本文選題：比格犬 + 周圍神經(jīng)損傷�。� 參考：《第四軍醫(yī)大學(xué)》2013年博士論文

【摘要】：周圍神經(jīng)損傷、再生及其功能修復(fù)是世界性臨床醫(yī)學(xué)難題。尤其是在臨床工作中對(duì)于短節(jié)段（30mm）神經(jīng)損傷，手術(shù)能夠直接將損傷的神經(jīng)對(duì)端吻合，使近端再生的神經(jīng)纖維能夠朝向遠(yuǎn)端發(fā)育生長(zhǎng)。但是特別對(duì)于長(zhǎng)節(jié)段（30mm）的神經(jīng)缺損，臨床上不能進(jìn)行無(wú)張力直接縫合，目前的國(guó)際上的治療金標(biāo)準(zhǔn)是將未損傷的非重要區(qū)域的自體神經(jīng)移植到受損傷區(qū)、來(lái)修復(fù)橋接損傷的神經(jīng)。但是，臨床上手術(shù)應(yīng)用自體神經(jīng)移植受到各種條件和因素限制：主要包括供體區(qū)必需進(jìn)行二次手術(shù)，但是供區(qū)神經(jīng)又存在來(lái)源不足，醫(yī)源性造成繼發(fā)的供區(qū)神經(jīng)功能喪失，以及供區(qū)神經(jīng)在組織的結(jié)構(gòu)和尺寸的大小上與需修補(bǔ)神經(jīng)匹配等問(wèn)題。綜上所述，必須有效研發(fā)能夠有效代替自體神經(jīng)的移植替代物，同時(shí)這是目前迫切需要解決的難題。近些年來(lái)，組織工程發(fā)展速度十分迅猛，制備組織工程周圍神經(jīng)早已經(jīng)成為新的研究熱點(diǎn)。運(yùn)用組織工程學(xué)方法制備組織工程移植物修復(fù)橋接長(zhǎng)節(jié)段神經(jīng)缺損收獲得越來(lái)越多的關(guān)注。組織工程移植物一般由三部分構(gòu)成：支架材料、種子細(xì)胞和相應(yīng)活性因子。其中支架材料作為種子細(xì)胞和相關(guān)神經(jīng)營(yíng)養(yǎng)因子的載體，是組成神經(jīng)損傷修復(fù)微環(huán)境的主要結(jié)構(gòu)，其結(jié)構(gòu)構(gòu)建和性能改進(jìn)是制約周圍神經(jīng)缺損修復(fù)的關(guān)鍵問(wèn)題，其組成結(jié)合及內(nèi)部微結(jié)構(gòu)是影響組織工程移植物修復(fù)長(zhǎng)節(jié)段神經(jīng)損傷的關(guān)鍵因素。最新的研究表明，內(nèi)部結(jié)構(gòu)具有定向微結(jié)構(gòu)的支架材料，在修復(fù)橋接神經(jīng)缺損時(shí)比無(wú)定向結(jié)構(gòu)的支架更加適于引導(dǎo)再生的神經(jīng)軸突定向生長(zhǎng)，從而促進(jìn)再生神經(jīng)纖維通過(guò)損傷區(qū)到達(dá)遠(yuǎn)端，進(jìn)一步驗(yàn)證了支架內(nèi)部結(jié)構(gòu)的物理引導(dǎo)作用對(duì)于神經(jīng)再生過(guò)程中的重要性，分析可能原因是因其內(nèi)部定向結(jié)構(gòu)模擬了正常神經(jīng)基底膜的軸向微管結(jié)構(gòu)。但是文獻(xiàn)報(bào)道的支架材料在原料組成及內(nèi)部結(jié)構(gòu)上，與神經(jīng)神經(jīng)基底膜的組成和結(jié)構(gòu)還有較大差距，需要進(jìn)一步研究和改進(jìn)。鑒于大量實(shí)驗(yàn)證實(shí)神經(jīng)基底膜微管結(jié)構(gòu)引導(dǎo)再生神經(jīng)軸突定向生長(zhǎng)的關(guān)鍵作用，如果能夠依據(jù)神經(jīng)組織的成分組成和結(jié)構(gòu)來(lái)選擇和制備組織工程支架，，使該支架具備一定的類似神經(jīng)組織的特性，有可能會(huì)獲得理想的移植替代修復(fù)結(jié)果。然而，截止目前為止，國(guó)內(nèi)外僅有少數(shù)具有類似結(jié)構(gòu)的支架研究被報(bào)道，而應(yīng)用其在實(shí)驗(yàn)動(dòng)物體內(nèi)長(zhǎng)節(jié)段周圍神經(jīng)缺損至今未見(jiàn)研究報(bào)道。 在本實(shí)驗(yàn)中，選用神經(jīng)基底膜基質(zhì)的主要材料——膠原-殼聚糖做為神經(jīng)支架的主要原料，應(yīng)用本實(shí)驗(yàn)組改良并獲得國(guó)家專利的梯度冷凍干燥技術(shù)，制備具有軸向微管結(jié)構(gòu)的三維多孔神經(jīng)支架材料，該支架在組成及內(nèi)部結(jié)構(gòu)方面高度模擬神經(jīng)基底膜。并從原料配比、冰醋酸濃度以及冷淋速度等三個(gè)方面對(duì)改良的梯度冷凍干燥技術(shù)工藝進(jìn)行橫向?qū)Ρ妊芯�，篩選出最佳的制備工藝指標(biāo)。同時(shí)為了使支架能夠滿足體內(nèi)移植的需求，應(yīng)用新型低細(xì)胞毒性交聯(lián)劑——京尼平進(jìn)行化學(xué)交聯(lián)，改善其機(jī)械強(qiáng)度和降解速度，并確定其最佳交聯(lián)參數(shù)。同時(shí)進(jìn)行相關(guān)生物力學(xué)測(cè)試，證實(shí)其具有良好的生物力學(xué)，適于體內(nèi)移植修復(fù)。最后，應(yīng)用免疫組織學(xué)、透射電鏡、神經(jīng)電生理、逆行示蹤技術(shù)等方法，從形態(tài)學(xué)和功能學(xué)兩方面綜合評(píng)價(jià)支架修復(fù)橋接格犬坐骨神經(jīng)30mm缺損效果，結(jié)果證實(shí)其修補(bǔ)缺損神經(jīng)效果接近于自體神經(jīng)移植。