周围神经再生修复早期缺氧的氧供方案的建立及应用

发布时间：2018-06-21 23:20

  本文选题：全氟三丁胺 + 神经缺损　； 参考：《第四军医大学》2017年博士论文

【摘要】：长节段周围神经损伤的治疗仍是一个世界性难题。目前,应用组织工程神经支架结合细胞修复神经缺损,被认为是替代自体神经移植治疗和克服自体神经移植缺陷的最有效方法。然而,由于神经损伤周围组织血管损伤造成的缺血、缺氧,植入组织工程支架内的细胞大量死亡,很大程度限制了细胞的功能,从而影响了其修复神经缺损效能的发挥。因此,在新生血管长入前,建立一种氧供系统为植入体内的细胞供氧有望成为修复长节段神经缺损的有效方法。本研究以全氟三丁胺为基础,构建了结合有雪旺细胞的氧供修复系统,在明确PFTBA对缺氧状态下雪旺细胞活性及功能有促进作用的基础上,我们提出了两种可用于长节段神经损伤的修复方案。(1)应用PFTBA纤维蛋白水凝胶保护早期缺氧状态下的雪旺细胞,并用其填充于可降解胶原-壳聚糖神经导管,构建雪旺细胞-PFTBA凝胶修复系统,修复大鼠长节段神经缺损,促进其神经再生。(2)构建基于同轴静电纺丝技术的壳-核结构,封装PFTBA,构建具有氧缓释功能的神经修复系统,桥接神经缺损,促进神经的再生和功能恢复。该研究提示:基于PFTBA构建的氧供修复系统极大的丰富了利用“氧”作为神经损伤修复的理论,为神经损伤修复开拓了新的领域。整个研究可分为以下三部分:第一部分:PFTBA纤维蛋白水凝胶氧供系统的构建及其对缺氧雪旺细胞的保护性研究背景:雪旺细胞作为一种经典的组织工程细胞,已经被越来越多的学者应用于周围神经损伤修复。然而,由于组织损伤后局部血管损伤造成的缺血,使得植入的雪旺细胞因缺氧而活性降低,影响了修复效果。因此,构建一种能为雪旺细胞早期供氧材料,保护其活性就显得即为重要。目的:构建PFTBA纤维蛋白水凝胶氧供系统,研究其对缺氧状态下雪旺细胞活性和功能的保护。方法:制备一种富含PFTBA的纤维蛋白水凝胶,使其作用于培养于缺氧环境中的雪旺细胞,从而观察检测其对雪旺细胞活性和功能的影响。首先,我们应用血气分析仪定量检测制备的PFTBA纤维蛋白水凝胶在培养基中的释氧能力。其次,我们将培养的雪旺细胞种植在含有这种水凝胶的培养基中,并将其置于正常氧或者缺氧环境中。再次,我们应用流式细胞仪对SCs对缺氧的耐受进行检测,同时应用CCK-8对细胞的增殖分化进行评估。进一步通过SCs的迁徙实验以及RT-PCR的检测对细胞功能进行研究。最后,我们应用PFTBA纤维蛋白水凝胶作为载体,对雪旺细胞的进行3D细胞培养,并采用活-死细胞染色评估细胞短期活性,采用RT-PCR检测评估细胞功能。结果:血气分析表明:PFTBA凝胶能够明显升高其周围培养基的氧浓度;PFTBA能够明显升高缺氧培养的雪旺细胞的CCK-8值,降低其凋亡率;PFTBA可以促进3D培养状态下雪旺细胞的活性,上调细胞NGF、BDNF、GDNF、VEGF、N-cam的mRNA表达水平。结论:PFTBA纤维蛋白水凝胶氧供系统对缺氧的雪旺细胞的活性和功能具有保护作用。第二部分:PFTBA纤维蛋白水凝胶氧供系统修复神经缺损的有效性研究背景:应用组织工程神经导管结合雪旺细胞来修复周围神经缺损,被认为是替代自体神经移植治疗和克服自体神经移植缺陷的最有效方法。然而,由于组织损伤后局部血管损伤造成的缺血、缺氧,神经导管内部的低氧环境很大程度上限制了雪旺细胞的活性,从而影响了其修复神经缺损效能的发挥。因此,应用含有携氧剂PFTBA的水凝胶作为雪旺细胞的载体,使其与神经导管复合,可能是一种更加有效和方便的解决方案,有望明显改善支架内部早期的缺氧环境,促进雪旺细胞的存活以及功能的发挥,从而促进外周受损神经的再生和功能恢复。目的:探讨PFTBA对植入体内的雪旺细胞活性的保护作用,研究PFTBA纤维蛋白水凝胶氧供系统修复神经缺损的有效性。方法:应用PFTBA水凝胶作为雪旺细胞的载体,与胶原-壳聚糖神经导管有机结合,构建PFTBA神经修复系统,修复大鼠15mm坐骨神经缺损,通过行为学分析、神经电生理检测、荧光金逆行示踪、靶器官及再生神经远端的形态学分析(Masson染色、甲苯胺蓝染色、免疫荧光染色、透射电镜等)等方法评价神经再生和功能恢复情况。结果:应用PFTBA水凝胶氧供系统修复长节段大鼠坐骨神经缺损时,复合雪旺细胞的PFTBA水凝胶的神经支架与不含有PFTBA水凝胶的神经导管相比,其雪旺细胞的存活率明显提升,其促进轴突顺利贯穿神经支架并支配远端靶器官。结论:PFTBA水凝胶可以明显改善神经导管内部早期的缺氧环境,促进雪旺细胞的存活以及功能的发挥,促进周围神经的再生和功能恢复。第三部分:基于同轴静电纺丝技术的PFTBA缓释修复系统的研究背景:周围神经损伤时,损伤的血管可以导致局部缺血、缺氧,而这一状态一直会持续至局部组织再血管化。因此,神经组织工程支架中心部位的细胞处于缺氧环境,严重限制着神经再生的修复。鉴于此,寻找可以解决局部供氧的材料有望改善局部再生微环境,保护植入雪旺细胞活性,从而促进神经缺损的修复效果。目的:探讨基于同轴静电纺丝技术的PFTBA缓释修复系统修复神经缺损的效能。方法:基于同轴静电纺丝技术,利用聚已内脂和壳聚糖,构建一种具有壳-核结构的封装有PFTBA的静电纺丝材料,并对其释氧特性进行评估。将雪旺细胞接种于该材料膜上,应用血气分析,CCK-8以及流式周期检测,评估缺氧环境或者正常氧环境中该壳-核结构对雪旺细胞的影响。其次,我们将最佳参数的PFTBA壳-核结构与PFTBA水凝胶作为雪旺细胞的载体,构建神经支架修复系统,修复大鼠17mm坐骨神经缺损,通过行为学分析、神经电生理检测、荧光金逆行示踪、靶器官及再生神经远端的形态学分析(HE染色、甲苯胺蓝染色、免疫荧光染色、透射电镜等)等方法评价神经再生和功能恢复情况。结果:PFTBA壳-核结构能够形成氧缓释状态,维持周围培养基一定的氧浓度至144h;该PFTBA壳-核结构纺丝对雪旺细胞无明显毒性,同时能够保护缺氧状态下雪旺细胞的活性;当应用该结构结合PFTBA水凝胶修复坐骨神经缺损时,其促进轴突顺利贯穿神经支架并支配远端靶器官。结论:基于PFTBA构建了结合有雪旺细胞的壳-核结构神经支架修复系统可显著加快神经再生,促进其功能的恢复。
[Abstract]:The treatment of peripheral nerve injury in the long segment is still a worldwide problem. At present, the use of tissue engineered nerve scaffolds to repair nerve defects with cells is considered as the most effective method to replace autologous nerve transplantation and to overcome the defects of autologous nerve transplantation. However, ischemia and hypoxia are caused by vascular injury in the peripheral tissue of the injured nerve. The number of cells implanted in the tissue engineering scaffold is largely dead, which greatly restricts the function of the cells and affects its effectiveness in repairing the nerve defects. Therefore, it is expected that the establishment of an oxygen supply system for the implantation of oxygen in the body is expected to be an effective method for repairing long segmental nerve defects before the growth of new blood vessels. On the basis of three dbrine, an oxygen supply repair system combined with Schwann cells was constructed. On the basis of identifying the effect of PFTBA on the activity and function of Schwann cells under anoxic state, we proposed two repair schemes for long segmental nerve damage. (1) the application of PFTBA fibrin hydrogel to protect