本文選題：復合支架 + 神經(jīng)��； 參考：《重慶醫(yī)科大學》2015年博士論文

【摘要】：周圍神經(jīng)缺損的修復與功能重建,一直是外科領域致力解決的難題之一。周圍神經(jīng)缺損,會導致肢體嚴重的感覺、運動障礙,致殘率極高,給患者、家庭及社會帶來沉重的經(jīng)濟負和精神上的痛苦,因此,需找到最佳的神經(jīng)修復材料迫在眉睫。自體神經(jīng)移植到目前仍是公認的神經(jīng)缺損修復的金標準,但存在供體神經(jīng)支配區(qū)永新的久性失神經(jīng)功能障礙,且神經(jīng)供體來源有限。多年來,無數(shù)學者致力于此,分別嘗試了多種生物材料(如靜脈、肌肉,去細胞神經(jīng)等)、人工合成材料(如硅膠管、聚羥乙酸等)材料,雖取得不少成就,但仍難以突破現(xiàn)實臨床應用的瓶頸,近年來,同種神經(jīng)異種在神經(jīng)缺損修復過程中取了得了較滿意的效果,但同種供體來源同樣有限,且成本費用極高,臨床應用受限,這促使我們努力尋找到廣闊的修復材料來源,為周圍神經(jīng)缺損提供更為有效的修復手段。該論文設計了人工合成神經(jīng)支架和異種去細胞組織工程神經(jīng)支架修復神經(jīng)缺損,希望能為周圍神經(jīng)缺損的修復提供新的思路。實驗分為以下兩個部分：1、將二氧化硅納米顆粒摻入到膠原蛋白溶液中,構建不同濃度的多孔狀膠原狀蛋白結構,尋找最適合周圍神經(jīng)再生所需的最理想支架。我們對制備支架的各項生物學特性進行了詳細研究,如形態(tài),化學成分,潤濕性,孔隙率,支架膨脹率和降解性。支架內(nèi)進行雪旺細胞的培養(yǎng),用于評價膠原/二氧化硅復合材料對神經(jīng)再生的生物活性的影響,并對支架內(nèi)雪旺細胞DNA含量進行測定。我們成功將二氧化硅納米顆粒摻入膠原構成支架,二氧化硅納米顆粒的摻入可以提高支架疏水性,降低孔隙率、膨脹率和降解速率。此外,雪旺細胞在多孔的支架內(nèi)比單純膠原更容易貼附、增殖。復合支架內(nèi)的細胞數(shù)量和DNA含量隨著納米顆粒濃度的增加而先增加后降低。與其它配比組相比,25微克/毫升的二氧化硅濃度適合細胞的貼附和增殖,其支架內(nèi)DNA含量最高。這些結果表明,摻入二氧化硅納米顆粒的多孔膠原支架有可能用作植入支架材料,促進周圍神經(jīng)的修復與再生。2、用化學去細胞方法萃取異種神經(jīng)支架AXN,體外培養(yǎng)大鼠BMSCs,與AXN構建組織工程化神經(jīng)；雌性Vistar大鼠60只,建立右側坐骨神經(jīng)10mm缺損修復模型,隨機分成3組(20只/組),A組：骨髓基質干細胞與異種去細胞神經(jīng)支架復合構建組織工程神經(jīng)橋接坐骨神經(jīng)缺損；B組：單純異種去細胞神經(jīng)支架橋接坐骨神經(jīng)缺損；C組：自體神經(jīng)移植修復坐骨神經(jīng)缺損組。分別于術后4周、12周進行干細胞的轉歸、神經(jīng)移植免疫學檢測、神經(jīng)電生理檢測、再生神經(jīng)組織學觀察、掃描電鏡觀察和患肢小腿三頭肌肌纖維橫徑測量等方法評判坐骨神經(jīng)功能恢復情況。該課題成功獲取了大鼠BMSCs,并用BrdU標記,免疫組織化學染色和流式細胞儀檢測培養(yǎng)的BMSCs表達CD44(+)、CD90(+)和CD34(-),BrdU具有較好的初始標記率,其可達88.36%。去細胞神經(jīng)與新鮮神經(jīng)相比較細胞和髓鞘被徹底清除,保持了原有的三維仿生結構,免疫原成份被清除,流式細胞儀對去細胞神經(jīng)支架組織MHC Ⅱ的檢測結果表明異種神經(jīng)經(jīng)化學去細胞處理后免疫原性明顯減弱,可供神經(jīng)移植使用。3.移植術后4周,流式細胞儀檢測外周血CD3+、CD4+和CD8+T細胞數(shù)量A、B、C組間均無統(tǒng)計學差異,取移植段切片熒光顯微鏡下可見再生纖維束狀排列,其間散在有BrdU標記的細胞核,S-100免疫組化染色檢測BrdU標記細胞S-100蛋白表達陽性；術后12周通過神經(jīng)電生理肌電圖檢測顯示,電刺激可通過移植的神經(jīng)支架到達遠端的效應感受器,記錄該神經(jīng)支配的肌肉所產(chǎn)生的運動誘發(fā)電位,通過移植神經(jīng)段的傳導速度B組動物較A組動物和C組動物略慢,A組和C組無統(tǒng)計學差異。神經(jīng)再生組織學檢查顯示有再生的神經(jīng)纖維通過移植段神經(jīng),移植段內(nèi)有縱行排列分布的SCs。A組、C組組織形態(tài)學、電生理檢測及小腿三頭肌肌纖維橫徑指標均優(yōu)于B組,A組與C組無顯著性差異。
[Abstract]:The repair and function reconstruction of peripheral nerve defect has always been one of the difficult problems to be solved in the field of surgery. The peripheral nerve defect can cause severe feeling, dyskinesia, high disability rate, heavy economic negative and mental pain for the patients, family and society. Therefore, it is urgent to find the best material for nerve repair. Autologous nerve transplantation is still a recognized gold standard for repair of nerve defects, but there is a permanent denervation dysfunction in the donor nerve area of Yongxin, and the source of the nerve donor is limited. Many scholars have tried this for years, and have tried a variety of biomaterials (such as static veins, muscles, cell nerves, etc.), and artificial synthetic materials (such as silicon, such as silicon) Although a lot of achievements have been made in rubber tube and polyoacetic acid, it is still difficult to break through the bottleneck of practical clinical application. In recent years, the same kind of nerve xenograft has taken a satisfactory effect in the process of nerve defect repair, but the source of the same donor is also limited, and the cost and cost are very high and the clinical application is limited. The restorative materials provide more effective repair methods for peripheral nerve defects. This paper designs artificial synthetic nerve scaffolds and xenoacellular tissue engineering nerve scaffolds to repair nerve defects, and hopes to provide new ideas for the repair of peripheral nerve defects. The experiment is divided into two parts: 1, silica nanoparticles The particles were added into the collagen solution to construct a porous protein structure with different concentrations to find the most ideal scaffold for the regeneration of peripheral nerves. We studied the biological properties of the scaffolds in detail, such as morphology, chemical composition, wettability, porosity, stent expansion rate and