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

【摘要】：周圍神經(jīng)缺損的修復(fù)與功能重建,一直是外科領(lǐng)域致力解決的難題之一。周圍神經(jīng)缺損,會(huì)導(dǎo)致肢體嚴(yán)重的感覺(jué)、運(yùn)動(dòng)障礙,致殘率極高,給患者、家庭及社會(huì)帶來(lái)沉重的經(jīng)濟(jì)負(fù)和精神上的痛苦,因此,需找到最佳的神經(jīng)修復(fù)材料迫在眉睫。自體神經(jīng)移植到目前仍是公認(rèn)的神經(jīng)缺損修復(fù)的金標(biāo)準(zhǔn),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(lái)源有限。多年來(lái),無(wú)數(shù)學(xué)者致力于此,分別嘗試了多種生物材料(如靜脈、肌肉,去細(xì)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(shí)臨床應(yīng)用的瓶頸,近年來(lái),同種神經(jīng)異種在神經(jīng)缺損修復(fù)過(guò)程中取了得了較滿意的效果,但同種供體來(lái)源同樣有限,且成本費(fèi)用極高,臨床應(yīng)用受限,這促使我們努力尋找到廣闊的修復(fù)材料來(lái)源,為周圍神經(jīng)缺損提供更為有效的修復(fù)手段。該論文設(shè)計(jì)了人工合成神經(jīng)支架和異種去細(xì)胞組織工程神經(jīng)支架修復(fù)神經(jīng)缺損,希望能為周圍神經(jīng)缺損的修復(fù)提供新的思路。實(shí)驗(yàn)分為以下兩個(gè)部分：1、將二氧化硅納米顆粒摻入到膠原蛋白溶液中,構(gòu)建不同濃度的多孔狀膠原狀蛋白結(jié)構(gòu),尋找最適合周圍神經(jīng)再生所需的最理想支架。我們對(duì)制備支架的各項(xiàng)生物學(xué)特性進(jìn)行了詳細(xì)研究,如形態(tài),化學(xué)成分,潤(rùn)濕性,孔隙率,支架膨脹率和降解性。支架內(nèi)進(jìn)行雪旺細(xì)胞的培養(yǎng),用于評(píng)價(jià)膠原/二氧化硅復(fù)合材料對(duì)神經(jīng)再生的生物活性的影響,并對(duì)支架內(nèi)雪旺細(xì)胞DNA含量進(jìn)行測(cè)定。我們成功將二氧化硅納米顆粒摻入膠原構(gòu)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(xì)胞在多孔的支架內(nèi)比單純膠原更容易貼附、增殖。復(fù)合支架內(nèi)的細(xì)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(xì)胞的貼附和增殖,其支架內(nèi)DNA含量最高。這些結(jié)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(jìn)周圍神經(jīng)的修復(fù)與再生。2、用化學(xué)去細(xì)胞方法萃取異種神經(jīng)支架AXN,體外培養(yǎng)大鼠BMSCs,與AXN構(gòu)建組織工程化神經(jīng)；雌性Vistar大鼠60只,建立右側(cè)坐骨神經(jīng)10mm缺損修復(fù)模型,隨機(jī)分成3組(20只/組),A組：骨髓基質(zhì)干細(xì)胞與異種去細(xì)胞神經(jīng)支架復(fù)合構(gòu)建組織工程神經(jīng)橋接坐骨神經(jīng)缺損；B組：?jiǎn)渭儺惙N去細(xì)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(fù)坐骨神經(jīng)缺損組。分別于術(shù)后4周、12周進(jìn)行干細(xì)胞的轉(zhuǎn)歸、神經(jīng)移植免疫學(xué)檢測(cè)、神經(jīng)電生理檢測(cè)、再生神經(jīng)組織學(xué)觀察、掃描電鏡觀察和患肢小腿三頭肌肌纖維橫徑測(cè)量等方法評(píng)判坐骨神經(jīng)功能恢復(fù)情況。該課題成功獲取了大鼠BMSCs,并用BrdU標(biāo)記,免疫組織化學(xué)染色和流式細(xì)胞儀檢測(cè)培養(yǎng)的BMSCs表達(dá)CD44(+)、CD90(+)和CD34(-),BrdU具有較好的初始標(biāo)記率,其可達(dá)88.36%。去細(xì)胞神經(jīng)與新鮮神經(jīng)相比較細(xì)胞和髓鞘被徹底清除,保持了原有的三維仿生結(jié)構(gòu),免疫原成份被清除,流式細(xì)胞儀對(duì)去細(xì)胞神經(jīng)支架組織MHC Ⅱ的檢測(cè)結(jié)果表明異種神經(jīng)經(jīng)化學(xué)去細(xì)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(shù)后4周,流式細(xì)胞儀檢測(cè)外周血CD3+、CD4+和CD8+T細(xì)胞數(shù)量A、B、C組間均無(wú)統(tǒng)計(jì)學(xué)差異,取移植段切片熒光顯微鏡下可見(jiàn)再生纖維束狀排列,其間散在有BrdU標(biāo)記的細(xì)胞核,S-100免疫組化染色檢測(cè)BrdU標(biāo)記細(xì)胞S-100蛋白表達(dá)陽(yáng)性；術(shù)后12周通過(guò)神經(jīng)電生理肌電圖檢測(cè)顯示,電刺激可通過(guò)移植的神經(jīng)支架到達(dá)遠(yuǎn)端的效應(yīng)感受器,記錄該神經(jīng)支配的肌肉所產(chǎn)生的運(yùn)動(dòng)誘發(fā)電位,通過(guò)移植神經(jīng)段的傳導(dǎo)速度B組動(dòng)物較A組動(dòng)物和C組動(dòng)物略慢,A組和C組無(wú)統(tǒng)計(jì)學(xué)差異。神經(jīng)再生組織學(xué)檢查顯示有再生的神經(jīng)纖維通過(guò)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(xué)、電生理檢測(cè)及小腿三頭肌肌纖維橫徑指標(biāo)均優(yōu)于B組,A組與C組無(wú)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R318.08;R651

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