Schwann fine in the early anoxic state Cell, and filled with degradable collagen chitosan nerve conduit, construct the -PFTBA gel repair system of Schwann cells, repair long segmental nerve defects in rats and promote nerve regeneration. (2) construct the shell nuclear structure based on coaxial electrospun technology, encapsulate PFTBA, construct the nerve repair system with oxygen release function, bridge nerve defect, promote nerve defect. The regeneration and functional recovery of the nervous system suggest that the oxygen supply repair system based on PFTBA greatly enriches the theory that oxygen is used as a nerve damage repair and opens up a new field for the repair of nerve damage. The whole study can be divided into three parts: the first part: the construction of PFTBA fibrin hydrogel oxygen supply system and the construction of oxygen supply system The protective research background of hypoxia Schwann cells: as a classic tissue engineering cell, Schwann cells have been used by more and more scholars to repair peripheral nerve damage. However, the ischemia caused by local vascular injury after tissue injury makes the implanted Schwann cells reduce the activity of hypoxia and affect the repair effect. Therefore, it is important to construct an early oxygen supply material for Schwann cells to protect its activity. Objective: to construct a PFTBA fibrin hydrogel oxygen supply system and to study the protection of the activity and function of Schwann cells under anoxic state. Method: to prepare a PFTBA rich fibrin hydrogel, which can be used in the cultivation of hypoxia. The effect of Schwann cells in the environment on the activity and function of Schwann cells was observed and detected. First, we used the blood gas analyzer to quantify the oxygen release capacity of the PFTBA fibrin hydrogel prepared in the medium. Secondly, we planted the cultured Schwann cells in the medium containing this hydrogel and put them in the positive. In the environment of oxygen or hypoxia. Again, we used flow cytometry to detect the tolerance of SCs to hypoxia, and evaluate the proliferation and differentiation of cells with CCK-8. Further study on the cell function through the Migration Experiment of SCs and the detection of RT-PCR. Finally, we used the PFTBA fibrin hydrogel as a carrier to snow the snow. The 3D cells were cultured and the cell viability was evaluated by live dead cell staining and RT-PCR detection was used to evaluate the cell function. Results: blood gas analysis showed that PFTBA gel could significantly increase the oxygen concentration in the surrounding medium, and PFTBA could significantly increase the CCK-8 value of Schwann cells in the hypoxia culture and reduce the apoptosis rate; PFTBA could reduce the apoptosis rate. To promote the activity of Schwann cells in 3D culture and up regulate the mRNA expression of NGF, BDNF, GDNF, VEGF, N-cam in cells. Conclusion: the oxygen supply system of PFTBA fibrin hydrogel has protective effect on the activity and function of the anoxic Schwann cells. The second part: the effectiveness of the PFTBA fibrin hydrogel oxygen supply system for the repair of nerve defects. Background: the use of tissue engineered nerve conduits to repair peripheral nerve defects with Schwann cells is considered the most effective way to replace autologous nerve transplantation and to overcome the defects of autologous nerve transplantation. However, ischemia, hypoxia, and the upper limit of the hypoxic environment inside the nerve conduit are limited by the local vascular injury after tissue injury. Therefore, the application of the hydrogel containing PFTBA as a carrier of Schwann cells and the combination of the nerve conduit with the nerve conduit may be a more effective and convenient solution to improve the early hypoxia environment in the stent and promote