that of the control group at each time point after operation, and the sciatic nerve index of the experimental group increased gradually with time, the difference was statistically significant (P 0.01). The motor nerve conduction velocity of sciatic nerve in the experimental group was lower than that in the control group at 3-28 days after transplantation, and the difference was statistically significant (P 0.01); the area of myelinated nerve fibers (um2) in the experimental group was significantly different from that in the control group at 7, 14, 21 and 28 days (P 0.01); the diameter of axons in the experimental group was significantly different from that in the control group at 7, 14 and 21 days. The results of RT-PCR showed that the expression of type II NRG-1 m RNA was significantly higher than that of the control group at 3-28 days after transplantation (P 0.01, respectively); Western blot showed that the expression of type II NRG-1 and type III NRG-1 protein was also significantly higher at 3-28 days after nerve transplantation (P 0.01, respectively). CONCLUSION: After nerve transplantation, the expression of NRG-1 m RNA and protein is up-regulated, and the expression of NRG-1 type II and NRG-1 type III is different. NRG-1 may be involved in the regulation of myelin regeneration after nerve transplantation. Methods: Fifty-four healthy male SD rats weighing 250-300 g were randomly divided into blank group (Blank), buffer control group (Model) and antisense oligonucleotide inhibition group (ASON), 18 rats in each group, divided into 6 time points according to 3, 7, 14, 21, 28 and 35 days after operation. On the day of operation and the third day after operation, PBS buffer was injected into the incision in Model group and NRG-1 antisense oligonucleotide was injected into ASON group. In blank group, the sciatic nerve was simply exposed and sutured. Nerve conduction velocity. Sciatic nerves of model group and ASON group were harvested and corresponding segments of sciatic nerves of Blank group were transplanted. The changes of myelin sheath regeneration were observed by transmission electron microscopy. The changes of type II NRG-1 m RNA were detected by RT-PCR, and the changes of type II NRG-1 protein were detected by Western blot. Results: The sciatic nerve index of ASON group was lower than that of Model group at all other time points except the 3rd day after transplantation, and the difference was statistically significant (P 0.01). Inhibition of NRG-1 expression in type II could slow down the sciatic nerve conduction velocity of rats at 14-35 days after transplantation. G-1 slowed the regeneration of autologous nerve graft. Morphological analysis of myelin sheath suggested that type II NRG-1 played a certain role in the process of myelin breakdown and Schwann cell myelination at 3-28 days after autologous nerve transplantation. RT-PCR and Western blot showed that the expression of M RNA and protein of type II NRG-1 was significantly lower than that of model group. In the process of nerve regeneration after autologous nerve transplantation in SD rats, P38-MAPK played a role in the process of myelin sheath disintegration and Schwann cell myelination. Lack of type II NRG-1 may affect the recovery of nerve function after autologous nerve transplantation. Experiment 3 P38-MAPK participated in the mechanism of NRG-1 regulating nerve regeneration after autologous nerve transplantation in rats. Objective:To study the transduction of P38 MAPK on NRG-1/Erb B signal system in the process of nerve autograft regeneration in rats.Fifty-four clean-grade healthy male SD rats weighing 250-300 g were randomly divided into blank group(Blank),buffer control group(Model) and P38 alpha MAPK inhibitory group(P38 alpha MAPK) with 18 rats in each group at 3,7,14,21,28,35 days after operation. The model group and P38 alpha MAPK inhibitor group were treated with retrograde sciatic nerve transplantation. PBS buffer was injected into the graft incision on the first day and the third day after transplantation. The P38 alpha MAPK inhibitor SB2035802 was injected into the P38 alpha MAPK inhibitor group. The changes of rat footprints were observed at different time points, SFI was calculated, MNCV was detected by electrophysiology, myelin regeneration was observed by transmission electron microscopy, myelin basic protein (MBP) m RNA was detected by RT-PCR, and P-P38 alpha MAPK protein was detected by Western blot. At the time point, the sciatic nerve index of P38 alpha MAPK inhibitor group was lower than that of Model group. MNCV value showed that the conduction velocity of P38 alpha MAPK inhibitor group was lower than that of Model group at 14, 21, 28 and 35 days after autologous nerve transplantation. There was no significant difference in the number of myelinated nerve fibers per unit area between the P38 alpha MAPK inhibitor group and the Model group; the diameter of axon: there was significant difference between the P38 alpha MAPK inhibitor group and the Model group on the 21st, 28 and 35 days (P 0.01); for the G-ratio, there was significant difference in the number of P38 alpha MAPK on the 28th and 35th days (P 0.01). Western blot and RT-PCR showed that after local injection of SB203580, the expression of M RNA: P38 alpha MAPK of MB P in the inhibition group was lower than that in the model group, and that of P-P38 alpha MAPK: P38 alpha MAPK in the inhibition group was lower than that in the model group. It is suggested that P38 alpha MAPK may be the downstream signal of NRG-1/Erb B signal transduction pathway in the autologous nerve transplantation model, and may play an important role in the recovery of nerve function and the regeneration and repair of myelin sheath.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622.3
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