本文選題：鯊魚皮 + 膠原海綿　； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：海戰(zhàn)時(shí)官兵可能遭受槍傷及彈片傷,海上作業(yè)人員常遇到各種碰撞傷與擦傷,海上漁民和海上養(yǎng)殖人員可能遭受各種刺傷與海洋生物咬傷及蟄傷等。由于海水的高滲、低溫、海洋弧菌等特殊性,傷口經(jīng)海水浸泡會(huì)加重受損程度,致使傷口難以愈合,嚴(yán)重時(shí)導(dǎo)致死亡。而且,海上傷員由于可能遠(yuǎn)離陸地,醫(yī)療資源有限,得到及時(shí)救治也會(huì)受各種條件限制。普通醫(yī)用敷料不具備防海水浸泡功能,現(xiàn)有防水敷料僅具有防水淋而不具有防浸泡功能,針對海水浸泡傷的促愈合敷料也鮮有報(bào)道。因此一款既能夠促進(jìn)海水浸泡傷口愈合,又能夠有效保護(hù)傷口免受海水浸泡的醫(yī)用敷料顯得尤為重要。傷口敷料應(yīng)具備的必要性能包括良好的柔韌性、耐久性、透氣性和鎖水性能等。多種膠原相關(guān)產(chǎn)品已應(yīng)用于促傷口愈合。Ι型膠原蛋白作為細(xì)胞外基質(zhì)的主要成分,其降解產(chǎn)物在傷口愈合肉芽形成階段具有細(xì)胞趨化功能,在傷口愈合過程中起著至關(guān)重要的作用。本文以青鯊魚皮膠原為研究對象,采用醋酸溶脹法分離純化得到Ι型膠原蛋白-鯊魚皮膠原蛋白(shark skin collagen,SSC),采用真空冷凍干燥技術(shù)制備鯊魚皮膠原蛋白海綿(shark skin collagen sponge,SSCS),采用UV、FT-IR、CD、SDS-PAGE及氨基酸組成分析等技術(shù),對SSC和SSCS的物化性質(zhì)進(jìn)行研究。結(jié)果表明SSC具有完整三螺旋結(jié)構(gòu)且純度較高;SSCS的孔隙率為83.57%;水蒸氣透過率為0.018732 g/h.cm2;斷裂強(qiáng)度和斷裂伸長率分別為1.788 N/mm和4.52%;在蒸餾水、生理鹽水、PBS、仿傷口滲透液中吸水率分別為86.96 g/g、15.4 g/g、13.13 g/g和16.18 g/g。采用體外自由基清除試驗(yàn)?zāi)Ｐ蜋z測SSC的抗氧化作用,結(jié)果顯示SSC對羥基自由基有顯著的清除作用,當(dāng)SSC濃度為20 mg/mL時(shí),清除率達(dá)到(84±2.1)%;對DPPH自由基的清除效果較弱,當(dāng)濃度為20 mg/mL時(shí),清除率為(18±1.2)%。SSC具有一定的總還原能力且呈現(xiàn)一定的劑量依賴關(guān)系,隨著濃度的增加逐漸升高,當(dāng)濃度為20 mg/mL時(shí),總還原力為0.112±0.002。將SSCS與篩選獲得的防海水浸泡PU膜通過醫(yī)用膠粘合制備得到鯊魚皮膠原海綿防海水浸泡敷貼(SSCS+PU),采用SD大鼠傷口愈合模型,分別研究SSCS促進(jìn)創(chuàng)傷及海水浸泡傷口的愈合作用,SSCS+PU防海水浸泡性能及促傷口愈合作用。結(jié)果顯示:(1)創(chuàng)傷愈合實(shí)驗(yàn),術(shù)后第3 d和第5 d,SSCS組愈合率分別為52.1%±0.057、57.1%±0.080,非常顯著高于陰性對照紗布(gauze,GZ)組的15.9%±0.083、28.4%±0.066(p㩳0.01),顯著高于陽性對照殼聚糖(chitosan,CS)組的22.2%±0.095、36.5%±0.033(p㩳0.05);組織病理學(xué)HE染色結(jié)果顯示SSCS能在愈合初期促進(jìn)微血管生長,愈合后期促進(jìn)肌肉生長和創(chuàng)傷組織的上皮化;CD31免疫組化結(jié)果顯示,術(shù)后第3 d,SSCS組新生血管數(shù)為38±4.73,顯著高于GZ組的26±3.6(p㩳0.05)和CS組的27±3.06(p㩳0.05);TGF-β免疫組化結(jié)果顯示,術(shù)后第3 d,SSCS組TGF-β陽性表達(dá)顆粒數(shù)為20.1%±3.2,顯著高于GZ組的6.7%±0.7(p㩳0.05)和CS組的10.7%±0.5(p㩳0.05)。EGF和b FGF免疫組化結(jié)果顯示,SSCS組第3 d EGF表達(dá)率和第5 d bFGF表達(dá)率顯著高于GZ組(p㩳0.05)。(2)海水浸泡傷愈合實(shí)驗(yàn)結(jié)果顯示,術(shù)后第3 d和第7 d,SSCS組愈合率分別為47.2%±0.08、81.7%±1.70,顯著高于GZ組的13.94%±5.50、49.31%±9.10和CS組的29.40%±1.10、59.25%±6.20(p㩳0.05);組織病理學(xué)結(jié)果顯示SSCS組在第3 d新生毛細(xì)血管顯著增多,第7 d新生膠原纖維較多并開始肌肉組織生長和上皮化;CD31免疫組化結(jié)果顯示,術(shù)后第3 d,SSCS組新生血管數(shù)為88.00±3.61,非常顯著高于GZ組的33.33±3.05(p㩳0.01)和CS組37.67±2.52(p㩳0.01),第5 d SSCS組新生血管數(shù)為67.00±6.00,顯著高于GZ組的52.67±3.51(p㩳0.05);TGF-β免疫組化結(jié)果顯示,術(shù)后第3 d、第5 d,SSCS組TGF-β陽性表達(dá)顆粒數(shù)為146.33±11.67、117.30±9.07,非常顯著高于GZ組的58.30±1.53、73.67±5.13和CS組的64.00±3.00、72.67±9.50(p㩳0.01);bFGF免疫組化結(jié)果顯示,SSCS組第3 d表達(dá)率顯著高于GZ組(p㩳0.05),EGF免疫組化結(jié)果顯示SSCS組第3 d表達(dá)率顯著高于GZ組(p㩳0.01)和CS組(p㩳0.05);RT-PCR檢測結(jié)果顯示,第3 d,SSCS組、CS組、GZ組TGF-βmRNA相對表達(dá)量分別為1.28±0.09、1.76±0.24、0.87±0.24,與陰性對照GZ組相比,CS組與SSCS組TGF-βm RNA表達(dá)量均顯著上調(diào)(p㩳0.05,p㩳0.01);第5 d,SSCS組、CS組、GZ組integrin-β1m RNA相對表達(dá)量分別為1.52±0.12、1.26±0.47、1.13±0.35,與陰性對照GZ組相比,SSCS組integrin-β1 mRNA表達(dá)量顯著上調(diào)(p㩳0.05);Western-blot結(jié)果顯示,在傷口愈合過程中,SSCS和CS能一定程度上調(diào)integrin-β1和α-SMA蛋白相對表達(dá);而在愈合后期則一定程度下調(diào)TGF-β1表達(dá)。