本文選題：殼聚糖 + 神經(jīng)再生��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：第一部分局部應(yīng)用殼聚糖-磷酸甘油-神經(jīng)生長(zhǎng)因子緩釋凝膠聯(lián)合靜脈導(dǎo)管治療大鼠面神經(jīng)損傷的實(shí)驗(yàn)研究目的：近年來(lái)生物材料及神經(jīng)導(dǎo)管技術(shù)在治療周圍神經(jīng)損傷中取得了一定的進(jìn)展,但其臨床效果并不理想。本實(shí)驗(yàn)的主要目的：1)研究殼聚糖-β-磷酸甘油(C/GP)凝膠是否可以在靜脈導(dǎo)管中起到支架作用；2)研究采用自體靜脈作為神經(jīng)導(dǎo)管,并在導(dǎo)管內(nèi)注入殼聚糖-β-磷酸甘油-神經(jīng)生長(zhǎng)因子緩釋凝膠(C/GP-NGF)修復(fù)大鼠面神經(jīng)缺損的效果。方法：首先,體外制備C/GP-NGF緩釋凝膠及C/GP凝膠,并觀察凝膠特性。采用掃描電鏡觀察C/GP-NGF及C/GP凝膠的超微結(jié)構(gòu)。采用NGF-ELISA試劑盒檢測(cè)NGF在12小時(shí)、1天、3天、5天、7天、9天及12天時(shí)的釋放量,并繪制NGF的體外釋放曲線。動(dòng)物實(shí)驗(yàn)共使用75只成年雌性Wistar大鼠(體重220克-250克),隨機(jī)分為5組(n=15)。首先制備大鼠右側(cè)面神經(jīng)上頰支缺損5mm動(dòng)物模型。A組(自體神經(jīng)移植組)：將切斷的神經(jīng)翻轉(zhuǎn)后縫合于神經(jīng)缺損處。B組-E組,采用大鼠自體頸外靜脈修復(fù)面神經(jīng)上頰支神經(jīng)缺損,并分別在靜脈導(dǎo)管中注入C/GP-NGF凝膠(C/GP-NGF組)、NGF溶液(NGF組)、C/GP凝膠(C/GP組)、及PBS溶液(PBS組)。所有大鼠均暴露左側(cè)面神經(jīng)頰支,但不做任何處理,作為對(duì)照。術(shù)后4周、8周及12周,評(píng)估各組大鼠觸須運(yùn)動(dòng)功能；同時(shí)記錄各組大鼠面神經(jīng)復(fù)合動(dòng)作電位(CMAPs);并分析CMAPs的最大振幅及再生神經(jīng)傳導(dǎo)速度。組織學(xué)檢查在術(shù)后4周、8周及12周,采用改良三色染色觀察再生神經(jīng)的形態(tài)：并在術(shù)后12周采用甲苯胺藍(lán)染色及透射電鏡的方法來(lái)分析再生神經(jīng)纖維直徑大小、髓鞘的厚度及再生軸突的密度。此外,本實(shí)驗(yàn)采用Tubulin與S-100雙重免疫熒光的方法來(lái)評(píng)估再生神經(jīng)纖維,并分析各組大鼠再生神經(jīng)中Tubulin陽(yáng)性顆粒的密度。結(jié)果：體外實(shí)驗(yàn)結(jié)果：C/GP凝膠在室溫下為液體,放置在37℃恒溫箱中30分鐘后變?yōu)槟z狀。掃描電鏡下發(fā)現(xiàn)C/GP凝膠及C/GP-NGF凝膠中含有大量的孔隙。C/GP-NGF凝膠在體外可以緩慢釋放NGF超過(guò)12天。動(dòng)物實(shí)驗(yàn)結(jié)果：所有大鼠術(shù)后右側(cè)觸須運(yùn)動(dòng)消失。自體神經(jīng)移植組中大鼠觸須運(yùn)動(dòng)恢復(fù)最快,但與C/GP-NGF組中大鼠觸須運(yùn)動(dòng)恢復(fù)速度相似,且差別無(wú)統(tǒng)計(jì)學(xué)意義(P 0.05)。C/GP-NGF組中大鼠觸須運(yùn)動(dòng)恢復(fù)速度較PBS組快,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。NGF組及C/GP組中大鼠觸須運(yùn)動(dòng)評(píng)分值與PBS組相似,且差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。電生理學(xué)檢查結(jié)果顯示：術(shù)后4周,自體神經(jīng)移植組及C/GP-NGF組中可以記錄到大鼠面神經(jīng)的CMAPs,但在其它組中記錄不到；術(shù)后8周及12周,所有實(shí)驗(yàn)組均可記錄到面神經(jīng)的CMAPs。