局部应用壳聚糖—磷酸甘油—神经生长因子缓释凝胶联合静脉导管治疗大鼠面神经损伤的实验研究

发布时间：2018-05-14 21:37

本文选题：壳聚糖 + 神经再生　；参考：《山东大学》2016年博士论文

【摘要】：第一部分局部应用壳聚糖-磷酸甘油-神经生长因子缓释凝胶联合静脉导管治疗大鼠面神经损伤的实验研究目的：近年来生物材料及神经导管技术在治疗周围神经损伤中取得了一定的进展,但其临床效果并不理想。本实验的主要目的：1)研究壳聚糖-β-磷酸甘油(C/GP)凝胶是否可以在静脉导管中起到支架作用；2)研究采用自体静脉作为神经导管,并在导管内注入壳聚糖-β-磷酸甘油-神经生长因子缓释凝胶(C/GP-NGF)修复大鼠面神经缺损的效果。方法：首先,体外制备C/GP-NGF缓释凝胶及C/GP凝胶,并观察凝胶特性。采用扫描电镜观察C/GP-NGF及C/GP凝胶的超微结构。采用NGF-ELISA试剂盒检测NGF在12小时、1天、3天、5天、7天、9天及12天时的释放量,并绘制NGF的体外释放曲线。动物实验共使用75只成年雌性Wistar大鼠(体重220克-250克),随机分为5组(n=15)。首先制备大鼠右侧面神经上颊支缺损5mm动物模型。A组(自体神经移植组)：将切断的神经翻转后缝合于神经缺损处。B组-E组,采用大鼠自体颈外静脉修复面神经上颊支神经缺损,并分别在静脉导管中注入C/GP-NGF凝胶(C/GP-NGF组)、NGF溶液(NGF组)、C/GP凝胶(C/GP组)、及PBS溶液(PBS组)。所有大鼠均暴露左侧面神经颊支,但不做任何处理,作为对照。术后4周、8周及12周,评估各组大鼠触须运动功能；同时记录各组大鼠面神经复合动作电位(CMAPs);并分析CMAPs的最大振幅及再生神经传导速度。组织学检查在术后4周、8周及12周,采用改良三色染色观察再生神经的形态：并在术后12周采用甲苯胺蓝染色及透射电镜的方法来分析再生神经纤维直径大小、髓鞘的厚度及再生轴突的密度。此外,本实验采用Tubulin与S-100双重免疫荧光的方法来评估再生神经纤维,并分析各组大鼠再生神经中Tubulin阳性颗粒的密度。结果：体外实验结果：C/GP凝胶在室温下为液体,放置在37℃恒温箱中30分钟后变为凝胶状。扫描电镜下发现C/GP凝胶及C/GP-NGF凝胶中含有大量的孔隙。C/GP-NGF凝胶在体外可以缓慢释放NGF超过12天。动物实验结果：所有大鼠术后右侧触须运动消失。自体神经移植组中大鼠触须运动恢复最快,但与C/GP-NGF组中大鼠触须运动恢复速度相似,且差别无统计学意义(P 0.05)。C/GP-NGF组中大鼠触须运动恢复速度较PBS组快,差别有统计学意义(P0.05)。NGF组及C/GP组中大鼠触须运动评分值与PBS组相似,且差别无统计学意义(P0.05)。电生理学检查结果显示：术后4周,自体神经移植组及C/GP-NGF组中可以记录到大鼠面神经的CMAPs,但在其它组中记录不到；术后8周及12周,所有实验组均可记录到面神经的CMAPs。C/GP-NGF组中CMAPs的振幅及神经传导速度与自体神经移植组相似,且差别无统计学意义(P0.05)。与C/GP-NGF组相比,NGF组、C/GP组及PBS组中大鼠CMAPs的振幅较小且神经传导速度较慢,差别有统计学意义(P0.05)。NGF组及C/GP组中大鼠CMAPs的振幅及神经传导速度与PBS组比较差别无统计学意义(P0.05)。三色染色结果显示：术后4周,再生神经横切面中可见再生轴突,但轴突较小且排列紊乱；术后8周及12周,再生神经不断成熟,且自体神经移植组及C/GP-NGF组中再生神经较其它三组成熟并且排列规整。此外,术后4周,C/GP-NGF组及C/GP组中大部分的壳聚糖凝胶已被吸收,神经横切面中仅见少量未被吸收的凝胶；术后8周,C/GP-NGF组及C/GP组再生神经横切面中可见极少量的颗粒状未被吸收的壳聚糖凝胶：术后12周,C/GP-NGF组及C/GP组再生神经横切面中未见壳聚糖凝胶。甲苯胺蓝染色及透射电镜结果显示：自体神经移植组中再生神经纤维直径较C/GP-NGF组大,且再生神经髓鞘较C/GP-NGF组厚,差别有统计学意义(P0.05); C/GP-NGF组中再生神经纤维直径较NGF组、C/GP组及PBS组中大,且髓鞘较厚,差别有统计学意义(P 0.05); C/GP-NGF组再生轴突密度与自体神经移植组相似,并且明显的比NGF组、C/GP组及PBS组高(P 0.05); NGF组及C/GP组再生神经的直径、髓鞘厚度及神经纤维密度与PBS组比较,差别无显著统计学差异(P0.05)。Tubulin及S-100双重免疫荧光结果显示,再生神经切片中轴突及髓鞘可以被特异性标记物染色。并且C/GP-NGF组中Tubulin阳性颗粒的密度与自体神经移植组相似,且比NGF组、C/GP组及PBS组高,差别有统计学意义(P0.05)。结论：C/GP凝胶在静脉导管中既可以作为支架维持静脉导管的管状结构,同时可以作为药物载体缓慢释放NGF。联合应用自体静脉导管及C/GP-NGF凝胶可以促进周围神经的再生。然而,静脉导管内单独使用C/GP凝胶或NGF并不能促进神经的再生。第二部分大鼠面神经下颌缘支的损伤对上颊支神经缺损修复的影响的实验研究目的：周围性面瘫是临床上常见的疾病。目前,面瘫后面神经功能的恢复并不理想,尤其是存在面神经缺损时预后效果更不满意。大鼠面神经上颊支缺损模型常用来研究面神经的损伤与修复。以往的研究中,为了观察面神经修复过程中大鼠触须功能的变化,面神经下颌缘支常被同时损伤。然而,下颌缘支的损伤是否对上颊支损伤后的修复有影响并不清楚。本实验研究的目的是观察大鼠面神经下颌缘支的损伤对上颊支神经的修复的影响。方法：Wistar大白鼠36只,体重220g-250g,随机分为2组。A组：大鼠右侧面神经上颊支损伤4mm,并用自体静脉导管修复神经缺损；同时切断面神经下颌缘支,并将神经断端结扎。B组：大鼠右侧面神经上颊支损伤4mmm,并用自体静脉修复神经缺损；下颌缘支未做处理。术后4周、8周及12周观察各组大鼠的触须功能的变化。术后8周及12周记录再生神经面神经复合动作电位(CMAPs)的变化。采用改良三色染色的方法,观察各组面神经上颊支再生过程中形态学的变化。采用甲苯胺蓝染色的方法,分析各组大鼠面神经上颊支再生神经纤维的个数、轴突的直径以及髓鞘的厚度。此外,采用Tubulin及S-100双重免疫荧光的方法来观察再生神经纤维的特异性。术后12周,取大鼠面部提上唇肌,并用Tubulin及a-银环蛇毒素标记神经肌肉接头(NMJ)。此外,术后12周,分别用神经示踪剂Dil及DiD于面神经上颊支及下颌缘支行神经逆行示踪；示踪后2周,取大鼠脑干面神经核团所在部位,计数各组大鼠面神经核团中被Dil及DiD示踪的神经元的数目。结果：A组中大鼠神经损伤术后右侧触须运动消失,评分为0分,术后3周到4周大鼠触须运动开始恢复,术后12周时大鼠触须运动评分为2.66±0.50。B组中大鼠神经损伤术后右侧触须运动与对侧相比无明显变化。A组中,再生面神经上颊支术后8周可以记录到CMAPs,且在12周时潜伏期缩短、振幅增大；B组中,再生面神经上颊支术后8周及12周均记录不到CMAPs.三色染色结果显示术后8周及12周A组中再生面神经较B组成熟,且甲苯胺蓝染色分析显示,与B组中再生神经纤维相比,A组中再生神经纤维密度较高、再生轴突直径较大、再生髓鞘较厚(P0.05)。Tubulin及S-100的特异性荧光染色结果表明,静脉导管中确为神经纤维；且A组中Tubulin阳性颗粒的密度较B组中高,差别有统计学意义(P0.05)。术后12周,A组中可见神经肌肉接头,且每个运动终板由一根神经支配；B组中也可见神经肌肉接头,但神经末梢与运动终板分离,或每个运动终板由多根神经支配。术后12周,各组面神经核团中均可见由Dil及DiD示踪的神经元。A组中,由Dil示踪的神经元数目较对照组中增多,而由DiD示踪的神经元的数目较对照组中减少,差别有统计学意义(P0.05)。B组中,由Dil示踪的神经元数目与对照组中比较无明显变化,而由DiD示踪的神经元数目较对照组中增多,差别有统计学意义(P0.05)。结论：大鼠面神经下颌缘支的损伤可以促进面神经上颊支的功能及形态的修复。