Schwann. The survival and function of the cells can promote the regeneration and functional recovery of the peripheral damaged nerves. Objective: To explore the protective effect of PFTBA on the activity of Schwann cells in the implanted body, and to study the effectiveness of the PFTBA fibrin hydrogel oxygen supply system to repair the nerve defects. Methods: the PFTBA hydrogel should be used as the carrier of Schwann cells and the glue. An organic combination of the chitosan nerve conduit was used to construct a PFTBA nerve repair system to repair the 15mm sciatic nerve defect in rats. By behavioral analysis, electrophysiological detection, retrograde tracing of fluorescent gold, the morphological analysis of the target organs and the distal nerve of the regenerated nerve (Masson staining, toluidine blue staining, immunofluorescence staining, transmission electron microscopy, etc.) Results: when the PFTBA hydrogel oxygen supply system was used to repair the sciatic nerve defect of the long segment of the sciatic nerve, the survival rate of the Schwann cells in the PFTBA hydrogel combined with the Schwann cells was significantly improved, and the axon promoted the neurite to penetrate the nerve scaffold smoothly. Conclusion: PFTBA hydrogel can obviously improve the early hypoxia environment in the internal nerve conduit, promote the survival and function of Schwann cells, promote the regeneration and function recovery of the peripheral nerve. The third part: the research background of the PFTBA slow release repair system based on the coaxial electrospun Technology: the peripheral nerve injury The damaged blood vessels can lead to local ischemia and hypoxia, and this state will continue until the local tissue revascularization. Therefore, the cells in the central part of the neural tissue engineering scaffold are in a hypoxic environment, which severely restricts the repair of nerve regeneration. To protect the activity of Schwann cells and promote the repair effect of nerve defect. Objective: To explore the efficacy of PFTBA sustained-release repair system based on coaxial electrostatic spinning technology to repair nerve defects. Method: Based on coaxial electrospun technology, a kind of chitosan and chitosan was used to construct a shell nuclear package with PFTBA electrospun. The effect of Schwann cells on this material was evaluated. Schwann cells were inoculated on the membrane. The effects of the shell and nuclear structure on the Schwann cells were evaluated by blood gas analysis, CCK-8 and flow cycle tests. Secondly, the PFTBA shell and nuclear structure of the best parameters and the PFTBA hydrogel were used as Schwann fine. Cell carrier, construction of neural scaffold repair system and repair of 17mm sciatic nerve defect in rats, through behavioral analysis, neurophysiological detection, retrograde tracing of fluorescent gold, morphological analysis of target organs and regenerated distal nerve (HE staining, toluidine blue staining, immunofluorescence staining, transmission electron microscopy, etc.) to evaluate nerve regeneration and functional recovery. Results: the PFTBA shell core structure can form oxygen release state and maintain a certain oxygen concentration to 144H in the surrounding medium; the PFTBA shell nuclear spinning has no obvious toxicity to Schwann cells, and can protect the activity of Schwann cells under anoxic state. When the structure is combined with PFTBA hydrogel to repair the sciatic nerve defect, it promotes the axis of the sciatic nerve. Conclusion: the shell nuclear scaffold repair system combined with Schwann cells based on PFTBA can significantly accelerate the regeneration of nerve and promote the recovery of its function.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R688
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本文编号：2050470