本文選題：全氟三丁胺 + 神經(jīng)缺損�。� 參考：《第四軍醫(yī)大學》2017年博士論文

【摘要】：長節(jié)段周圍神經(jīng)損傷的治療仍是一個世界性難題。目前,應用組織工程神經(jīng)支架結(jié)合細胞修復神經(jīng)缺損,被認為是替代自體神經(jīng)移植治療和克服自體神經(jīng)移植缺陷的最有效方法。然而,由于神經(jīng)損傷周圍組織血管損傷造成的缺血、缺氧,植入組織工程支架內(nèi)的細胞大量死亡,很大程度限制了細胞的功能,從而影響了其修復神經(jīng)缺損效能的發(fā)揮。因此,在新生血管長入前,建立一種氧供系統(tǒng)為植入體內(nèi)的細胞供氧有望成為修復長節(jié)段神經(jīng)缺損的有效方法。本研究以全氟三丁胺為基礎(chǔ),構(gòu)建了結(jié)合有雪旺細胞的氧供修復系統(tǒng),在明確PFTBA對缺氧狀態(tài)下雪旺細胞活性及功能有促進作用的基礎(chǔ)上,我們提出了兩種可用于長節(jié)段神經(jīng)損傷的修復方案。(1)應用PFTBA纖維蛋白水凝膠保護早期缺氧狀態(tài)下的雪旺細胞,并用其填充于可降解膠原-殼聚糖神經(jīng)導管,構(gòu)建雪旺細胞-PFTBA凝膠修復系統(tǒng),修復大鼠長節(jié)段神經(jīng)缺損,促進其神經(jīng)再生。(2)構(gòu)建基于同軸靜電紡絲技術(shù)的殼-核結(jié)構(gòu),封裝PFTBA,構(gòu)建具有氧緩釋功能的神經(jīng)修復系統(tǒng),橋接神經(jīng)缺損,促進神經(jīng)的再生和功能恢復。該研究提示:基于PFTBA構(gòu)建的氧供修復系統(tǒng)極大的豐富了利用“氧”作為神經(jīng)損傷修復的理論,為神經(jīng)損傷修復開拓了新的領(lǐng)域。整個研究可分為以下三部分:第一部分:PFTBA纖維蛋白水凝膠氧供系統(tǒng)的構(gòu)建及其對缺氧雪旺細胞的保護性研究背景:雪旺細胞作為一種經(jīng)典的組織工程細胞,已經(jīng)被越來越多的學者應用于周圍神經(jīng)損傷修復。然而,由于組織損傷后局部血管損傷造成的缺血,使得植入的雪旺細胞因缺氧而活性降低,影響了修復效果。因此,構(gòu)建一種能為雪旺細胞早期供氧材料,保護其活性就顯得即為重要。目的:構(gòu)建PFTBA纖維蛋白水凝膠氧供系統(tǒng),研究其對缺氧狀態(tài)下雪旺細胞活性和功能的保護。方法:制備一種富含PFTBA的纖維蛋白水凝膠,使其作用于培養(yǎng)于缺氧環(huán)境中的雪旺細胞,從而觀察檢測其對雪旺細胞活性和功能的影響。首先,我們應用血氣分析儀定量檢測制備的PFTBA纖維蛋白水凝膠在培養(yǎng)基中的釋氧能力。其次,我們將培養(yǎng)的雪旺細胞種植在含有這種水凝膠的培養(yǎng)基中,并將其置于正常氧或者缺氧環(huán)境中。再次,我們應用流式細胞儀對SCs對缺氧的耐受進行檢測,同時應用CCK-8對細胞的增殖分化進行評估。進一步通過SCs的遷徙實驗以及RT-PCR的檢測對細胞功能進行研究。最后,我們應用PFTBA纖維蛋白水凝膠作為載體,對雪旺細胞的進行3D細胞培養(yǎng),并采用活-死細胞染色評估細胞短期活性,采用RT-PCR檢測評估細胞功能。結(jié)果:血氣分析表明:PFTBA凝膠能夠明顯升高其周圍培養(yǎng)基的氧濃度;PFTBA能夠明顯升高缺氧培養(yǎng)的雪旺細胞的CCK-8值,降低其凋亡率;PFTBA可以促進3D培養(yǎng)狀態(tài)下雪旺細胞的活性,上調(diào)細胞NGF、BDNF、GDNF、VEGF、N-cam的mRNA表達水平。結(jié)論:PFTBA纖維蛋白水凝膠氧供系統(tǒng)對缺氧的雪旺細胞的活性和功能具有保護作用。第二部分:PFTBA纖維蛋白水凝膠氧供系統(tǒng)修復神經(jīng)缺損的有效性研究背景:應用組織工程神經(jīng)導管結(jié)合雪旺細胞來修復周圍神經(jīng)缺損,被認為是替代自體神經(jīng)移植治療和克服自體神經(jīng)移植缺陷的最有效方法。