[Abstract]:Partial application of Chitosan Phosphate glycerol nerve growth factor sustained-release gel in combination with venous catheter in the treatment of facial nerve injury in rats: biological materials and nerve conduit technology have made some progress in the treatment of peripheral nerve injury in recent years, but the effect of its presence on the bed is not ideal. The main purpose of this experiment is the main purpose of this experiment. 1) study whether chitosan beta phosphate glycerol (C/GP) gel can play a scaffolding role in the venous catheter; 2) the effect of autologous vein as a nerve conduit and chitosan beta glycerol neuro growth factor sustained release gel (C/GP-NGF) was injected into the catheter to repair the facial nerve defect in rats. Method: first, to prepare C/G in vitro P-NGF slow-release gel and C/GP gel were used to observe the gel properties. The ultrastructure of C/GP-NGF and C/GP gel was observed by scanning electron microscope. NGF-ELISA kit was used to detect the release of NGF at 12 hours, 1 days, 3 days, 7 days, 9 days and 12 days, and the release curves of NGF in vitro were plotted. A total of 75 adult female Wistar rats were used in animal experiments (body weight 2). 20 grams of -250 grams), randomly divided into 5 groups (n=15). First, to prepare the 5mm animal model.A group (autologous nerve graft) on the right lateral buccal branch of the right side of the rat (autologous nerve graft): after the severed nerve was turned into the.B group -E of the nerve defect, the nerve defect of the facial nerve was repaired by the external jugular vein of the rat, and the C/GP-N was injected into the venous catheter respectively. GF gel (group C/GP-NGF), NGF solution (group NGF), C/GP gel (group C/GP), and PBS solution (group PBS). All rats were exposed to the left lateral nerve buccal branch, but no treatment was done as a control. 4 weeks, 8 weeks and 12 weeks after operation, the motor function of the tentacles was evaluated in each group. The compound action potential (CMAPs) of the facial nerve was recorded at the same time and CMAPs was analyzed. The maximum amplitude and regenerative nerve conduction velocity. The histological examination was performed at 4, 8 and 12 weeks after the operation. The modified tricolor staining was used to observe the morphology of the regenerative nerve. The diameter of the regenerated nerve fibers, the thickness of the myelin sheath and the density of the regenerated axon were analyzed by the method of toluidine blue staining and transmission electron microscopy at 12 weeks after the operation. The Tubulin and S-100 double immunofluorescence methods were used to evaluate the regenerated nerve fibers, and the density of Tubulin positive particles in the regenerated nerve of the rats was analyzed. Results: the results of the experiment in vitro: the C/GP gel was liquid at room temperature, and placed at the 37 C incubator