(3)防海水浸泡實(shí)驗(yàn)結(jié)果顯示,SSCS+PU能夠有效防止海水對傷口浸泡,有效防浸泡時(shí)間大于4 h;術(shù)后第3 d和第5d,SSCS組愈合率分別為25.90%±0.14、38.02%±0.10,非常顯著高于陰性對照GZ組8.17%±0.02、15.17%±0.06(p㩳0.01);組織病理學(xué)結(jié)果顯示SSCS組第5 d炎癥細(xì)胞較少,傷口邊緣有更多成纖維細(xì)胞生成,第8 d生長較多微血管和膠原纖維并開始上皮化;CD31免疫組化結(jié)果顯示,術(shù)后第5 d,SSCS組新生血管數(shù)為38.00±7.81,顯著高于GZ組15.00±2.00和CS組20.33±4.04(p㩳0.05);TGF-β免疫組化結(jié)果顯示,術(shù)后第5 d,SSCS組TGF-β陽性表達(dá)顆粒數(shù)為60.00±6.24,非常顯著高于GZ組31.00±3.61(p㩳0.01);EGF和bFGF免疫組化結(jié)果顯示,SSCS組第5 d EGF和bFGF表達(dá)率顯著高于GZ組(p㩳0.05)。結(jié)果提示SSCS和SSCS防海水浸泡敷貼能夠顯著提高創(chuàng)傷及海水浸泡傷口的愈合速率,SSCS防海水浸泡敷貼可有效防海水浸泡至少4 h;組織病理學(xué)實(shí)驗(yàn)結(jié)果表明SSCS和SSCS防海水浸泡敷貼均可促進(jìn)創(chuàng)傷組織肉芽組織、微血管和膠原纖維的生長,以及成纖維細(xì)胞的增殖與遷移;免疫組化實(shí)驗(yàn)結(jié)果表明SSCS和SSCS防海水浸泡敷貼可在愈合初期顯著促進(jìn)CD31、TGF-β、EGF等生長因子的表達(dá)。根據(jù)《醫(yī)療器械生物學(xué)評價(jià)》(GBT 16886-2000),開展SSCS的生物安全性評價(jià)試驗(yàn)。采用CCK8檢測其細(xì)胞毒性,新西蘭兔血檢測其血液相容性,小鼠模型檢測其急性毒性最大耐受量,新西蘭兔模型研究其皮膚刺激性,豚鼠模型檢測其致敏性。結(jié)果顯示,SSCS無細(xì)胞毒性,100%SSCS浸提液對M3T3的增值率為164%±0.308,對L929增值率為150%±0.22,表明高濃度SSCS浸提液對L929和M3T3具有增殖作用;溶血率為0.27%,無溶血性,無急性毒性,無皮膚刺激性及致敏性,符合國家醫(yī)用敷料相關(guān)標(biāo)準(zhǔn)。本研究發(fā)現(xiàn)SSC可有效促進(jìn)創(chuàng)傷及海水浸泡創(chuàng)傷的愈合,在傷口愈合初期可促進(jìn)相關(guān)生長因子的陽性表達(dá),可有效促進(jìn)新生血管生成和肉芽組織的形成,顯著促進(jìn)傷口愈合。同時(shí)SSCS防海水浸泡敷貼可有效防海水浸泡,對海上作業(yè)人員的醫(yī)療救治具有重要意義。
[Abstract]:Military officers and soldiers may suffer from gunshot wounds and bullet wounds during sea battles. Marine workers often encounter various injuries and abrasions. Marine fishermen and marine farmed may suffer from various stabs and marine bites and stings. Due to the special nature of sea water, low temperature, and Vibrio marine Vibrio, the wound will aggravate the damage by seawater immersion and cause the wound. It is difficult to heal and cause death when it is serious. Moreover, the casualty of the sea is limited by the possible distance from the land, the medical resources are limited and the timely treatment will be limited. The ordinary medical dressings do not have the anti seawater immersion function. The existing waterproof dressings are only waterproof and do not have the anti soaking function, and the healing dressing for seawater immersion wound is also used for healing dressing. There are few reports. Therefore, a medical dressing which can promote wound healing and effectively protect the wound from seawater immersion is particularly important. The necessary properties of the wound dressing include good flexibility, durability, air permeability and