然而,由于組織損傷后局部血管損傷造成的缺血、缺氧,神經(jīng)導管內(nèi)部的低氧環(huán)境很大程度上限制了雪旺細胞的活性,從而影響了其修復神經(jīng)缺損效能的發(fā)揮。因此,應用含有攜氧劑PFTBA的水凝膠作為雪旺細胞的載體,使其與神經(jīng)導管復合,可能是一種更加有效和方便的解決方案,有望明顯改善支架內(nèi)部早期的缺氧環(huán)境,促進雪旺細胞的存活以及功能的發(fā)揮,從而促進外周受損神經(jīng)的再生和功能恢復。目的:探討PFTBA對植入體內(nèi)的雪旺細胞活性的保護作用,研究PFTBA纖維蛋白水凝膠氧供系統(tǒng)修復神經(jīng)缺損的有效性。方法:應用PFTBA水凝膠作為雪旺細胞的載體,與膠原-殼聚糖神經(jīng)導管有機結(jié)合,構(gòu)建PFTBA神經(jīng)修復系統(tǒng),修復大鼠15mm坐骨神經(jīng)缺損,通過行為學分析、神經(jīng)電生理檢測、熒光金逆行示蹤、靶器官及再生神經(jīng)遠端的形態(tài)學分析(Masson染色、甲苯胺藍染色、免疫熒光染色、透射電鏡等)等方法評價神經(jīng)再生和功能恢復情況。結(jié)果:應用PFTBA水凝膠氧供系統(tǒng)修復長節(jié)段大鼠坐骨神經(jīng)缺損時,復合雪旺細胞的PFTBA水凝膠的神經(jīng)支架與不含有PFTBA水凝膠的神經(jīng)導管相比,其雪旺細胞的存活率明顯提升,其促進軸突順利貫穿神經(jīng)支架并支配遠端靶器官。結(jié)論:PFTBA水凝膠可以明顯改善神經(jīng)導管內(nèi)部早期的缺氧環(huán)境,促進雪旺細胞的存活以及功能的發(fā)揮,促進周圍神經(jīng)的再生和功能恢復。第三部分:基于同軸靜電紡絲技術(shù)的PFTBA緩釋修復系統(tǒng)的研究背景:周圍神經(jīng)損傷時,損傷的血管可以導致局部缺血、缺氧,而這一狀態(tài)一直會持續(xù)至局部組織再血管化。因此,神經(jīng)組織工程支架中心部位的細胞處于缺氧環(huán)境,嚴重限制著神經(jīng)再生的修復。鑒于此,尋找可以解決局部供氧的材料有望改善局部再生微環(huán)境,保護植入雪旺細胞活性,從而促進神經(jīng)缺損的修復效果。目的:探討基于同軸靜電紡絲技術(shù)的PFTBA緩釋修復系統(tǒng)修復神經(jīng)缺損的效能。方法:基于同軸靜電紡絲技術(shù),利用聚已內(nèi)脂和殼聚糖,構(gòu)建一種具有殼-核結(jié)構(gòu)的封裝有PFTBA的靜電紡絲材料,并對其釋氧特性進行評估。將雪旺細胞接種于該材料膜上,應用血氣分析,CCK-8以及流式周期檢測,評估缺氧環(huán)境或者正常氧環(huán)境中該殼-核結(jié)構(gòu)對雪旺細胞的影響。其次,我們將最佳參數(shù)的PFTBA殼-核結(jié)構(gòu)與PFTBA水凝膠作為雪旺細胞的載體,構(gòu)建神經(jīng)支架修復系統(tǒng),修復大鼠17mm坐骨神經(jīng)缺損,通過行為學分析、神經(jīng)電生理檢測、熒光金逆行示蹤、靶器官及再生神經(jīng)遠端的形態(tài)學分析(HE染色、甲苯胺藍染色、免疫熒光染色、透射電鏡等)等方法評價神經(jīng)再生和功能恢復情況。結(jié)果:PFTBA殼-核結(jié)構(gòu)能夠形成氧緩釋狀態(tài),維持周圍培養(yǎng)基一定的氧濃度至144h;該PFTBA殼-核結(jié)構(gòu)紡絲對雪旺細胞無明顯毒性,同時能夠保護缺氧狀態(tài)下雪旺細胞的活性;當應用該結(jié)構(gòu)結(jié)合PFTBA水凝膠修復坐骨神經(jīng)缺損時,其促進軸突順利貫穿神經(jīng)支架并支配遠端靶器官。結(jié)論:基于PFTBA構(gòu)建了結(jié)合有雪旺細胞的殼-核結(jié)構(gòu)神經(jīng)支架修復系統(tǒng)可顯著加快神經(jīng)再生,促進其功能的恢復。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R688
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本文編號：2050470