【摘要】：周圍神經(jīng)損傷是臨床上常見(jiàn)的病癥,其修復(fù)與再生是神經(jīng)科學(xué)領(lǐng)域的研究熱點(diǎn)。當(dāng)損傷距離較短時(shí),周圍神經(jīng)能自我修復(fù),但是長(zhǎng)距離缺損,必須借助神經(jīng)移植物才能完成修復(fù)。作為“金標(biāo)準(zhǔn)”的自體神經(jīng)移植存在來(lái)源匱乏、供體與損傷神經(jīng)尺寸不匹配等不足,因此尋求合適的人工神經(jīng)移植物,引導(dǎo)、促進(jìn)神經(jīng)再生,加快功能重建是科研人員努力的目標(biāo)。理想的人工神經(jīng)移植物必須具備良好的生物相容性和與植入組織匹配的力學(xué)性能。大量研究表明蠶絲的絲素蛋白具有良好的生物相容性,但蠶絲在脫膠過(guò)程中力學(xué)性能大幅度下降。為構(gòu)建符合要求的人工神經(jīng)移植物,本課題以天然蠶絲為基本原料,將傳統(tǒng)的編織法與靜電紡絲法相結(jié)合,制備兼具良好生物相容性和力學(xué)性能的具有復(fù)合結(jié)構(gòu)的絲素蛋白神經(jīng)導(dǎo)管(CSF-NGCs),對(duì)導(dǎo)管進(jìn)行生物安全性評(píng)價(jià),并將其用于修復(fù)大鼠10 mm坐骨神經(jīng)缺損,通過(guò)一系列方法評(píng)價(jià)其修復(fù)效果。主要研究?jī)?nèi)容和結(jié)論如下:利用靜電紡絲法制備CSF-NGCs的內(nèi)層和外層,借助掃描電子顯微鏡(SEM)研究了溶液濃度、電壓、推進(jìn)速度對(duì)靜電紡納米纖維形貌和尺寸的影響,篩選出最優(yōu)工藝參數(shù)為:溶液濃度:18%,電壓:21 kV,推進(jìn)速度:0.2 mL/h。用自改裝的編織機(jī)編織絲素纖維網(wǎng),通過(guò)改變攜紗器的運(yùn)轉(zhuǎn)速度和編織物向上提拉速度,獲得具有不同編織角和編織密度的絲素網(wǎng)。測(cè)試所制備導(dǎo)管的拉伸性能、抗手術(shù)縫合線強(qiáng)度以及抗壓性能,分析了編織角、編織密度對(duì)三種力學(xué)性能的影響。通過(guò)比較最終用于后續(xù)實(shí)驗(yàn)的CSF-NGCs拉伸時(shí)最大荷重為16.3 N,將導(dǎo)管拉破時(shí)拉力與2倍管壁厚度的比值Fpull-out/2t值是3.5N/mm,抗壓試驗(yàn)中形變50%時(shí)對(duì)應(yīng)的荷重達(dá)2.1 N,各項(xiàng)指標(biāo)都與文獻(xiàn)報(bào)道結(jié)果相當(dāng),同時(shí)明顯優(yōu)于單純靜電紡絲素導(dǎo)管。后續(xù)縫合術(shù)和體內(nèi)試驗(yàn)證實(shí)了這一點(diǎn)。根據(jù)實(shí)驗(yàn)操作要求與力學(xué)性能測(cè)試結(jié)果,確定編織最佳工藝參數(shù)為:攜紗器運(yùn)行速度:3.6 rpm,編織物上提速度:4.5 cm/min。測(cè)定CSF-NGCs的壁厚、表面形貌、孔隙率、吸水性、通透性,結(jié)果表明CSF-NGCs具有三維多孔納米結(jié)構(gòu),吸水性、滲透性良好。對(duì)CSF-NGCs進(jìn)行體外模擬降解,分析了降解過(guò)程中絲素力學(xué)性能、蛋白結(jié)構(gòu)、質(zhì)量等的變化,結(jié)果表明CSF-NGCs在體外蛋白酶XIV溶液中可降解。通過(guò)靜電紡絲法制備了靜電紡絲素納米纖維膜,將膜與雪旺氏細(xì)胞及背根神經(jīng)節(jié)共培養(yǎng),培養(yǎng)3天和5天時(shí)拍照,并進(jìn)行掃描電鏡和免疫熒光染色觀察,MTT法檢測(cè)細(xì)胞活力,同時(shí)用實(shí)時(shí)PCR檢測(cè)雪旺氏細(xì)胞BDNF和NGF mRNA的表達(dá)及釋放情況。結(jié)果表明靜電紡絲素納米纖維膜與周圍神經(jīng)組織、細(xì)胞具有良好的生物相容性,為神經(jīng)導(dǎo)管的構(gòu)建及體內(nèi)研究奠定了基礎(chǔ)。按照GB/T 16886規(guī)定的方法,通過(guò)遺傳實(shí)驗(yàn)(包括Ames試驗(yàn)、小鼠骨髓嗜多染紅細(xì)胞(PCE)微核試驗(yàn)、小鼠精子畸變實(shí)驗(yàn))、體外細(xì)胞毒性實(shí)驗(yàn)、皮內(nèi)實(shí)驗(yàn)、遲發(fā)型超敏反應(yīng)試驗(yàn)、皮下植入局部反應(yīng)試驗(yàn)、急性全身毒性實(shí)驗(yàn)對(duì)所制備的CSF-NGCs進(jìn)行了生物安全性評(píng)價(jià),結(jié)果表明CSF-NGCs無(wú)遺傳毒性和細(xì)胞毒性、對(duì)皮膚無(wú)刺激性和潛在致敏性、無(wú)急性全身毒性作用,生物相容性好,可生物降解,符合GB/T 16886關(guān)于醫(yī)療器械生物學(xué)評(píng)價(jià)的相關(guān)標(biāo)準(zhǔn)。將制備的CSF-NGCs用于橋接SD大鼠坐骨神經(jīng)10 mm缺損(導(dǎo)管組),另設(shè)自體組和缺損組。術(shù)后1、3、6個(gè)月,以Catwalk步態(tài)儀檢測(cè)、分析其運(yùn)動(dòng)功能的恢復(fù)情況;術(shù)后3、6個(gè)月行熱痛覺(jué)測(cè)定、運(yùn)用光鏡觀察靶肌形態(tài)并利用圖像分析系統(tǒng)進(jìn)行計(jì)量分析;術(shù)后6個(gè)月進(jìn)行電生理檢測(cè),對(duì)再生神經(jīng)進(jìn)行免疫組化染色,運(yùn)用光鏡和電鏡技術(shù)觀察再生神經(jīng)形貌并進(jìn)行統(tǒng)計(jì)分析。結(jié)果:移植術(shù)后,隨著時(shí)間推移,導(dǎo)管組和自體組各方面功能都在逐步恢復(fù),自體組恢復(fù)較快,導(dǎo)管組恢復(fù)較慢,3個(gè)月時(shí)兩組間存在較大差異,但術(shù)后6個(gè)月,所有檢測(cè)項(xiàng)目結(jié)果與自體組的差異都不具統(tǒng)計(jì)學(xué)意義,而與缺損組間差異顯著。