water locking. Type collagen is the main component of extracellular matrix, and its degradation product has cellular chemotaxis in the stage of wound healing granulation, and plays a vital role in the healing process. Shark skin collagen, SSC), using vacuum freeze-drying technology to prepare collagen sponge of shark skin (shark skin collagen sponge, SSCS), using UV, FT-IR, CD, amino acid composition analysis and other techniques. The water vapor permeation rate was 0.018732 g/h.cm2, the breaking strength and elongation at break were 1.788 N/mm and 4.52%, respectively. The water absorption rate in distilled water, saline, PBS, and imitated wound fluid was 86.96 g/g, 15.4 g/g, 13.13 g/g and 16.18 g/g., and the antioxidant activity of SSC was detected by the free radical scavenging test model in vitro, and the results showed SSC. The scavenging effect of the hydroxyl radical is significant. When the concentration of SSC is 20 mg/mL, the clearance rate is (84 + 2.1)%, and the clearance effect of DPPH free radical is weak. When the concentration is 20 mg/mL, the clearance rate is (18 + 1.2)%.SSC with a certain total reduction ability and presents a certain dose dependence, with the increase of concentration, when the concentration is 20. At the time of mg/mL, the total reducing force was 0.112 + 0.002., and SSCS and the screened seawater immersion PU membrane were prepared by adhesive to prepare the shark skin collagen sponge anti seawater immersion application (SSCS+PU). The wound healing model of SD rats was used to study the healing effect of SSCS to promote wound and seawater immersion wound, and SSCS+PU to prevent seawater immersion and to prevent seawater immersion. The results showed that: (1) the wound healing experiment, third D and fifth d after operation, the healing rate of group SSCS were 52.1% + 0.057,57.1% + 0.080 respectively, which was significantly higher than the 15.9% + 0.083,28.4% + 0.066 (P? 0.01) of the negative control gauze (gauze, GZ) group, which was significantly higher than the 22.2% + 0.095,36.5% + 0.033 (P? 0) of the positive control chitosan group (chitosan, CS). 5): histopathological HE staining showed that SSCS could promote microvascular growth in the early healing, promote muscle growth and epithelialization in the later period of healing; CD31 immunohistochemical results showed that third d after operation, 38 + 4.73 in SSCS group, significantly higher than 26 + 3.6 (P? 0.05) in group GZ and 27 + 3.06 (P? 0.05) in CS group, and TGF- beta immunohistochemistry The results showed that third d after operation, the number of TGF- beta positive particles in group SSCS was 20.1% + 3.2, which was significantly higher than that in group GZ, 6.7% + 0.7 (P? 0.05) and 10.7% + 0.5 (P? 0.05).EGF and B FGF in CS group. The expression rate and expression rate of third D (third d) in SSCS group were significantly higher than that in the group (0.05). (2) the experimental results of seawater immersion wound healing showed the operation After third D and seventh D, the healing rate of group SSCS was 47.2% + 0.08,81.7% + 1.70, respectively, significantly higher than 13.94% + 5.50,49.31% + 9.10 in GZ group and 29.40% + 1.10,59.25% + 6.20 in CS group (P? 0.05). Histopathological results showed that SSCS group increased significantly in third D newborn capillaries, seventh D new collagen fibers were more and began to muscle tissue growth and epithelium. CD31 immunohistochemical results showed that the number of neovascularization in group SSCS was 88 + 3.61 after operation, and the number of new blood vessels in group SSCS was 88 + 3.61, which was significantly higher than that in group GZ, 33.33 + 3.05 (P? 0.01) and CS group 37.67 + 2.52 (P? 0.01). The number of new vessels in fifth D SSCS group was 67 + 6, significantly higher than GZ group 52.67 + 3.51 (P?). TGF- beta immunohistochemical results showed after operation The positive expression of GF- beta was 146.33 + 11.67117.30 + 9.07, which was significantly higher than 58.30 + 1.53,73.67 + 5.13 in GZ group and 64 + 3.00,72.67 + 9.50 (P? 0.01) in group CS. The immunohistochemical results of bFGF showed that the expression rate of third D in SSCS group was significantly higher than that in GZ group (P? 0.05). And CS group (P? 0.05); RT-PCR detection results showed that the relative expression of TGF- beta mRNA in the third D, SSCS, CS and GZ groups was 1.28 + 0.09,1.76 + 0.24, respectively, compared with the negative control GZ group (0.05, 0.01); Fifth 0.12,1.26 + 0.47,1.13 + 0.35, compared with the negative control group GZ, the expression of integrin- beta 1 mRNA in the SSCS group was significantly up (P? 0.05). Western-blot results showed that SSCS and CS could increase the relative expression of integrin- beta 1 and alpha -SMA protein to a certain extent during the wound healing process, while in the later period of healing, the expression of beta 1 was down to a certain extent. (3) anti seawater immersion The results of the bubble test showed that SSCS+PU could effectively prevent seawater immersion, and the effective immersion time was more than 4 h, and the healing rate of third D and 5D after operation was 25.90% + 0.14,38.02% + 0.10 respectively, which was significantly higher than that of negative control GZ group 8.17% + 0.02,15.17% + 0.06 (P? 0.01), and histopathological results showed that SSCS Group Fifth D inflammatory cells were less. More fibroblasts were produced