【作者】 赵琳；

【导师】 宋建星；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 一、研究背景创面愈合是一个复杂的过程,在创面的修复形成过程中,成纤维细胞(Fibroblast,Fbs)、部分细胞生长因子及胶原(procollagen, PC)等是关键因素。皮肤缺损后,Fbs参与了愈合的全过程,其中Fbs增殖能力的加强、胶原蛋白合成的增高及细胞生长因子分泌含量的变化等是最为重要的生物学特性改变之一,通过不同途径改善Fbs生物学特性可以进一步促进创面加速愈合。除了组织本身的生长代谢特点外,创面的外部环境对组织愈合也有着非同寻常的意义,其中创面的无菌条件、周围的湿润环境等外部因素是影响其愈合的关键。根据临床调查,目前创面一般不会被直接暴露在外界环境中,而是需要敷料进行包扎,所以敷料对创面形成的环境直接影响着其愈合进程。目前人们发现应用细菌纤维素(Bacterial Cellulose, BC)合成的生物敷料克服了以往敷料的缺点,具有众多有益于创面愈合的特点,包括含水率高、吸水性强、抗张强度大、生物适应性好、抑制创面细菌生长、可促进创面愈合等显著优点,是当今生物材料研究的热点之一,同时BC作为一种新型生物合成敷料在医学界也有了很大的发展。Axcd就是由醋酸杆菌属(Acetobacter aceti, Aa)中的木醋杆菌(Acetobacterxy-linum, Ax)在特定条件下产生的BC构成,其结构与植物纤维素的结构基本相同,属于天然纤维素。由于其独特的优点,弥补了目前其它创面敷料的缺点,使其在众多敷料中脱颖而出。本课题旨在以Axcd为研究对象,主要研究内容分二大部分,前一部分为硕士期间所完成的初步研究,包括动物实验研究,以此部分课题作为铺垫,重点研究Axcd对Fbs及其它相关胶原、细胞生长因子的影响,以期探讨Axcd促进创面愈合的可能机制,为下一步临床应用打下坚实的基础。二、研究目的1、了解Axcd对手术创面的治疗效果及安全性,研究其抑制感染创面细菌作用机理及促进创面愈合的能力。2、探讨Axcd促进人皮肤成纤维细胞(human skin fibroblast, HSFb)的增殖、胶原合成能力变化、转化生长因子β1 (transforming growth factor-β1,TGF-β1)、γ-干扰素(interferon gamma, IFN-γ)及碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)分泌含量的变化,可能与其促进创面愈合的机制有关。三、研究内容和方法1、选用9只小型猪,完全随机分为3组,每组3只,每只小猪制作感染创面模型6个,制备10分钟内各创面接种金黄色葡萄球菌(Staphylococcus aureus, SA)和大肠杆菌(Escherichia coli, EC)混合菌液,Axcd为实验组、新霉素黄连素溶液抗菌纱布为对照组一、凡士林纱布为对照组二,观察其创面用三种敷料覆盖后促进愈合、控制伤口感染方面的疗效和安全性。2、采用消化分离法进行Fbs的原代培养,通过形态学和免疫组织化学法鉴定Fbs。第6代纯化的Fbs随机分为2组,1组与木醋杆菌纤维素敷料共同培养,1组设为空白对照组。培养24、48、72h后,经MTT法检测每孔的光吸收值(OD值)。应用流式细胞仪检测细胞周期变化,应用碱水解法检测培养液中羟脯氨酸的含量变化,检测2组培养72h后的培养上清液中Ⅰ型与Ⅲ型胶原含量及其比值的变化。相应ELISA试剂盒分别检测24h及48h后细胞上清液中TGF—β1、IFN—γ及bFGF的分泌含量变化。四、研究结果1、从术后第1天起每隔4天测定一次细菌计数,结果表明用药后第5d、9d、13d及17d的细菌累计抑制率实验组较对照组一和二明显高。经创面直接观察和病理组织切片观察,发现实验组创面比对照组一和二用药后创面愈合时间提前3-5天。Axcd对动物血化验和肝、肾功能均无明显影响,未见局部致敏和全身症状。2\Axcd加入到Fbs中培养24、48、72h后,经MTT法检测,实验组与空白对照组OD值比较差异有显著性统计学意义(P<0.05)。细胞周期时项分析结果显示实验组细胞周期S+G2/M期细胞百分率及增殖指数PI值较空白组增高(P<0.05),并且在不同时间段时其成纤维细胞培养液中羟脯氨酸含量均明显高于空白组(P<0.05)。实验组与空白组比较,培养上清液中Ⅰ型与Ⅲ型胶原含量增高,Ⅰ型与Ⅲ型胶原比值下降(P<0.05)。相应ELISA试剂盒检测TGF—β1含量：培养24h细胞TGF—β1分泌含量空白对照组为89.27±9.85 pg/ml,实验组为134.75±12.40 pg/ml,48h细胞TGF—β1含量空白对照组为128.48±10.53pg/ml,实验组为188.32±15.68pg/ml。ELISA检测IFN—γ分泌含量：培养24 h细胞IFN—γ含量空白对照组为108.35±10.24pg/ml,实验组为73.56±8.73pg/ml,48 h细胞IFN—γ含量空白对照组为130.25±14.50pg/ml,实验组为85.78±13.62pg/ml。ELISA检测bFGF分泌含量：培养24 h细胞bFGF含量空白对照组为30.53±6.32pg/ml,实验组为48.35±8.87pg/ml,48 h细胞bFGF含量空白对照组为42.65±5.45pg/ml,实验组为65.20±7.36pg/ml。五、研究结论Axcd所具有的柔软、光滑、湿润、制作随意性及紧密贴合性等特点,使其应用范围大大增加,并且减少了感染性创面细菌的侵入。其对创面SA和EC的抑制率在起效时间和强度上都优于其它两组敷料,能有效促进HSFb的增殖和胶原的合成,对体外培养的成纤维细胞TGF—β1及bFGF分泌含量有显著上调作用,对IFN—γ分泌含量有下调作用,可能与其促进创面愈合的机制有关。Axcd与其他敷料相比能更好的抑制创面细菌生长、有效促进创面愈合,是目前临床上较为理想的创面敷料。更多还原