術(shù)后6個(gè)月具體數(shù)據(jù):(1)自體組、導(dǎo)管組、缺損組步態(tài)規(guī)律指數(shù)RI分別為:85%、79%、29%(95%以上為正常),坐骨神經(jīng)功能指數(shù)SFI分別為-55、-58、-85(0為完全恢復(fù),-100為完全損傷);(2)用爪痛測(cè)試儀記錄大鼠熱痛閾潛伏期,非術(shù)側(cè)、自體組、導(dǎo)管組平均潛伏期分別為7.78 s、9.15 s、10.23 s,缺損組大鼠腳基本不動(dòng),所以時(shí)間都超過(guò)設(shè)定值20.1 s;(3)電生理檢測(cè):自體組、導(dǎo)管組的腓腸肌復(fù)合肌動(dòng)作電位(CMAP)波幅分別相當(dāng)于非術(shù)側(cè)的72.7%和65.3%,導(dǎo)管組、自體組和非術(shù)側(cè)的神經(jīng)傳導(dǎo)速度分別為28.10±4.03 m/s、35.57±3.49 m/s和50.00±3.18 m/s,缺損組未記錄到CMAP;(4)自體組、導(dǎo)管組、缺損組腓腸肌濕重比分別為0.64、0.57、0.18,肌纖維平均橫截面積分別相當(dāng)于非術(shù)側(cè)的82.9%、74.8%、9.8%;(5)免疫組化結(jié)果顯示導(dǎo)管組和自體組的軸突排布都比較亂,直徑也較非術(shù)側(cè)小,缺損組只有極少量神經(jīng)纖維;(6)電鏡觀察結(jié)果:導(dǎo)管組和自體組的有髓神經(jīng)纖維都比較緊密,雖然髓鞘厚度不及非術(shù)側(cè),但有完整的基底膜。非術(shù)側(cè)、自體組、導(dǎo)管組髓鞘厚度分別為:1.38?m、0.78?m、0.65?m,神經(jīng)纖維直徑分別為2.31?m、1.62?m、1.51?m,缺損組幾乎無(wú)有髓神經(jīng)。綜上所述,各實(shí)驗(yàn)結(jié)果表明術(shù)后6個(gè)月,導(dǎo)管組大鼠運(yùn)動(dòng)功能、感覺(jué)功能都恢復(fù)良好,修復(fù)效果和自體組相當(dāng)。本課題的研究工作為制備人工神經(jīng)導(dǎo)管提供了新思路,新方法,相關(guān)結(jié)果為新制備導(dǎo)管在組織工程領(lǐng)域的進(jìn)一步研究與應(yīng)用提供了理論基礎(chǔ)和依據(jù)。
[Abstract]:Peripheral nerve injury is a common clinical disease, and its repair and regeneration is a hot spot in the field of neuroscience. When the injury distance is short, the peripheral nerve can repair itself, but the long distance defect must be repaired with the aid of the nerve graft. There is a shortage of sources, donor and injury as the "gold standard" of autologous nerve transplantation. Therefore, it is the goal of researchers to seek suitable artificial nerve graft, guide, promote nerve regeneration and accelerate functional reconstruction. Ideal artificial nerve graft must have good biocompatibility and the ability to match the implanted tissue. A large number of studies show silk fibroin albumen. It has good biocompatibility, but the mechanical properties of silk in the process of degumming are greatly reduced. In order to construct the artificial neural grafts that meet the requirements, this subject uses natural silk as the basic raw material to combine the traditional knitting method with the electrostatic spinning method to prepare a composite silk egg with a composite structure with good biocompatibility and mechanical properties. The white nerve conduit (CSF-NGCs) was used to evaluate the biological safety of the catheter and to repair the 10 mm sciatic nerve defect of the rat and evaluate the repair effect by a series of methods. The main contents and conclusions are as follows: the inner and outer layers of CSF-NGCs were prepared by the electrostatic spinning method, and the concentration of the solution was studied by scanning electron microscope (SEM). The effect of voltage and propulsion speed on the morphology and size of electrospun nanofibers was selected. The optimum process parameters were as follows: solution concentration: 18%, voltage: 21 kV, propulsion speed: 0.2 mL/h. weave silk fibroin with self modified braiding machine, and obtain different knitting angles and weaving angles by changing the speed of the yarn carrying device and the speed of the knitting fabric to the up drawing speed. The silk net of the woven density. Test the tensile properties, the strength of the surgical suture and the compressive strength. The influence of the braiding angle and the braiding density on the three mechanical properties was analyzed. The maximum weight of the three kinds of mechanical properties was 16.3 N when the final CSF-NGCs was used for