相關(guān)具體內(nèi)容如下： 第一部分神經(jīng)支架的制備工藝參數(shù)確定 目的：制備并構(gòu)建組成、結(jié)構(gòu)高度仿真的組織工程神經(jīng)支架。 方法：以膠原-殼聚糖為原料，應(yīng)用改良的梯度冷凍干燥技術(shù)制備仿真支架，掃描電鏡觀察其結(jié)構(gòu)，評(píng)測(cè)支架孔徑、孔隙率等基本性能，優(yōu)化其制備參數(shù)。 結(jié)果：本實(shí)驗(yàn)中同時(shí)采用不同冷淋速度方法來(lái)制備各種孔徑大小和結(jié)構(gòu)的支架材料。選擇速度為2×10~(-5)m/s的冷凝速度時(shí)，支架材料內(nèi)部的微管直徑逐漸增大，效果達(dá)到最佳，平均孔徑為37.34±13.24μm，其內(nèi)部微管呈軸向平行規(guī)律排列。在綜合考慮仿生神經(jīng)支架內(nèi)部結(jié)構(gòu)和孔徑的前提條件下，證實(shí)冰醋酸濃度為3mg/ml，冷淋速度為2×10~(-5)m/s為制備最佳參數(shù)。同時(shí)進(jìn)一步確定以膠原：殼聚糖（C：CH）=3：1制備的神經(jīng)支架材料具有最佳的三維仿生結(jié)構(gòu)和良好的性能，孔徑在24μm～102μm之間，平均孔徑為49.85±19.85μm；孔隙率90%以上，上述結(jié)構(gòu)在神經(jīng)再生過(guò)程中，可以起到瘢痕屏障的保護(hù)作用，能夠在不影響神經(jīng)營(yíng)養(yǎng)物質(zhì)互相交通的情況下，有效阻止瘢痕纖維的長(zhǎng)入，保護(hù)再生纖維的順利向遠(yuǎn)端通過(guò)。同時(shí)還能夠滿足理想支架對(duì)孔徑的要求：小到能夠物引導(dǎo)再生神經(jīng)軸突的定向生長(zhǎng)；大到能夠支持足夠有效的血管化及相關(guān)再生支持細(xì)胞的不斷滲入。 第二部分支架材料改性及生物力學(xué)評(píng)測(cè) 目的：通過(guò)改進(jìn)支架的機(jī)械性能及生物降解性，達(dá)到適應(yīng)體內(nèi)移植需求，并進(jìn)行生物力性評(píng)價(jià)為體內(nèi)移植提供實(shí)驗(yàn)依據(jù)和基礎(chǔ)。 方法：選用低生物毒性交聯(lián)劑——京尼平進(jìn)行化學(xué)交聯(lián)改性，通過(guò)測(cè)定交聯(lián)率、機(jī)械拉力、降解率等指標(biāo)檢驗(yàn)交聯(lián)參數(shù)對(duì)CCH支架性能的影響。 結(jié)果：應(yīng)用低生物毒性交聯(lián)劑——京尼平（1wt%）交聯(lián)48h，結(jié)果顯示：未交聯(lián)的支架材料在純PBS液中共孵育8w后重量減少29.6±4.8%。而經(jīng)過(guò)Genipin交聯(lián)的支架材料在相同條件下重量?jī)H減少原來(lái)的17.9±4.2%，重量丟失明顯少于未交聯(lián)組。在溶菌酶溶液組中，未交聯(lián)的支架重量減少36.3±5.2%，明顯多于交聯(lián)組支架的20.1±4.6%。未交聯(lián)和經(jīng)Genipin交聯(lián)后的CCH支架分別在干燥、濕潤(rùn)的狀態(tài)下進(jìn)行拉力實(shí)驗(yàn)，結(jié)果顯示：干燥組的拉伸應(yīng)力均高于濕潤(rùn)組，而相應(yīng)的拉伸應(yīng)變則為干燥狀態(tài)下小于濕的狀態(tài)，證明交聯(lián)處理能夠改善支架材料的生物力學(xué)性能，同時(shí)使支架材料在神經(jīng)軸突修復(fù)再生過(guò)程中能夠保持穩(wěn)定的內(nèi)部結(jié)構(gòu)，有效的配合神經(jīng)軸突的再生。 第三部分支架材料修復(fù)比格犬坐骨神經(jīng)缺損有效性評(píng)價(jià) 目的：評(píng)價(jià)支架材料修復(fù)長(zhǎng)階段神經(jīng)缺損的有效性。 方法：應(yīng)用免疫組織學(xué)、透射電鏡、神經(jīng)電生理、逆行示蹤技術(shù)等方法，從形態(tài)學(xué)和功能學(xué)兩方面綜合評(píng)價(jià)支架材料橋接比格犬坐骨神經(jīng)30mm的修復(fù)效果。 結(jié)果：術(shù)后12w在支架材料組,盡管新生髓鞘比較纖細(xì)，但外形結(jié)構(gòu)完整，在髓鞘周圍可見(jiàn)基底膜完整的雪旺細(xì)胞，同時(shí)還可以觀察到完整的再生血管結(jié)構(gòu)和良好排列的髓鞘板層結(jié)構(gòu)，修復(fù)效果接近自體神經(jīng)移植組。同時(shí)神經(jīng)電生理、逆行示蹤等方法，從功能學(xué)的不同側(cè)面綜合支持材料橋接神經(jīng)損傷的修復(fù)效果。在術(shù)后24w支架材料組的運(yùn)動(dòng)神經(jīng)傳導(dǎo)速度、潛伏期和波幅與自體神經(jīng)移植結(jié)果接近，兩組之間差異無(wú)顯著性意義；逆行示蹤標(biāo)記后，在脊髓前角和背根神經(jīng)節(jié)可檢測(cè)到與自體神經(jīng)移植組數(shù)量相當(dāng)?shù)臒晒饨饦?biāo)記陽(yáng)性神經(jīng)元，膠原-殼聚糖支架橋接的坐骨神經(jīng)缺損功能性修復(fù)效果接近自體神經(jīng)移植。