【Abstract】 BackgroundThe healing of wound surface is a complicated courses.Fibroblast (Fbs),cytokine and procollagen (PC) and some part of cell growth factor are the key point among the wound surface reparation cource.Fbs participate the healing whole course after cutaneous deficiency,then the most important biology changes are the strengthen of Fbs’s reproduct-ive activity and increace of collagen protein synthetic and the content changes of cytoki-ne.Wound surface can be accelerate healing through improve Fbs’s bionomics. Both the organism’s metabolism features and milieu exterieur of wound surface have great significance to healing.Sterility of wound surface and moisten of surroundings are all the key point for wound healing.According to the clinical investigation,wound surface need wrapped by dressing and will not be exposed in the surroundings directly. So the healing of wound surface is influenced by surroundings of dressing. Now,people found dressing which synthesized by Bacterial Cellulose (BC) overcome the upper defects and also have some good features,including high water ratio,high hydrophilism,high tensile strength and high biocompatibility,which can refrain the growth of wound surface bacterium and promote wound surface healing et al.The using of BC will have widespread application perspective in medicine and biomedical engineering territory,and the study of BC is a hot spot biomaterial investigation nowadays.Meanwhile BC will have great development as a neotype biosynthesis dressing in medical world.Axcd’s main composition is Bacterial Cellulose by produced Acetobacter xylinum (Ax) which contain in Acetobacter aceti (Aa).Its construction identical to plant cellulose which belong to native cellulose.Axcd have outshine others in many dressings because of its distinctive merits.Axcd is the main study object in this study,and its main study content include two part.The first study include pro-tow part (study of animal experiment) during master period.The influence of Fbs and PC and some cell growth factor are the mose emphasis study.In order to approach the mechanism of promote healing by Axcd.Objective1. To understand Axcd’s therapeutic efficacy and safety of operation wound surface(depth reach deep layer of dermis), including study its mechanism action of refrain bacterium from infected wound surface and ability of hasten wound surface healing.2.To approch human skin fibroblast’s (HSFb) proliferation and synthetical ability of collogen and the excete content changes of transforming growth factor-β1 (TGF-β1) and interferon gamma (IFN-γ) and basic fibroblast growth factor (bFGF) by Axcd,that may be related with promote wound surface healing.Contents and methods1.9 small pigs were divided into 3 groups random completely,each groups have 3 pigs,and 6 models of animal infection wound surface were made on every pigs,every wound surface were inoculated combining bacterium including Staphylococcus aureus (SA)and Escherichia coli(EC).Experimental group,control group 1 and 2 include Axcd ,antibiosis gauze which contain neomycin-berberine solution and petrolatum gauze respectively.To observe their security and therapeutic effect of hasten wound surface and ability of control wound infection. 