C/GP-NGF組中CMAPs的振幅及神經(jīng)傳導(dǎo)速度與自體神經(jīng)移植組相似,且差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。與C/GP-NGF組相比,NGF組、C/GP組及PBS組中大鼠CMAPs的振幅較小且神經(jīng)傳導(dǎo)速度較慢,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。NGF組及C/GP組中大鼠CMAPs的振幅及神經(jīng)傳導(dǎo)速度與PBS組比較差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。三色染色結(jié)果顯示：術(shù)后4周,再生神經(jīng)橫切面中可見(jiàn)再生軸突,但軸突較小且排列紊亂；術(shù)后8周及12周,再生神經(jīng)不斷成熟,且自體神經(jīng)移植組及C/GP-NGF組中再生神經(jīng)較其它三組成熟并且排列規(guī)整。此外,術(shù)后4周,C/GP-NGF組及C/GP組中大部分的殼聚糖凝膠已被吸收,神經(jīng)橫切面中僅見(jiàn)少量未被吸收的凝膠；術(shù)后8周,C/GP-NGF組及C/GP組再生神經(jīng)橫切面中可見(jiàn)極少量的顆粒狀未被吸收的殼聚糖凝膠：術(shù)后12周,C/GP-NGF組及C/GP組再生神經(jīng)橫切面中未見(jiàn)殼聚糖凝膠。甲苯胺藍(lán)染色及透射電鏡結(jié)果顯示：自體神經(jīng)移植組中再生神經(jīng)纖維直徑較C/GP-NGF組大,且再生神經(jīng)髓鞘較C/GP-NGF組厚,差別有統(tǒng)計(jì)學(xué)意義(P0.05); C/GP-NGF組中再生神經(jīng)纖維直徑較NGF組、C/GP組及PBS組中大,且髓鞘較厚,差別有統(tǒng)計(jì)學(xué)意義(P 0.05); C/GP-NGF組再生軸突密度與自體神經(jīng)移植組相似,并且明顯的比NGF組、C/GP組及PBS組高(P 0.05); NGF組及C/GP組再生神經(jīng)的直徑、髓鞘厚度及神經(jīng)纖維密度與PBS組比較,差別無(wú)顯著統(tǒng)計(jì)學(xué)差異(P0.05)。Tubulin及S-100雙重免疫熒光結(jié)果顯示,再生神經(jīng)切片中軸突及髓鞘可以被特異性標(biāo)記物染色。并且C/GP-NGF組中Tubulin陽(yáng)性顆粒的密度與自體神經(jīng)移植組相似,且比NGF組、C/GP組及PBS組高,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：C/GP凝膠在靜脈導(dǎo)管中既可以作為支架維持靜脈導(dǎo)管的管狀結(jié)構(gòu),同時(shí)可以作為藥物載體緩慢釋放NGF。聯(lián)合應(yīng)用自體靜脈導(dǎo)管及C/GP-NGF凝膠可以促進(jìn)周圍神經(jīng)的再生。然而,靜脈導(dǎo)管內(nèi)單獨(dú)使用C/GP凝膠或NGF并不能促進(jìn)神經(jīng)的再生。第二部分大鼠面神經(jīng)下頜緣支的損傷對(duì)上頰支神經(jīng)缺損修復(fù)的影響的實(shí)驗(yàn)研究目的：周圍性面癱是臨床上常見(jiàn)的疾病。目前,面癱后面神經(jīng)功能的恢復(fù)并不理想,尤其是存在面神經(jīng)缺損時(shí)預(yù)后效果更不滿意。大鼠面神經(jīng)上頰支缺損模型常用來(lái)研究面神經(jīng)的損傷與修復(fù)。以往的研究中,為了觀察面神經(jīng)修復(fù)過(guò)程中大鼠觸須功能的變化,面神經(jīng)下頜緣支常被同時(shí)損傷。然而,下頜緣支的損傷是否對(duì)上頰支損傷后的修復(fù)有影響并不清楚。本實(shí)驗(yàn)研究的目的是觀察大鼠面神經(jīng)下頜緣支的損傷對(duì)上頰支神經(jīng)的修復(fù)的影響。方法：Wistar大白鼠36只,體重220g-250g,隨機(jī)分為2組。