[Abstract]:Peripheral nerve injury, regeneration and functional repair are the world's clinical medical problems. Especially in clinical work, the operation can directly anastomosed the injured nerve to the injured nerve to the distal segment (30mm) nerve injury, so that the nerve fibers regenerated near the end can grow toward the distal end, but especially for the long segment (30mm) nerve defect. There is no tension free direct suture on the bed. The current international standard of treatment is to transplant autologous nerves from undamaged areas to the damaged area to repair the injured nerve. However, the application of autologous nerve graft in clinical surgery is limited by various conditions and factors: two times, mainly including the donor area, are required. The operation, but the source of the donor nerve has a shortage of sources, iatrogenic loss of the donor nerve function secondary to the donor area, and the matching of nerve in the structure and size of the donor nerve in the tissue and size. In summary, it is necessary to develop an effective substitute for the autologous transplanting substitutes. At the same time, it is urgently needed. In recent years, the development of tissue engineering has been developing rapidly, and the preparation of tissue engineering peripheral nerve has become a new research hotspot. Tissue engineering method is used to prepare tissue engineering graft for repairing long segmental nerve defects. Scaffold materials, seed cells and corresponding active factors, in which scaffold material is the carrier of seed cells and related neurotrophic factors, is the main structure to form neural damage repair microenvironment. Its structure construction and performance improvement are the key problems that restrict the repair of peripheral nerve defect. Its composition and internal microstructure are the influence of tissue workers. The latest research shows that the internal structure has a directional microstructural scaffold material, which is more suitable for guiding the regeneration of the axon to direct the growth of the axon in repairing the bridged nerve defect than the non directional scaffold, thus promoting the regeneration of the regenerated nerve fibers to the distal end through the damaged area. The importance of the physical guidance of the internal structure of the stent to the process of nerve regeneration is verified. The possible reason is that the axial microtubule structure of the normal nerve basement membrane is simulated because of its internal orienting structure. However, the composition and structure of the scaffold material in the material composition and internal structure of the material and the nerve nerve basement membrane are reported in the literature. There is still a big gap, and further