鲨鱼皮胶原蛋白防海水浸泡生物医用材料研究

发布时间：2018-06-24 00:09

本文选题：鲨鱼皮 + 胶原海绵　；参考：《上海海洋大学》2017年硕士论文

【摘要】：海战时官兵可能遭受枪伤及弹片伤,海上作业人员常遇到各种碰撞伤与擦伤,海上渔民和海上养殖人员可能遭受各种刺伤与海洋生物咬伤及蛰伤等。由于海水的高渗、低温、海洋弧菌等特殊性,伤口经海水浸泡会加重受损程度,致使伤口难以愈合,严重时导致死亡。而且,海上伤员由于可能远离陆地,医疗资源有限,得到及时救治也会受各种条件限制。普通医用敷料不具备防海水浸泡功能,现有防水敷料仅具有防水淋而不具有防浸泡功能,针对海水浸泡伤的促愈合敷料也鲜有报道。因此一款既能够促进海水浸泡伤口愈合,又能够有效保护伤口免受海水浸泡的医用敷料显得尤为重要。伤口敷料应具备的必要性能包括良好的柔韧性、耐久性、透气性和锁水性能等。多种胶原相关产品已应用于促伤口愈合。Ι型胶原蛋白作为细胞外基质的主要成分,其降解产物在伤口愈合肉芽形成阶段具有细胞趋化功能,在伤口愈合过程中起着至关重要的作用。本文以青鲨鱼皮胶原为研究对象,采用醋酸溶胀法分离纯化得到Ι型胶原蛋白-鲨鱼皮胶原蛋白(shark skin collagen,SSC),采用真空冷冻干燥技术制备鲨鱼皮胶原蛋白海绵(shark skin collagen sponge,SSCS),采用UV、FT-IR、CD、SDS-PAGE及氨基酸组成分析等技术,对SSC和SSCS的物化性质进行研究。结果表明SSC具有完整三螺旋结构且纯度较高;SSCS的孔隙率为83.57%;水蒸气透过率为0.018732 g/h.cm2;断裂强度和断裂伸长率分别为1.788 N/mm和4.52%;在蒸馏水、生理盐水、PBS、仿伤口渗透液中吸水率分别为86.96 g/g、15.4 g/g、13.13 g/g和16.18 g/g。采用体外自由基清除试验模型检测SSC的抗氧化作用,结果显示SSC对羟基自由基有显著的清除作用,当SSC浓度为20 mg/mL时,清除率达到(84±2.1)%;对DPPH自由基的清除效果较弱,当浓度为20 mg/mL时,清除率为(18±1.2)%。SSC具有一定的总还原能力且呈现一定的剂量依赖关系,随着浓度的增加逐渐升高,当浓度为20 mg/mL时,总还原力为0.112±0.002。将SSCS与筛选获得的防海水浸泡PU膜通过医用胶粘合制备得到鲨鱼皮胶原海绵防海水浸泡敷贴(SSCS+PU),采用SD大鼠伤口愈合模型,分别研究SSCS促进创伤及海水浸泡伤口的愈合作用,SSCS+PU防海水浸泡性能及促伤口愈合作用。结果显示:(1)创伤愈合实验,术后第3 d和第5 d,SSCS组愈合率分别为52.1%±0.057、57.1%±0.080,非常显著高于阴性对照纱布(gauze,GZ)组的15.9%±0.083、28.4%±0.066(p㩳0.01),显著高于阳性对照壳聚糖(chitosan,CS)组的22.2%±0.095、36.5%±0.033(p㩳0.05);组织病理学HE染色结果显示SSCS能在愈合初期促进微血管生长,愈合后期促进肌肉生长和创伤组织的上皮化;CD31免疫组化结果显示,术后第3 d,SSCS组新生血管数为38±4.73,显著高于GZ组的26±3.6(p㩳0.05)和CS组的27±3.06(p㩳0.05);TGF-β免疫组化结果显示,术后第3 d,SSCS组TGF-β阳性表达颗粒数为20.1%±3.2,显著高于GZ组的6.7%±0.7(p㩳0.05)和CS组的10.7%±0.5(p㩳0.05)。EGF和b FGF免疫组化结果显示,SSCS组第3 d EGF表达率和第5 d bFGF表达率显著高于GZ组(p㩳0.05)。