2. Fibroblasts were obtained from the digested and aparted method of primary culture and identified by morphology and immunohistochemistry.The sixth purified fibroblasts were divided into two groups at random and cultured with Axcd and sham respectively. MTT methods was used to measure OD number of each hold after being cultured for 24,48,72h respectively. Cell cycle was measured by flow cytometry.The hydroxyproline in the cultural medium was determined by alkaline hydrolysis.The amount of typeⅠandⅢprocollagen(PCⅠ,PCⅢ)in the supernatants and the PCⅠto PCⅢratio were determined. The excrete content of TGF-β1 and IFN-γand bFGF in cultured human fibroblasts was detected by relative ELISA kits.Results1.After made animal infection wound surface models,determine the bacterial count every other 4 days after the first day of operation,the consequences indicate that total bacterium inhibition ratio after the 5d,9d,13d and 17d respectively.The effect of Axcd’s total bacterium inhibition ratio is better compare with other two groups.The healing time of wound surface which use Axcd is ahead of other two groups about 3-5d by obersavation of ordinary and pathological section.Axcd have not influence to animal blood chemical examination,liver and renal function,local sensitization and general symptom.2. Fibroblasts was cultured with Axcd,compared with blank groups,OD numbers were obviously different (P<0.05). Employing the flow cytometry technique we observed the cell cycle of S+G2/M stage and the proliferation index (PI) were increased (P<0.05).Co-mpared with the other group the hydroxyproline in the cultural medium was increased (P<0.05),when the cells were stimulated by Axcd.To compare the sham group,PC I, PCIII amounts (P<0.05),and decreased ratio of PC I to PCⅢ(P<0.05).ELlSA kits: TGF-β1 content of cell cultured with sham in 24hr is 89.27±9.85 pg/ml, with Axcd is 134.75±12.40 pg/ml, and with sham in 48hr is 128.48±10.53 pg/ml, with Axcd is 188.32±15.68pg/ml. IFN-γcontent of cell cultured with sham in 24hr is 108.35±10. 24pg/ml, with Axcd is 73.56±8.73 pg/ml, and with sham in 48hr is 130.25±14.50 pg /ml, with Axcd is 85.78±13.62pg/ml. bFGF content of cell cultured with sham in 24hr is30.53±6.32 pg/ml, with Axcd is 48.35±8.87 pg/ml, and with sham in 48hr is 42.65±5.45pg/ml, with Axcd is 65.20±7.36pg/ml.ConclusionAxcd have great applicate scopes because of many features,such as slick,moisten, and can make various kinds shapes according to wound surface’s position and size,and have the ability reduce invation of bacterium in wound surface.The inhibiton ration of Axcd outweigh other two groups to SA and EC.Axcd have the better bacteriostatic action and ability of promote wound surface healing compare with other dressings. Axcd can enhance the growth of HSFb and the collage synthesis and content of TGF-β1 and bFGF of cultured human fibroblasts,but the content of IFN-γis down regulation.It might be beneficial to ranulation tissue formation during wound healing.All in all,it is the ideal wound dressing in clinical at present.更多还原