【摘要】：周圍神經(jīng)特別是臂叢等近端神經(jīng)損傷,由于損傷部位距離靶器官遠(yuǎn)和軸突再生速度緩慢,遠(yuǎn)端神經(jīng)和肌肉長時(shí)間處于失神經(jīng)支配狀態(tài),發(fā)生慢性變性與纖維化,影響功能恢復(fù)。采用正常神經(jīng)“寄養(yǎng)”遠(yuǎn)端神經(jīng),能使失神經(jīng)的靶器官及時(shí)獲得神經(jīng)再支配,減輕其慢性變性。但是,肢體近端神經(jīng)損傷后遠(yuǎn)端常沒有可供“寄養(yǎng)”的正常神經(jīng)。文獻(xiàn)報(bào)道和我們先前的研究發(fā)現(xiàn)胚胎神經(jīng)干細(xì)胞移植至損傷的周圍神經(jīng)遠(yuǎn)端能存活、發(fā)育成神經(jīng)元,并發(fā)出軸突再支配遠(yuǎn)端肌肉、減輕肌肉失神經(jīng)萎縮。因此,本課題在先前研究基礎(chǔ)上,以大鼠脛神經(jīng)損傷為實(shí)驗(yàn)動物模型,研究移植胚胎脊髓細(xì)胞“寄養(yǎng)”損傷的神經(jīng)遠(yuǎn)端能否減輕遠(yuǎn)端靶器官失神經(jīng)慢性變性?結(jié)束細(xì)胞“寄養(yǎng)”后遠(yuǎn)端神經(jīng)是否再次發(fā)生早期失神經(jīng)變性?細(xì)胞“寄養(yǎng)”能否促進(jìn)延期神經(jīng)修復(fù)后軸突再生和功能恢復(fù)?第一部分不同培養(yǎng)代數(shù)的胚胎脊髓細(xì)胞移植至周圍神經(jīng)的存活與分化【目的】體外培養(yǎng)的神經(jīng)干細(xì)胞移植至損傷的周圍神經(jīng)遠(yuǎn)端能存活與軸突生長,預(yù)防失神經(jīng)肌萎縮。本實(shí)驗(yàn)研究不同培養(yǎng)時(shí)間的胚胎脊髓細(xì)胞移植至損傷神經(jīng)遠(yuǎn)端后的存活與分化�！痉椒ā咳≡�13.5天轉(zhuǎn)基因大鼠(GFP)胚胎脊髓組織,制備胚胎脊髓細(xì)胞懸液(原代,P0),培養(yǎng)3天后傳代(P1),連續(xù)培養(yǎng)至P3代。移植前,體外鑒定細(xì)胞成分。另取30只SD雌性大鼠,脛神經(jīng)切斷、遠(yuǎn)端變性7天后,動物隨機(jī)分成3組,分別注射P0、P1、P3細(xì)胞至脛神經(jīng)遠(yuǎn)端。動物飼養(yǎng)3個月后取移植區(qū)神經(jīng)節(jié)段行免疫組織化學(xué)染色鑒定細(xì)胞的存活及分化情況�！窘Y(jié)果】體外鑒定結(jié)果顯示原代胚胎脊髓細(xì)胞大部分是表達(dá)β-tubulin陽性的神經(jīng)元前體細(xì)胞。隨著傳代次數(shù)的增加,神經(jīng)元前體細(xì)胞比例逐漸降低,表達(dá)GFAP陽性的膠質(zhì)細(xì)胞比例增加;細(xì)胞移植三個月后的結(jié)果與體外結(jié)果一致:隨著傳代次數(shù)的增加,存活的細(xì)胞表達(dá)NEUN和CHAT的比例逐漸降低�！窘Y(jié)論】胚胎脊髓細(xì)胞富含神經(jīng)元和神經(jīng)元前體細(xì)胞,隨著傳代次數(shù)的增加,神經(jīng)元的比例逐漸減少,因此,移植細(xì)胞至損傷的神經(jīng)遠(yuǎn)端最好用原代胚胎脊髓細(xì)胞。第二部分胚胎脊髓細(xì)胞“寄養(yǎng)”對遠(yuǎn)端神經(jīng)和肌肉慢性變性的影響【目的】先前的研究證明原代胚胎脊髓細(xì)胞移植至預(yù)變性的周圍神經(jīng)遠(yuǎn)端能存活、有較高的神經(jīng)元比例。本部分進(jìn)一步研究:1.移植原代胚胎脊髓細(xì)胞“寄養(yǎng)”損傷的脛神經(jīng)遠(yuǎn)端對遠(yuǎn)端神經(jīng)和肌肉慢性變性的影響;2.切除細(xì)胞移植區(qū)、結(jié)束“寄養(yǎng)”后遠(yuǎn)端神經(jīng)內(nèi)雪旺細(xì)胞的功能狀態(tài)�！痉椒ā咳�30只SD雌性大鼠,脛神經(jīng)切斷變性7天后,隨機(jī)分成2組:細(xì)胞移植“寄養(yǎng)”組:注射原代胚胎脊髓細(xì)胞3ul(10萬/ul)至脛神經(jīng)遠(yuǎn)端。對照組:注射等量神經(jīng)培養(yǎng)液至相同部位。大鼠飼養(yǎng)3月,每組10只大鼠行電生理、遠(yuǎn)端神經(jīng)電鏡、肌肉HE、免疫熒光染色等,比較兩組神經(jīng)軸突計(jì)數(shù)、腓腸肌肌萎縮、神經(jīng)營養(yǎng)因子和髓鞘蛋白表達(dá)等的差別;剩余每組5只大鼠,再次手術(shù),實(shí)驗(yàn)組切除細(xì)胞移植區(qū)5mm神經(jīng),對照組切除相同位置神經(jīng),2周后遠(yuǎn)端神經(jīng)行神經(jīng)營養(yǎng)因子及髓鞘蛋白的檢測,比較兩組差別�！窘Y(jié)果】實(shí)驗(yàn)組遠(yuǎn)側(cè)段神經(jīng)可見髓鞘包繞的再生軸突,其腓腸肌肌萎縮程度更輕、可見再生神經(jīng)支配的運(yùn)動終板,電刺激產(chǎn)生動作電位;對照組遠(yuǎn)側(cè)段神經(jīng)未見再生的有髓神經(jīng)纖維,腓腸肌萎縮明顯、未見神經(jīng)支配的運(yùn)動終板,電刺激沒有動作電位產(chǎn)生。兩組神經(jīng)營養(yǎng)因子BDNF,GDNF,NGF,NT-3的表達(dá)無顯著差別;實(shí)驗(yàn)組髓鞘蛋白的表達(dá)顯著高于對照組;結(jié)束“寄養(yǎng)”2周后,實(shí)驗(yàn)組髓鞘蛋白的表達(dá)水平下降,神經(jīng)營養(yǎng)因子BDNF、GDNF的表達(dá)水平顯著上升�！窘Y(jié)論】移植胚胎脊髓細(xì)胞“寄養(yǎng)”損傷的脛神經(jīng)遠(yuǎn)端,移植細(xì)胞能存活、發(fā)育成神經(jīng)元和運(yùn)動神經(jīng)元,發(fā)出軸突,與肌肉建立有功能的神經(jīng)肌肉接頭,減輕遠(yuǎn)端神經(jīng)和肌肉慢性失神經(jīng)變性。結(jié)束“寄養(yǎng)”后,遠(yuǎn)端神經(jīng)再次發(fā)生失神經(jīng)變性,多種神經(jīng)營養(yǎng)因子的表達(dá)升高。第三部分:胚胎脊髓細(xì)胞“寄養(yǎng)”遠(yuǎn)端神經(jīng)促進(jìn)延期神經(jīng)修復(fù)后軸突再生【目的】研究移植胚胎脊髓細(xì)胞“寄養(yǎng)”損傷的遠(yuǎn)端神經(jīng)能否促進(jìn)神經(jīng)延期修復(fù)后軸突再生和功能恢復(fù)。