(2)海水浸泡伤愈合实验结果显示,术后第3 d和第7 d,SSCS组愈合率分别为47.2%±0.08、81.7%±1.70,显著高于GZ组的13.94%±5.50、49.31%±9.10和CS组的29.40%±1.10、59.25%±6.20(p㩳0.05);组织病理学结果显示SSCS组在第3 d新生毛细血管显著增多,第7 d新生胶原纤维较多并开始肌肉组织生长和上皮化;CD31免疫组化结果显示,术后第3 d,SSCS组新生血管数为88.00±3.61,非常显著高于GZ组的33.33±3.05(p㩳0.01)和CS组37.67±2.52(p㩳0.01),第5 d SSCS组新生血管数为67.00±6.00,显著高于GZ组的52.67±3.51(p㩳0.05);TGF-β免疫组化结果显示,术后第3 d、第5 d,SSCS组TGF-β阳性表达颗粒数为146.33±11.67、117.30±9.07,非常显著高于GZ组的58.30±1.53、73.67±5.13和CS组的64.00±3.00、72.67±9.50(p㩳0.01);bFGF免疫组化结果显示,SSCS组第3 d表达率显著高于GZ组(p㩳0.05),EGF免疫组化结果显示SSCS组第3 d表达率显著高于GZ组(p㩳0.01)和CS组(p㩳0.05);RT-PCR检测结果显示,第3 d,SSCS组、CS组、GZ组TGF-βmRNA相对表达量分别为1.28±0.09、1.76±0.24、0.87±0.24,与阴性对照GZ组相比,CS组与SSCS组TGF-βm RNA表达量均显著上调(p㩳0.05,p㩳0.01);第5 d,SSCS组、CS组、GZ组integrin-β1m RNA相对表达量分别为1.52±0.12、1.26±0.47、1.13±0.35,与阴性对照GZ组相比,SSCS组integrin-β1 mRNA表达量显著上调(p㩳0.05);Western-blot结果显示,在伤口愈合过程中,SSCS和CS能一定程度上调integrin-β1和α-SMA蛋白相对表达;而在愈合后期则一定程度下调TGF-β1表达。(3)防海水浸泡实验结果显示,SSCS+PU能够有效防止海水对伤口浸泡,有效防浸泡时间大于4 h;术后第3 d和第5d,SSCS组愈合率分别为25.90%±0.14、38.02%±0.10,非常显著高于阴性对照GZ组8.17%±0.02、15.17%±0.06(p㩳0.01);组织病理学结果显示SSCS组第5 d炎症细胞较少,伤口边缘有更多成纤维细胞生成,第8 d生长较多微血管和胶原纤维并开始上皮化;CD31免疫组化结果显示,术后第5 d,SSCS组新生血管数为38.00±7.81,显著高于GZ组15.00±2.00和CS组20.33±4.04(p㩳0.05);TGF-β免疫组化结果显示,术后第5 d,SSCS组TGF-β阳性表达颗粒数为60.00±6.24,非常显著高于GZ组31.00±3.61(p㩳0.01);EGF和bFGF免疫组化结果显示,SSCS组第5 d EGF和bFGF表达率显著高于GZ组(p㩳0.05)。结果提示SSCS和SSCS防海水浸泡敷贴能够显著提高创伤及海水浸泡伤口的愈合速率,SSCS防海水浸泡敷贴可有效防海水浸泡至少4 h;组织病理学实验结果表明SSCS和SSCS防海水浸泡敷贴均可促进创伤组织肉芽组织、微血管和胶原纤维的生长,以及成纤维细胞的增殖与迁移;免疫组化实验结果表明SSCS和SSCS防海水浸泡敷贴可在愈合初期显著促进CD31、TGF-β、EGF等生长因子的表达。根据《医疗器械生物学评价》(GBT 16886-2000),开展SSCS的生物安全性评价试验。采用CCK8检测其细胞毒性,新西兰兔血检测其血液相容性,小鼠模型检测其急性毒性最大耐受量,新西兰兔模型研究其皮肤刺激性,豚鼠模型检测其致敏性。结果显示,SSCS无细胞毒性,100%SSCS浸提液对M3T3的增值率为164%±0.308,对L929增值率为150%±0.22,表明高浓度SSCS浸提液对L929和M3T3具有增殖作用;溶血率为0.27%,无溶血性,无急性毒性,无皮肤刺激性及致敏性,符合国家医用敷料相关标准。本研究发现SSC可有效促进创伤及海水浸泡创伤的愈合,在伤口愈合初期可促进相关生长因子的阳性表达,可有效促进新生血管生成和肉芽组织的形成,显著促进伤口愈合。同时SSCS防海水浸泡敷贴可有效防海水浸泡,对海上作业人员的医疗救治具有重要意义。
[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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;TS254.9

【参考文献】