【摘要】：雖然顯微外科技術(shù)已經(jīng)有了很大的進(jìn)步和發(fā)展,但是對(duì)于周?chē)窠?jīng)損傷和再生的病理生理機(jī)制我們?nèi)匀蝗狈ι钊肓私�。周�(chē)窠?jīng)缺損的治療仍然是外科醫(yī)生的一個(gè)重大難題。準(zhǔn)確的掌握神經(jīng)損傷修復(fù)的解剖學(xué)、病理生理學(xué)和外科重建技術(shù)是對(duì)神經(jīng)損傷修復(fù)治療的先決條件。對(duì)于大于5cm的神經(jīng)缺失,直接的神經(jīng)吻和會(huì)導(dǎo)致過(guò)大的張力,導(dǎo)致治療的失敗,需要進(jìn)行自體神經(jīng)移植或者應(yīng)用套管技術(shù)。對(duì)于套管技術(shù),雖然在生物醫(yī)學(xué)工程方面取得了很大的創(chuàng)新和突破,但是由于免疫耐受的因素,仍然無(wú)法有效的應(yīng)用于臨床治療。自體神經(jīng)移植依然是治療神經(jīng)損傷的金標(biāo)準(zhǔn),但是移植后修復(fù)的過(guò)程慢,壞死率高,是移植中常常遇到的問(wèn)題。近年來(lái),許多研究者發(fā)現(xiàn)NRG-1參與了神經(jīng)損傷的修復(fù),并且發(fā)揮著重要調(diào)節(jié)的作用。本研究中,為了探索NRG-1在神經(jīng)移植后修復(fù)過(guò)程中的調(diào)節(jié)作用,我們采用SD大鼠自體神經(jīng)移植模型,首先觀察了NRG-1的表達(dá)變化及移植神經(jīng)的修復(fù)過(guò)程,然后通過(guò)反義寡核苷酸技術(shù),抑制NRG-1在自體神經(jīng)移植模型中的表達(dá),并采用SB203580特異性抑制P38αMAPK激活,并分別觀察神經(jīng)移植后髓鞘修復(fù)及神經(jīng)功能恢復(fù)的過(guò)程,進(jìn)一步分析NRG-1參與調(diào)控神經(jīng)移植后髓鞘修復(fù)的作用及其信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。實(shí)驗(yàn)分為三部分:實(shí)驗(yàn)一NRG-1在大鼠自體神經(jīng)移植再生過(guò)程中的變化目的:研究NRG-1在大鼠自體坐骨神經(jīng)移植再生過(guò)程中的變化方法:采用清潔級(jí)健康雄性SD大鼠,40只,體重250-300g隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,每組20只,按手術(shù)后3、7、14、21、28天分為5個(gè)時(shí)間點(diǎn)。實(shí)驗(yàn)組行坐骨神經(jīng)倒轉(zhuǎn)自體移植術(shù),對(duì)照組單純暴露坐骨神經(jīng)后縫合處理,在不同時(shí)間點(diǎn)觀察大鼠足跡的變化,計(jì)算坐骨神經(jīng)指數(shù)(SFI),電生理檢測(cè)運(yùn)動(dòng)神經(jīng)傳導(dǎo)速度(MNCV)變化,之后分別切取實(shí)驗(yàn)組移植段坐骨神經(jīng)和對(duì)照組移植對(duì)應(yīng)段的坐骨神經(jīng),透射電鏡觀察移植后的神經(jīng)端髓鞘再生的變化,并通過(guò)RT-PCR技術(shù)檢測(cè)II型和III型NRG-1 m RNA的表達(dá)變化,Western blot技術(shù)檢測(cè)NRG-1蛋白表達(dá)變化。