research and improvement are needed. In view of the fact that a large number of experiments have proved the key role of the nerve basement membrane microtubule structure to guide the directional growth of the axon, it is possible to select and prepare a tissue engineering scaffold based on the composition and structure of the nerve tissue, so that the scaffold has some characteristics similar to the nerve tissue. However, only a small number of scaffolding studies with similar structures have been reported at home and abroad, and there have been no reports on the application of it to the long segment of peripheral nerve defects in experimental animals.
In this experiment, the main material of the nerve basement membrane matrix, collagen chitosan, was used as the main raw material of the nerve scaffold. The experimental group was used to improve and obtain the national patent gradient freeze drying technology to prepare the three-dimensional porous nerve scaffold with the axial microtubule structure. The scaffold was high mould in the composition and internal structure. The modified gradient freeze-drying technology was compared in three aspects, such as the ratio of raw materials, glacial acetic acid concentration and the speed of cold drenching. In order to meet the needs of the body transplantation, a new type of low cytotoxic crosslinking agent, geniping, should be used to make the stent meet the needs of the body transplantation. Chemical crosslinking was performed to improve the mechanical strength and degradation rate, and to determine the best cross-linking parameters. At the same time, the biomechanical tests were carried out to prove that it had good biomechanics and suitable for the transplantation in vivo. Finally, the methods of immunohistochemistry, transmission electron microscopy, neuroelectrophysiology, retrograde tracing technique were used in the two party of morphology and function. The results showed that the repair of the bridged canine sciatic nerve 30mm defect was close to that of the autologous nerve graft.
Part 1 Determination of preparation parameters of neural scaffolds
Objective: to prepare and construct tissue-engineered neural scaffolds with high degree of organization and structure.
Methods: using the collagen chitosan as the raw material, the modified gradient freeze drying technology was used to prepare the simulation scaffold. The structure was observed by scanning electron microscope, and the basic properties of the scaffold diameter and porosity were evaluated, and the parameters of the preparation were optimized.