for 30 minutes and became gelatinous. The C/GP gel and C/GP-NGF were found under scanning electron microscope. The gel contained a large number of pore.C/GP-NGF gels that could slowly release NGF for more than 12 days in vitro. Animal experiment results: all rats' right tentacle movement disappeared after operation. The motion recovery of the tentacles in the autologous nerve graft group was the fastest, but it was similar to the recovery speed of the tentacle movement in the C/GP-NGF group, and the difference was not statistically significant (P 0.05). The motion recovery rate of the tentacles in the C/GP-NGF group was faster than that in the PBS group. The difference was statistically significant (P0.05) in the group.NGF and the C/GP group, the score of the tentacle movement was similar to that of the PBS group (P0.05). The electrophysiological examination showed that 4 weeks after the operation, the facial nerve could be recorded in the autologous transplanting group and the C/GP-NGF group. CMAPs, but not in other groups, 8 and 12 weeks after the operation, all the experimental groups could record the amplitude and the nerve conduction velocity of CMAPs in the CMAPs.C/GP-NGF group of the facial nerve, and the difference was not statistically significant (P0.05). The amplitude of CMAPs in the NGF group, the C/GP group and the PBS group was smaller than that of the C/GP-NGF group. The nerve conduction velocity was slow, and the difference was statistically significant (P0.05) in group.NGF and group C/GP, the amplitude of CMAPs and the nerve conduction velocity were not significantly different from those in the PBS group (P0.05). The results of tricolor staining showed that the regenerated axons were seen in the regenerated nerve transverse section at 4 weeks after the operation, but the axons were small and arranged in disorder; 8 and 12 weeks after the operation. The regenerative nerve matured continuously, and the regenerative nerve in the autologous nerve graft group and the C/GP-NGF group was mature and orderly compared with the other three groups. In addition, 4 weeks after the operation, most of the chitosan gels in group C/GP-NGF and C/GP were absorbed, only a small amount of non absorbed gel was found in the transverse section of the nerve; 8 weeks after the operation, the regenerated nerves in group C/GP-NGF and C/GP group were regenerated. A small amount of granular and non absorbed chitosan gel was seen in the transverse section: 12 weeks after the operation, no chitosan gel was found in the regenerated nerve transversal surface in group C/GP-NGF and C/GP. The results of toluidine blue staining and transmission electron microscopy showed that the diameter of the regenerated nerve fibers in the autologous nerve graft group was larger than that in the C/ GP-NGF group, and the regenerated nerve myelin sheath was C/GP-NGF Group thickness, the difference was statistically significant (P0.05). The diameter of regenerated nerve fibers in group C/GP-NGF was larger than that in group NGF, group C/GP and PBS, and the myelin sheath was thicker, and the difference was statistically significant (P 0.05). The regenerated axon density in the C/GP-NGF