胚胎脊髓细胞“寄养”远端神经促进延期神经修复后轴突再生

发布时间：2018-08-07 14:31

【摘要】：周围神经特别是臂丛等近端神经损伤,由于损伤部位距离靶器官远和轴突再生速度缓慢,远端神经和肌肉长时间处于失神经支配状态,发生慢性变性与纤维化,影响功能恢复。采用正常神经“寄养”远端神经,能使失神经的靶器官及时获得神经再支配,减轻其慢性变性。但是,肢体近端神经损伤后远端常没有可供“寄养”的正常神经。文献报道和我们先前的研究发现胚胎神经干细胞移植至损伤的周围神经远端能存活、发育成神经元,并发出轴突再支配远端肌肉、减轻肌肉失神经萎缩。因此,本课题在先前研究基础上,以大鼠胫神经损伤为实验动物模型,研究移植胚胎脊髓细胞“寄养”损伤的神经远端能否减轻远端靶器官失神经慢性变性?结束细胞“寄养”后远端神经是否再次发生早期失神经变性?细胞“寄养”能否促进延期神经修复后轴突再生和功能恢复?第一部分不同培养代数的胚胎脊髓细胞移植至周围神经的存活与分化【目的】体外培养的神经干细胞移植至损伤的周围神经远端能存活与轴突生长,预防失神经肌萎缩。本实验研究不同培养时间的胚胎脊髓细胞移植至损伤神经远端后的存活与分化。【方法】取孕13.5天转基因大鼠(GFP)胚胎脊髓组织,制备胚胎脊髓细胞悬液(原代,P0),培养3天后传代(P1),连续培养至P3代。移植前,体外鉴定细胞成分。另取30只SD雌性大鼠,胫神经切断、远端变性7天后,动物随机分成3组,分别注射P0、P1、P3细胞至胫神经远端。动物饲养3个月后取移植区神经节段行免疫组织化学染色鉴定细胞的存活及分化情况。【结果】体外鉴定结果显示原代胚胎脊髓细胞大部分是表达β-tubulin阳性的神经元前体细胞。随着传代次数的增加,神经元前体细胞比例逐渐降低,表达GFAP阳性的胶质细胞比例增加;细胞移植三个月后的结果与体外结果一致:随着传代次数的增加,存活的细胞表达NEUN和CHAT的比例逐渐降低。【结论】胚胎脊髓细胞富含神经元和神经元前体细胞,随着传代次数的增加,神经元的比例逐渐减少,因此,移植细胞至损伤的神经远端最好用原代胚胎脊髓细胞。第二部分胚胎脊髓细胞“寄养”对远端神经和肌肉慢性变性的影响【目的】先前的研究证明原代胚胎脊髓细胞移植至预变性的周围神经远端能存活、有较高的神经元比例。本部分进一步研究:1.移植原代胚胎脊髓细胞“寄养”损伤的胫神经远端对远端神经和肌肉慢性变性的影响;2.切除细胞移植区、结束“寄养”后远端神经内雪旺细胞的功能状态。【方法】取30只SD雌性大鼠,胫神经切断变性7天后,随机分成2组:细胞移植“寄养”组:注射原代胚胎脊髓细胞3ul(10万/ul)至胫神经远端。对照组:注射等量神经培养液至相同部位。大鼠饲养3月,每组10只大鼠行电生理、远端神经电镜、肌肉HE、免疫荧光染色等,比较两组神经轴突计数、腓肠肌肌萎缩、神经营养因子和髓鞘蛋白表达等的差别;剩余每组5只大鼠,再次手术,实验组切除细胞移植区5mm神经,对照组切除相同位置神经,2周后远端神经行神经营养因子及髓鞘蛋白的检测,比较两组差别。【结果】实验组远侧段神经可见髓鞘包绕的再生轴突,其腓肠肌肌萎缩程度更轻、可见再生神经支配的运动终板,电刺激产生动作电位;对照组远侧段神经未见再生的有髓神经纤维,腓肠肌萎缩明显、未见神经支配的运动终板,电刺激没有动作电位产生。两组神经营养因子BDNF,GDNF,NGF,NT-3的表达无显著差别;实验组髓鞘蛋白的表达显著高于对照组;结束“寄养”2周后,实验组髓鞘蛋白的表达水平下降,神经营养因子BDNF、GDNF的表达水平显著上升。【结论】移植胚胎脊髓细胞“寄养”损伤的胫神经远端,移植细胞能存活、发育成神经元和运动神经元,发出轴突,与肌肉建立有功能的神经肌肉接头,减轻远端神经和肌肉慢性失神经变性。结束“寄养”后,远端神经再次发生失神经变性,多种神经营养因子的表达升高。第三部分:胚胎脊髓细胞“寄养”远端神经促进延期神经修复后轴突再生【目的】研究移植胚胎脊髓细胞“寄养”损伤的远端神经能否促进神经延期修复后轴突再生和功能恢复。【方法】20只SD雌性大鼠,胫神经切断、预变性7天后动物随机分成2组。实验组与对照组处理同实验二。3个月后,再次手术,实验组切除细胞移植区域5mm胫神经,对照组切除5mm胫神经,胫神经远端与同侧新鲜切断的腓总神经近端行端端缝合。动物饲养3月后,行荧光金逆行标记、电生理检测、神经电镜和肌肉染色等,比较实验组与对照组脊髓运动神经元数量、远段有髓神经纤维计数及肌肉电生理功能恢复上的差别。【结果】腓总神经近端交叉缝合胫神经远端术后3个月,实验组脊髓内再生的神经元数量、远端神经内有髓轴突计数、腓肠肌湿重和截面积及电生理结果优于对照组,差异有统计学意义。【结论】胚胎脊髓细胞移植“寄养”损伤胫神经远端可促进延期交叉缝合后轴突生长和肌肉功能恢复。
[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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R745

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