degradation. The culture of Schwann cells was used to evaluate the effect of collagen / silica composite on the biological activity of nerve regeneration and to determine the DNA content of Schwann cells in the scaffold. We successfully mixed silica nanoparticles into the scaffolds. The incorporation of silica nanoparticles can improve the hydrophobicity of the scaffolds, reduce the porosity, and expand. In addition, Schwann cells are more easily attached and proliferate in porous scaffolds than pure collagen. The number of cells and DNA content in the composite scaffold increase first and then decrease with the increase of the concentration of nanoparticles. Compared with the other matching groups, the concentration of 25 microgram / ml of two silicon oxide is suitable for cell attachment and proliferation, and its scaffold The content of internal DNA is the highest. These results suggest that the porous collagen scaffold doped with silica nanoparticles may be used as a scaffold material to promote the repair and regeneration of peripheral nerve,.2, AXN, BMSCs in vitro, and tissue engineering nerve in vitro, and 60 female Vistar rats. The right sciatic nerve 10mm defect repair model was established and divided randomly into 3 groups (20 rats / groups). Group A: bone marrow stromal cells and xenogeneic nerve scaffolds to construct tissue engineering nerve bridging sciatic nerve defect; group B: simple xenoacellular nerve scaffold bridged sciatic deity defect; group C: autologous nerve graft for the repair of sciatic deity After 4 weeks and 12 weeks, the changes of stem cells, neural transplantation immunology, neurophysiological test, regenerative nerve histology, scanning electron microscopy and transverse diameter measurement of triceps muscle fiber were used to evaluate the recovery of sciatic nerve function. The BMSCs of rats was successfully obtained, and the BrdU standard was used. BMSCs expressed CD44 (+), CD90 (+) and CD34 (-), and BrdU had better initial labeling rate, which could reach 88.36%. to cell nerve and fresh nerve. The cells and myelin sheath were thoroughly removed, and the original three-dimensional biomimetic structure was maintained. The immunogen components were cleared and flow cytometry was used. The results of the detection of MHC II in the cellular nerve scaffold showed that the immunogenicity of the xenogeneic nerve was obviously weakened after the chemical removal of the cell. 4 weeks after the transplantation of.3., the flow cytometry was used to detect the number of CD3+, CD4+ and CD8+T cells in the peripheral blood, A, B, C, and the fluorescence microscope of the transplantation section was seen under the fluorescence microscope. The regenerated fibers were arranged in a fascicular arrangement, scattered in the nucleus with BrdU markers, and S-100 immunohistochemical staining was used to detect the positive expression of S-100 protein in the BrdU labeled cells. Electroelectromyography detected by the electrophysiological electromyography at 12 weeks after the operation showed that the electrical stimulation could reach the distal effector through the transplantation of the nerve scaffold to record the muscle of the innervated muscle. The motor evoked potential of the B group was slightly slower than that of the A group and the C group through the conduction velocity of the transplanted nerve segment. There was no statistical difference between the group A and the C group. The regeneration of the nerve regeneration histology showed that the regenerated nerve fibers passed the segmental nerve, the SCs.A group was arranged in a longitudinal arrangement, the morphology of the C group, the electrophysiological test and the calf. The transverse diameter index of triceps muscle fiber was better than that of group B, and there was no significant difference between group A and group C.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R318.08;R651

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