group was similar to that of the autologous nerve graft group, and was significantly higher than the NGF group, C/GP group and PBS group (P 0.05); NGF group and regeneration group were regenerated. There was no significant difference in the diameter of nerve, the thickness of myelin sheath and the density of nerve fiber compared with the PBS group (P0.05). The double immunofluorescence of.Tubulin and S-100 showed that the axon and myelin sheath in the regenerated nerve section could be stained by specific markers. And the density of Tubulin positive particles in the C/GP-NGF group was similar to that of the autologous nerve graft group. Compared with group NGF, group C/GP and group PBS, the difference was statistically significant (P0.05). Conclusion: C/GP gel can be used as the stent to maintain the tubular structure of the venous catheter in the venous catheter, and can be used as a drug carrier for the slow release of NGF. and the combination of autologous venous catheter and C/GP-NGF gel to promote the regeneration of peripheral nerve. However, the vein can be promoted. The use of C/GP gel or NGF alone in the catheter does not promote the regeneration of the nerve. Experimental study on the effects of the injury of the mandibular branch of the second part of the facial nerve on the repair of the upper buccal nerve defect: peripheral facial paralysis is a common clinical disease. At present, the recovery of nerve function behind facial paralysis is not ideal, especially in the presence of facial nerve. The prognosis of the defect is more unsatisfactory. The model of the facial nerve defect in the facial nerve is often used to study the injury and repair of the facial nerve. In the previous study, the mandibular branch of the facial nerve was often damaged in order to observe the changes in the function of the tentacles during the repair of the facial nerve. The objective of this study was to observe the effect of the injury of the mandibular branch of the facial nerve on the repair of the upper buccal nerve. Methods: 36 Wistar rats, weight 220g-250g, were randomly divided into 2 groups of.A groups: the right lateral buccal branch of the right side of the rat was damaged by 4mm, and the autologous venous catheter was used to repair the nerve defect; at the same time cut off the nerve. The mandibular marginal branch of the facial nerve was ligated and ligated in group.B: the upper right lateral buccal branch of the right side of the rat was damaged by 4mmm, and the nerve defect was repaired with autologous vein. The mandibular branch was not treated. The changes of the tentacle function of the rats were observed at 4 weeks, 8 and 12 weeks after operation. The regenerative nerve facial nerve compound action potential (CMAPs) was recorded at 8 and 12 weeks after operation. Changes. The morphological changes during the regeneration of the upper buccal branches of each facial nerve were observed by modified tricolor staining. The number of regenerated nerve fibers, the diameter of axon and the thickness of the myelin sheath were analyzed by toluidine blue staining. In addition, the Tubulin and S-100 double immunofluorescence methods were used. The specificity of the regenerated nerve fiber was observed. 