【方法】20只SD雌性大鼠,脛神經(jīng)切斷、預(yù)變性7天后動物隨機(jī)分成2組。實(shí)驗(yàn)組與對照組處理同實(shí)驗(yàn)二。3個月后,再次手術(shù),實(shí)驗(yàn)組切除細(xì)胞移植區(qū)域5mm脛神經(jīng),對照組切除5mm脛神經(jīng),脛神經(jīng)遠(yuǎn)端與同側(cè)新鮮切斷的腓總神經(jīng)近端行端端縫合。動物飼養(yǎng)3月后,行熒光金逆行標(biāo)記、電生理檢測、神經(jīng)電鏡和肌肉染色等,比較實(shí)驗(yàn)組與對照組脊髓運(yùn)動神經(jīng)元數(shù)量、遠(yuǎn)段有髓神經(jīng)纖維計(jì)數(shù)及肌肉電生理功能恢復(fù)上的差別�！窘Y(jié)果】腓總神經(jīng)近端交叉縫合脛神經(jīng)遠(yuǎn)端術(shù)后3個月,實(shí)驗(yàn)組脊髓內(nèi)再生的神經(jīng)元數(shù)量、遠(yuǎn)端神經(jīng)內(nèi)有髓軸突計(jì)數(shù)、腓腸肌濕重和截面積及電生理結(jié)果優(yōu)于對照組,差異有統(tǒng)計(jì)學(xué)意義�！窘Y(jié)論】胚胎脊髓細(xì)胞移植“寄養(yǎng)”損傷脛神經(jīng)遠(yuǎn)端可促進(jìn)延期交叉縫合后軸突生長和肌肉功能恢復(fù)。
[Abstract]:The peripheral nerve especially the brachial plexus and other proximal nerve injuries, because the injured part is far away from the target organ and the axon regeneration speed is slow, the distal nerve and the muscle are in the denervated state for a long time, and the chronic denaturation and fibrosis will occur, and the function recovery is affected by the normal nerve "sending" the distal nerve to make the target organ of the denervated in time. It has been reported and our previous study found that the transplantation of embryonic neural stem cells to the distal nerve of the injured peripheral nerve can survive, develop into the deity element, and emit the axon to control the distal muscles and reduce the distal muscles. Therefore, on the basis of previous studies, we use the rat tibial nerve injury as an experimental animal model to study whether the distal nerve distal to the distal target organ can reduce the chronic denaturation of the distal target organ degenerative nerve in the transplanted embryonic spinal cord cells, and the distal denervation of the distal nerve is again after the end of the cell "foster". Can the cell "foster" promote axonal regeneration and function recovery after delayed nerve repair? The first part of the cultured embryonic spinal cord cells transplanted to the peripheral nerve survival and differentiation. [Objective] the transplantation of neural stem cells in vitro to the injured peripheral nerve can survive and axon growth and prevent denervation. In this experiment, the survival and differentiation of embryonic spinal cord cells with different culture times were transplanted to the distal end of the injured nerve. [Methods] the embryonic spinal cord tissue of transgenic rats (GFP) of 13.5 days of pregnancy was taken to prepare the embryonic spinal cord cell suspension (P0), and then cultured for 3 days (P1) and continuously cultured to the P3 generation. Before transplantation, the cell components were identified in vitro. Another 30 was obtained. In only SD female rats, the tibial nerve was cut off and the distal denaturation was 7 days later. The animals were randomly divided into 3 groups. The animals were injected with P0, P1, and P3 cells to the distal tibial nerve respectively. The