Results: in this experiment, different cooling rate methods were used to prepare the scaffold materials of various sizes and structures. The microtubule diameter of the stent material increased gradually when the velocity of 2 * 10~ (-5) m/s was selected. The average diameter was 37.34 + 13.24 mu m, and the internal microtubule was arranged in the axial parallel law. Under the condition of the internal structure and aperture of the bionic nerve scaffold, it was confirmed that the concentration of glacial acetic acid was 3mg/ml and the cooling rate was 2 * 10~ (-5) m/s as the best parameter. At the same time, it was further determined that the scaffold materials prepared with collagen: C:CH =3:1 had the best three-dimensional bionic structure and good performance, and the pore size was 24 mu. Between M and 102 mu, the average pore size is 49.85 + 19.85 mu m and the porosity is more than 90%. In the process of nerve regeneration, the structure can play a protective role in the scar barrier. It can effectively prevent the long entry of scar fibers and protect the regenerated fibers from the distal end without affecting the mutual traffic of the nerve nutrients. The requirements for the aperture of the ideal scaffold are satisfied: small enough to guide the directional growth of the regeneration of the axon; large enough to support sufficient and effective vascularization and the continuous infiltration of the associated regenerative support cells.
The second part is the modification and biomechanical evaluation of scaffold materials.
Objective: to improve the mechanical performance and biodegradability of the scaffold to meet the needs of transplantation in vivo, and to provide the basis and basis for the evaluation of biological force for the body transplantation.
Methods: a low biological toxic crosslinking agent, genipine, was modified by chemical crosslinking. The effects of crosslinking parameters on the performance of CCH scaffold were tested by measuring the cross-linking rate, mechanical pull and degradation rate.
Results: using a low biotoxic crosslinking agent, genipine (1wt%) crosslinked 48h, the results showed that the weight of the stents without cross linking was reduced by 29.6 + 4.8%. in the pure PBS solution for 8W and the weight of the scaffold crosslinked by Genipin only decreased by 17.9 + 4.2% in the same condition, and the weight loss was obviously less than that of the non crosslinked group. In the enzyme solution group, the weight of the non crosslinked scaffold decreased by 36.3 + 5.2%, obviously more than 20.1 + 4.6%. of the crosslinked scaffold and the CCH scaffold after Genipin crosslinking, and the tensile tests were carried out in the dry and wet state respectively. The results showed that the tensile stress of the drying group was higher than that of the wet group, while the corresponding tensile strain was in the dry state. It is proved that the cross-linking treatment can improve the biomechanical properties of the scaffold material and can maintain the stable internal structure during the repair and regeneration of the axon, and effectively cooperate with the regeneration of the axon.
Third parts of scaffold materials for repairing beagle dogs' sciatic nerve defects
Objective: To evaluate the effectiveness of scaffold materials in repairing long-term nerve defects.
Methods: with the methods of immunohistochemistry, transmission electron microscopy, neuroelectrophysiology, and retrograde tracing technique, the effect of the scaffold material bridging the 30mm of the sciatic nerve of beagle dogs was evaluated from two aspects of morphology and function.
Results: after the operation, 12W in the scaffold material group, although the newborn myelin sheath was thin, but the shape and structure was complete, the intact Schwann cells in the basement membrane were visible around the myelin sheath, and the complete regenerative vascular structure and the well arranged myelin lamellar structure were also observed. The repair effect was close to the autologous nerve transplantation group. In the 24W stent material group, the motor nerve conduction velocity, the latency and amplitude were close to the results of autologous nerve transplantation, and there was no significant difference between the two groups. The retrograde tracer markers were in the anterior horn of the spinal cord and the dorsal root ganglion. The effect of collagen chitosan scaffold bridged on the functional repair of the sciatic nerve defect was close to that of the autologous nerve graft.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08

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1 Shinji Yoshii;Katsuhiro Mabe;Katsuhiko Nosho;Hiroyuki Yamamoto;Hiroshi Yasui;Hiroyuki Okuda;Akira Suzuki;Masahiro Fujita;Toshihiro Sato;;Submucosal hematoma is a highly suggestive finding for amyloid light-chain amyloidosis:Two case reports[J];World Journal of Gastrointestinal Endoscopy;2012年09期

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