結(jié)果:術(shù)后各個(gè)時(shí)間點(diǎn)的實(shí)驗(yàn)組坐骨神經(jīng)指數(shù)均低于對(duì)照組,且實(shí)驗(yàn)組坐骨神經(jīng)指數(shù)隨時(shí)間進(jìn)行逐漸增加,差異有統(tǒng)計(jì)學(xué)意義(P0.01);在自體神經(jīng)移植的3-28天實(shí)驗(yàn)組的坐骨神經(jīng)運(yùn)動(dòng)神經(jīng)傳導(dǎo)速度小于對(duì)照組,并且有統(tǒng)計(jì)學(xué)差異(P均0.01);實(shí)驗(yàn)組有髓鞘神經(jīng)纖維的面積(um2)在第7、14、21、28天與對(duì)照組比較有明顯的統(tǒng)計(jì)學(xué)差異(P0.01),實(shí)驗(yàn)組軸突直徑在第7、14、21天與對(duì)照組比較有明顯的統(tǒng)計(jì)學(xué)差異(P0.01);RT-PCR結(jié)果分析顯示,在移植的3-28天II型NRG-1 m RNA表達(dá)均較對(duì)照組增高,且差異有統(tǒng)計(jì)學(xué)意義(P分別0.01);Western blot結(jié)果顯示在神經(jīng)移植術(shù)后第3-28天,II型NRG-1和III型NRG-1蛋白表達(dá)也明顯增高,且有顯著性差異(P分別0.01)。結(jié)論:在神經(jīng)移植后,NRG-1 m RNA及蛋白表達(dá)上調(diào),II型NRG-1和III型NRG-1表達(dá)改變不同。NRG-1可能參與了神經(jīng)移植后髓鞘再生的調(diào)節(jié)過(guò)程。實(shí)驗(yàn)二II型NRG-1在大鼠自體神經(jīng)移植再生過(guò)程中的作用目的:研究II型NRG-1在大鼠自體坐骨神經(jīng)移植再生過(guò)程中可能的作用。方法:采用清潔級(jí)健康雄性SD大鼠,54只,體重250-300g隨機(jī)分為空白組(Blank)、緩沖液對(duì)照組(Model)和反義寡核苷酸抑制組(ASON),每組18只,按手術(shù)后3、7、14、21、28、35天分為6個(gè)時(shí)間點(diǎn)。緩沖液對(duì)照組和反義寡核苷酸抑制組行自體坐骨神經(jīng)倒轉(zhuǎn)移植術(shù),在術(shù)后當(dāng)日和第三天,Model組給予手術(shù)切口局部注射PBS緩沖液,ASON組給予注射II型NRG-1反義寡核苷酸;空白組單純暴露坐骨神經(jīng)后縫合處理。在不同時(shí)間點(diǎn)觀察大鼠足跡的變化,計(jì)算坐骨神經(jīng)指數(shù),電生理檢測(cè)運(yùn)動(dòng)神經(jīng)傳導(dǎo)速度。之后分別切取Model組及ASON組移植段坐骨神經(jīng)和Blank組移植對(duì)應(yīng)段的坐骨神經(jīng),透射電鏡觀察移植后的神經(jīng)端髓鞘再生的變化,RT-PCR技術(shù)檢測(cè)II型NRG-1 m RNA的變化,以及通過(guò)Western blot技術(shù)檢測(cè)II型NRG-1蛋白變化,分析II型NRG-1在自體神經(jīng)移植中可能的作用。結(jié)果:發(fā)現(xiàn)術(shù)后ASON組除第3天以外,其余各個(gè)時(shí)間點(diǎn)的ASON組坐骨神經(jīng)指數(shù)均低于Model組的結(jié)果且差異有統(tǒng)計(jì)學(xué)意義(P0.01),在移植的14-35天,抑制II型NRG-1的表達(dá),可減慢大鼠坐骨神經(jīng)傳導(dǎo)速度。說(shuō)明缺少I(mǎi)I型NRG-1減緩自體移植神經(jīng)的再生。髓鞘的形態(tài)學(xué)分析提示II型NRG-1在自體神經(jīng)移植3-28天,對(duì)髓鞘的崩解,雪旺細(xì)胞髓鞘化的過(guò)程中均發(fā)揮著一定的調(diào)節(jié)作用。RT-PCR和Western blot顯示:II型NRG-1的m RNA和蛋白的表達(dá)均較Model組明顯減低。