the subsequent experiment. The ratio of the tensile force to the 2 times the thickness of the tube wall when the catheter was pulled out was Fpull. The -out/2t value is 3.5N/mm, and the corresponding load of the compression test is 2.1 N when the deformation is 50%. Each index is equivalent to the literature report, and it is obviously superior to the pure electrospun silk conduit. The following suture and in vivo test confirm this point. According to the experimental operation requirements and the mechanical performance test results, the best weaving process parameters are determined to be carried out. The running speed of the yarn is 3.6 rpm, the speed of lifting on the fabric: 4.5 cm/min. to determine the wall thickness, surface morphology, porosity, water absorption and permeability of CSF-NGCs. The results show that CSF-NGCs has three-dimensional porous nanostructure, water absorbability and permeability. The mechanical properties, protein structure and quality of the fibroin in the degradation process are analyzed in vitro. The results showed that the CSF-NGCs was degradable in the protease XIV solution in vitro. Electrospun silk fibroin nanofibrous membrane was prepared by electrostatic spinning method. The membrane was co cultured with Schwann cell and dorsal root ganglion and cultured for 3 days and 5 days. The scanning electron microscope and immunofluorescence staining were used to detect the activity of the cell, and the cell viability was detected by MTT method. The expression and release of BDNF and NGF mRNA in Schwann cells were detected by real time PCR. The results showed that the electrospun fibroin nanofiber membrane had good biocompatibility with the peripheral nerve tissue and cells. It laid the foundation for the construction of nerve conduit and in vivo research. According to the method of GB/T 16886, the genetic experiment (including Ames test, small) Mouse bone marrow polychromatic erythrocyte (PCE) micronucleus test, mouse sperm aberration test, in vitro cytotoxicity test, intradermal experiment, delayed type hypersensitivity test, subcutaneous implantation local reaction test and acute systemic toxicity test were used to evaluate the biological safety of the prepared CSF-NGCs. The results showed that CSF-NGCs had no hereditary toxicity and cytotoxicity. No irritation and potential sensitivities to the skin, no acute systemic toxicity, good biocompatibility and biodegradability, which accords with the related standards of GB/T 16886 on biological evaluation of medical instruments. The prepared CSF-NGCs was used to bridge the 10 mm defect of the sciatic nerve of SD rats (catheter group), and also set up the autologous group and the defect group. After 1,3,6 months, Catwalk A gait instrument was used to detect the recovery of the motor function. After 3,6 months, the heat pain was measured, the shape of the target muscle was observed by light microscope and the image analysis system was used for measurement and analysis. The electrophysiological examination was carried out for 6 months after the operation, the regenerated nerve was immunohistochemical