on the edge of the wound. Eighth D grew more microvessels and collagen fibers and began to be epithelialization. CD31 immunohistochemical results showed that fifth d after operation and 38 + 7.81 in group SSCS were significantly higher than that in group GZ 15 + 2 and CS group 20.33 + (P? 0.05); TGF- beta immunohistochemical results showed fifth d after operation and TGF- beta Yang in SSCS group. The number of sexual expression particles was 60 + 6.24, which was significantly higher than that in group GZ 31 + 3.61 (P? 0.01). The immunohistochemical results of EGF and bFGF showed that the expression rate of fifth D EGF and bFGF in SSCS group was significantly higher than that in the GZ group (P? 0.05). The results suggested that SSCS and SSCS anti seawater application could significantly improve the healing rate of wound and seawater immersion wound. The paste could effectively prevent seawater immersion at least 4 h. The results of histopathological experiments showed that SSCS and SSCS anti seawater application could promote the growth of tissue granulation tissue, microvascular and collagen fibers, and the proliferation and migration of fibroblasts. The results of immunohistochemistry showed that SSCS and SSCS in seawater immersion application could be significant at the early stage of healing. Promote the expression of growth factors such as CD31, TGF- beta and EGF. According to the biological evaluation of medical instruments (GBT 16886-2000), the biosafety evaluation test of SSCS was carried out. CCK8 was used to detect its cytotoxicity. The blood compatibility of New Zealand rabbit blood was detected. The mice model tested its acute toxicity maximum tolerance. The New Zealand rabbit model studied its skin irritation. The results showed that SSCS had no cytotoxicity. The value of 100%SSCS was 164% + 0.308 to M3T3 and 150% + 0.22 to L929. It showed that the high concentration SSCS extract had a proliferation effect on L929 and M3T3; the hemolysis rate was 0.27%, no hemolysis, no acute toxicity, no skin irritation and sensitization. It accords with the country. This study found that SSC can effectively promote the healing of trauma and seawater immersion wound. It can promote the positive expression of related growth factors at the beginning of wound healing. It can effectively promote the formation of neovascularization and granulation tissue and promote the healing of the wound. At the same time, SSCS anti seawater soaking application can effectively prevent seawater immersion. Bubble is of great significance to the medical treatment of marine workers.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08;TS254.9

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