結(jié)論:II型NRG-1在SD大鼠自體神經(jīng)后神經(jīng)再生的的過(guò)程中,在移植的3-28天,對(duì)髓鞘的崩解,雪旺細(xì)胞髓鞘化的過(guò)程中均發(fā)揮著一定的調(diào)節(jié)作用。缺少I(mǎi)I型NRG-1可能影響移植后神經(jīng)功能的恢復(fù)。實(shí)驗(yàn)三P38-MAPK參與NRG-1調(diào)控大鼠自體神經(jīng)移植再生過(guò)程的機(jī)制研究目的:P38 MAPK在大鼠自體神經(jīng)移植再生過(guò)程中對(duì)NRG-1/Erb B信號(hào)系統(tǒng)的轉(zhuǎn)導(dǎo)作用方法:采用清潔級(jí)健康雄性SD大鼠,54只,體重250-300g隨機(jī)分為空白組(Blank)、緩沖液對(duì)照組(Model)和P38αMAPK抑制組(P38αMAPK),每組18只,按手術(shù)后3、7、14、21、28、35天分為6個(gè)時(shí)間點(diǎn)。Model組和P38αMAPK抑制組行自體坐骨神經(jīng)倒轉(zhuǎn)移植術(shù),在術(shù)后當(dāng)日和第三天,Model組給予移植切口局部注射PBS緩沖液,P38αMAPK抑制組給予注射P38αMAPK抑制劑SB2035802;空白組單純暴露坐骨神經(jīng)后縫合不對(duì)其其他實(shí)驗(yàn)處理。在不同時(shí)間點(diǎn)觀察大鼠足跡的變化,計(jì)算SFI,電生理檢測(cè)MNCV,透射電鏡觀察移植后的神經(jīng)端髓鞘再生的變化,RT-PCR技術(shù)檢測(cè)髓鞘堿性蛋白(MBP)m RNA的變化,以及通過(guò)Western blot技術(shù)檢測(cè)P-P38αMAPK蛋白變化。結(jié)果:SD大鼠的SFI值在移植的7、14、21、28、35五個(gè)時(shí)間點(diǎn),P38αMAPK抑制組坐骨神經(jīng)指數(shù)均低于Model組。MNCV值顯示:在自體神經(jīng)移植的14、21、28、35天P38αMAPK抑制組的傳導(dǎo)速度小于Model組。在電鏡的髓鞘觀察和分析中有髓鞘神經(jīng)纖維的面積(um2):P38αMAPK抑制組在第28、35天出現(xiàn)與Model組明顯的統(tǒng)計(jì)學(xué)差異(P0.01),而前期無(wú)明顯統(tǒng)計(jì)學(xué)差異;單位面積的有髓鞘神經(jīng)纖維的數(shù)量:P38αMAPK抑制組與Model組無(wú)明顯的統(tǒng)計(jì)學(xué)差異;軸突的直徑:在第21、28、35天出現(xiàn)P38αMAPK抑制組與Model組明顯的統(tǒng)計(jì)學(xué)差異(P0.01);對(duì)于G-ratio,在第28天和35天P38αMAPK抑制組均較Model組高且有明顯的統(tǒng)計(jì)學(xué)差異(P0.01)。Western blot和RT-PCR顯示:大鼠切口局部注射SB203580后,MBP的m RNA:P38αMAPK抑制組的表達(dá)較Model組減少;P-P38αMAPK:P38αMAPK抑制組均較Model組少。結(jié)論:在SD大鼠自體神經(jīng)移植后,抑制P38αMAPK的激活,會(huì)出現(xiàn)大鼠神經(jīng)功能恢復(fù)減慢,有髓鞘神經(jīng)纖維的面積和軸突的直徑會(huì)減小,G-ratio值增加。提示在自體神經(jīng)移植模型中,P38αMAPK可能為NRG-1/Erb B信號(hào)轉(zhuǎn)導(dǎo)途徑的下游信號(hào),并對(duì)神經(jīng)功能的恢復(fù)和髓鞘的再生修復(fù)發(fā)揮著促進(jìn)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R622.3

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