survival and differentiation of the cells were identified by immuno histochemical staining in the ganglion segment of the transplanted region for 3 months. [results] the results of in vitro identification showed the large part of the primary embryonic spinal cord cells. The percentage of neuron precursor cells gradually decreased and the proportion of GFAP positive glial cells increased with the increase of the number of passages, and the result of cell transplantation three months after transplantation was consistent with the results in vitro: the proportion of NEUN and CHAT in the surviving cells decreased gradually as the number of passages increased. [Conclusion] the embryonic spinal cord cells are rich in neurons and neuron progenitor cells. With the increase of the number of passages, the proportion of neurons decreases gradually. Therefore, the best use of the primary embryonic spinal cord cells to the injured nerve distal to the injured nerve cells. The effect of "mailing" on the second part of the embryonic spinal cord cells on the chronic degeneration of the distal nerve and muscle [Objective] previous studies have shown that the transplantation of primary embryonic spinal cord cells to the premodified distal peripheral nerve can survive and have a higher proportion of neurons. This part further studies the effect of the distal nerve and muscle degeneration of the distal nerve to the distal nerve of the 1. transplanted embryonic spinal cord cells with "foster" injury; 2. excision of the cell transplantation area, The functional state of Schwann cells in the distal nerve after "foster" was completed. [Methods] 30 SD female rats were taken, and the tibial nerve was cut and denatured for 7 days, and randomly divided into 2 groups: cell transplantation "foster" group: injection of primary embryonic spinal cord cells (100 thousand /ul) to the distal tibial nerve. The control group was injected with equal amount of nerve culture to the same site. Rats were fed to the same area. In March, 10 rats in each group were treated with electrophysiology, distal nerve electron microscopy, muscle HE, and immunofluorescence staining. The difference between the two groups of axon counts, gastrocnemius muscle atrophy, neurotrophic factor and myelin protein expression were compared. The remaining 5 rats in each group were reoperated, the experimental group excised the 5mm nerve in the cell transplantation area, and the control group excised the same position nerve. After 2 weeks, the distal nerve DNF and myelin protein were detected and compared between the two groups. [results] the regenerative axons were seen in the distal segment of the nerve in the experimental group. The extent of the gastrocnemius muscle atrophy was lighter, the regenerative nerve innervated motor endplate was seen, the electrical stimulation produced the action potential, and the control group