A組：大鼠右側(cè)面神經(jīng)上頰支損傷4mm,并用自體靜脈導(dǎo)管修復(fù)神經(jīng)缺損；同時(shí)切斷面神經(jīng)下頜緣支,并將神經(jīng)斷端結(jié)扎。B組：大鼠右側(cè)面神經(jīng)上頰支損傷4mmm,并用自體靜脈修復(fù)神經(jīng)缺損；下頜緣支未做處理。術(shù)后4周、8周及12周觀察各組大鼠的觸須功能的變化。術(shù)后8周及12周記錄再生神經(jīng)面神經(jīng)復(fù)合動(dòng)作電位(CMAPs)的變化。采用改良三色染色的方法,觀察各組面神經(jīng)上頰支再生過(guò)程中形態(tài)學(xué)的變化。采用甲苯胺藍(lán)染色的方法,分析各組大鼠面神經(jīng)上頰支再生神經(jīng)纖維的個(gè)數(shù)、軸突的直徑以及髓鞘的厚度。此外,采用Tubulin及S-100雙重免疫熒光的方法來(lái)觀察再生神經(jīng)纖維的特異性。術(shù)后12周,取大鼠面部提上唇肌,并用Tubulin及a-銀環(huán)蛇毒素標(biāo)記神經(jīng)肌肉接頭(NMJ)。此外,術(shù)后12周,分別用神經(jīng)示蹤劑Dil及DiD于面神經(jīng)上頰支及下頜緣支行神經(jīng)逆行示蹤；示蹤后2周,取大鼠腦干面神經(jīng)核團(tuán)所在部位,計(jì)數(shù)各組大鼠面神經(jīng)核團(tuán)中被Dil及DiD示蹤的神經(jīng)元的數(shù)目。結(jié)果：A組中大鼠神經(jīng)損傷術(shù)后右側(cè)觸須運(yùn)動(dòng)消失,評(píng)分為0分,術(shù)后3周到4周大鼠觸須運(yùn)動(dòng)開(kāi)始恢復(fù),術(shù)后12周時(shí)大鼠觸須運(yùn)動(dòng)評(píng)分為2.66±0.50。B組中大鼠神經(jīng)損傷術(shù)后右側(cè)觸須運(yùn)動(dòng)與對(duì)側(cè)相比無(wú)明顯變化。A組中,再生面神經(jīng)上頰支術(shù)后8周可以記錄到CMAPs,且在12周時(shí)潛伏期縮短、振幅增大；B組中,再生面神經(jīng)上頰支術(shù)后8周及12周均記錄不到CMAPs.三色染色結(jié)果顯示術(shù)后8周及12周A組中再生面神經(jīng)較B組成熟,且甲苯胺藍(lán)染色分析顯示,與B組中再生神經(jīng)纖維相比,A組中再生神經(jīng)纖維密度較高、再生軸突直徑較大、再生髓鞘較厚(P0.05)。Tubulin及S-100的特異性熒光染色結(jié)果表明,靜脈導(dǎo)管中確為神經(jīng)纖維；且A組中Tubulin陽(yáng)性顆粒的密度較B組中高,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。術(shù)后12周,A組中可見(jiàn)神經(jīng)肌肉接頭,且每個(gè)運(yùn)動(dòng)終板由一根神經(jīng)支配；B組中也可見(jiàn)神經(jīng)肌肉接頭,但神經(jīng)末梢與運(yùn)動(dòng)終板分離,或每個(gè)運(yùn)動(dòng)終板由多根神經(jīng)支配。術(shù)后12周,各組面神經(jīng)核團(tuán)中均可見(jiàn)由Dil及DiD示蹤的神經(jīng)元。A組中,由Dil示蹤的神經(jīng)元數(shù)目較對(duì)照組中增多,而由DiD示蹤的神經(jīng)元的數(shù)目較對(duì)照組中減少,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。B組中,由Dil示蹤的神經(jīng)元數(shù)目與對(duì)照組中比較無(wú)明顯變化,而由DiD示蹤的神經(jīng)元數(shù)目較對(duì)照組中增多,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：大鼠面神經(jīng)下頜緣支的損傷可以促進(jìn)面神經(jīng)上頰支的功能及形態(tài)的修復(fù)。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R745.12

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