12 weeks after the operation, the upper lip muscle of the rat was extracted and the neuromuscular junction (NMJ) was marked with Tubulin and a- venin toxin. In addition, the nerve tracer Dil and DiD were used for retrograde tracing of the upper buccal branch of the facial nerve and the marginal mandibular branch of the facial nerve at 12 weeks after the operation, and 2 weeks after the tracer, the nucleus of the brain dry facial nucleus of the rat was taken. At the site, the number of neurons traced by Dil and DiD in the nucleus of the facial nerve in rats was counted. Results: the right tentacle movement disappeared after the nerve injury in the rats in group A, the score was 0 points, and the tentacle movement began to recover from 3 to 4 weeks after the operation, and the score of the tentacle movement of the rats in the 2.66 + 0.50.B group after 12 weeks was right after the operation. In group.A, there was no significant change in the lateral tentacle movement compared with the contralateral side. CMAPs could be recorded at 8 weeks after the upper buccal branch of the regenerative facial nerve, and the latency shortened and the amplitude increased at the 12 week. In group B, the regenerated facial nerve of the A group was not recorded at 8 and 12 weeks after the operation of the regenerated facial nerve at 8 and 12 weeks, and the regenerated facial nerve was more than B in the group of A and 12 weeks after the operation. Compared with the regenerated nerve fibers in the B group, the density of the regenerated nerve fibers in the A group was higher than that in the B group, and the regenerated axon diameter was larger. The specific fluorescent staining results of the regenerated myelin sheath (P0.05).Tubulin and S-100 showed that the venous catheterization was indeed a deity fiber, and the density of the Tubulin positive particles in the A group was more than that in the B group. The difference was statistically significant (P0.05). 12 weeks after the operation, the neuromuscular junction was seen in group A, and each motor endplate was dominated by one nerve, and the neuromuscular junction was also seen in group B, but the nerve endings were separated from the motor endplates or each motor endplate was dominated by multiple nerves. 12 weeks after the operation, all the groups of facial nerve nuclei were seen from Dil and DiD. The number of neurons traced by Dil in the tracer.A group was more than that in the control group, but the number of neurons traced by DiD was less than that in the control group, and the number of neurons traced by Dil was not significantly different from that in the control group, but the number of neurons traced by DiD was more than that in the control group, and the number of neurons traced by DiD was more than that in the control group. The difference was statistically significant (P0.05). Conclusion: the injury of the mandibular ramus of facial nerve in rats can promote the repair of the function and morphology of the buccal branches of the facial nerve.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R745.12

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