had no regeneration of the distal segment nerve. Myelinated nerve fibers, gastrocnemius atrophy obvious, no innervated motor endplate, electrical stimulation without action potential. The expression of two groups of neurotrophic factors BDNF, GDNF, NGF, NT-3 was not significantly different; the expression of myelin protein in the experimental group was significantly higher than that in the control group; after the end of "mailing", the expression level of myelin protein in the experimental group decreased. [Conclusion] the expression level of the management culture factor BDNF, GDNF is significantly increased. [Conclusion] the transplanted embryonic spinal cord cells "foster" injury of the distal tibial nerve, the transplanted cells can survive, develop into neurons and motor neurons, send out axons, establish a functional neuromuscular junction with the muscles, and reduce the chronic degenerative nerve and muscle denaturation of the distal nerve. After "foster", the distal nerve is degenerated again, and the expression of a variety of neurotrophic factors increases. The third part: the "foster" distal nerve of the embryonic spinal cord cells promote the regeneration of the axon after the delayed nerve repair [Objective] to study whether the distal nerve of the transplanted embryonic spinal cord cells "foster" injured injured nerve can promote the delayed repair of the nerve. Axonal regeneration and functional recovery. [Methods] 20 SD female rats were divided into 2 groups randomly. After 7 days of predegeneration, the animals were randomly divided into 2 groups. The experimental group and the control group were treated with the same experiment for two.3 months, reoperation, the experimental group excised the 5mm tibial nerve in the cell transplantation area, the control group excised the tibial nerve, the distal tibial nerve and the general peroneal cutting of the same side of the same side. The proximal end suture of the nerve was performed. After the animals were raised in March, the fluorescent gold retrograde labeling, electrophysiological examination, neural electron microscopy and muscle staining were used to compare the number of spinal motor neurons in the experimental group and the control group, the count of the myelinated nerve fibers in the far segment and the recovery of the muscle electrophysiological function. [results] the proximal and cross suture of the tibial nerve in the peroneal nerve. 3 months after the distal operation, the number of neurons regenerated in the spinal cord in the experimental group, the count of the axons in the distal nerve, the wet weight of the gastrocnemius and the electrophysiological results were superior to those of the control group, and the difference was statistically significant. Muscle function recovery.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R745

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