staining, and the morphologies of the regenerated nerve were observed by light microscopy and electron microscopy. Statistical analysis. Results: after the transplantation, the functions of the catheter group and the autologous group were gradually restored with time. The autologous group recovered quickly and the catheter group recovered slowly. There was a big difference between the two groups at 3 months, but 6 months after the operation, the difference between the results of all the test items and the autologous group was not statistically significant, but the difference from the defect group was not significant. 6 months after operation specific data: (1) the gait regularity index RI of autologous group, catheter group and defect group were 85%, 79%, 29% (95% above normal), the sciatic nerve function index SFI was -55, -58, -85 (0 was completely recovered, -100 was completely damaged); (2) the latent period of rat thermal pain threshold was recorded by claw pain tester, non operative, autologous group and catheter group were recorded. The average latency was 7.78 s, 9.15 s, 10.23 s, and the foot of the defect group was basically not moving, so the time exceeded the set value of 20.1 s; (3) electrophysiological test: the autologous group, the gastrocnemius muscle action potential (CMAP) amplitude of the catheter group was equivalent to the non operative 72.7% and 65.3%, the catheter group, the autologous group and the non lateral nerve conduction velocity were respectively 28.10 + 4.03 m/s, 35.57 + 3.49 m/s and 50 + 3.18 m/s, the defect group did not record CMAP; (4) the wet weight ratio of the gastrocnemius muscle in the autologous group, the catheter group and the defect group was 0.64,0.57,0.18, the average cross section of the muscle fiber was equivalent to 82.9%, 74.8%, 9.8% of the non operative side, and the results of the exempting group showed that the axon arrangement in the catheter group and the autologous group were all in disorder. There were only a few nerve fibers in the defect group. (6) the results of electron microscope observation: the medullary nerve fibers in the catheter group and the autologous group were close, although the thickness of the myelin sheath was less than the non operative side, but there was a complete basement membrane. The non operative, autologous and catheter groups were 1.38? M, 0.78? M, 0.65? M, and the diameter of nerve fibers, respectively. For 2.31? M, 1.62? M, 1.51? M, there was almost no myelinated nerve in the defect group. In summary, the experimental results showed that the motor function and sensory function of the rats in the catheter group recovered well and the effect was equivalent to the autologous group at 6 months after the operation. The research work of this subject provided new ideas, new methods, and related results for the preparation of artificial nerve conduits. The tube provides a theoretical basis and basis for further research and application in the field of tissue engineering.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R318.08;R745

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本文編號(hào)：2155057