【作者】 赵丽；

【导师】 董福生； 任贵云；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2004， 硕士

【摘要】 目的：研制表皮生长因子-透明质酸复合膜，通过动物实验观察其对皮肤切口愈合的影响，探讨表皮生长因子-透明质酸复合膜抑制瘢痕的作用机制，为临床应用提供实验依据。方法：1. 表皮生长因子膜、透明质酸膜及表皮生长因子-透明质酸复合膜的制备：透明质酸、表皮生长因子、聚乙烯醇配制前以分析天平称量。在超净台内，先用无菌生理盐水配制聚乙烯醇溶液，待完全溶解后，再分别放入表皮生长因子、透明质酸、表皮生长因子和透明质酸，用玻璃棒搅拌配制成均匀的凝胶，然后将凝胶平铺于玻璃板上，置低温干燥箱干燥后，制备成表皮生长因子膜，透明质酸膜，表皮生长因子-透明质酸复合膜，再用同样的方法制成聚乙烯醇膜，在超净台内切割成长5.5cm、宽1.0cm的膜。2. 72只SD雄性大鼠，随机分成四组，全麻下于背部脊柱两侧旁1cm处，平行脊柱分别做长约5cm的切口，切开皮肤、皮下组织，深达肌层，3～0丝线缝合。将表皮生长因子-透明质酸复合膜，表皮生长因子膜，透明质酸膜和聚乙烯醇膜（对照）无菌生理盐水浸湿后分别贴于四组动物的伤口，外用无菌纱布包扎。每日更换外敷膜1次，至术后14天。分别于术<WP=4>后3、5、7、9、14、21天每组处死3只动物，留取标本，做HE染色，masson染色，EGF、HA免疫组化染色及PCIII含量测定。结果：1.大体观察：术后3日，聚乙烯醇膜聚乙烯醇膜对照组，部分伤口有血迹，结痂，不易清洁，创周红肿；EGF-HA复合膜治疗组，伤口清洁，渗液较少，部分伤口有少许结痂，炎症反应轻，创周无明显红肿；EGF膜治疗组，表皮愈合倾向明显；HA膜治疗组类似于EGF-HA复合膜治疗组。术后5日，聚乙烯醇膜聚乙烯醇膜对照组，伤口大部分结痂，创周稍红肿，缝线处反应较重；EGF-HA复合膜治疗组表皮层愈合，无红肿，部分伤口有轻微缝线反应；EGF膜治疗组和HA膜治疗组类似于EGF-HA复合膜治疗组。术后7日，聚乙烯醇膜聚乙烯醇膜对照组，部分伤口处仍有血痂，部分缝线脱落，部分伤口仍有较重缝线反应；EGF-HA复合膜治疗组伤口愈合良好，表面清洁干净，部分缝线脱落；EGF膜治疗组和HA膜治疗组类似于EGF-HA复合膜治疗组。术后9、14、21天，聚乙烯醇膜聚乙烯醇膜对照组，伤口愈合良好，可见到明显的愈合线；EGF-HA复合膜治疗组伤口愈合良好，无明显的愈合线；EGF膜治疗组和HA膜治疗组类似于EGF-HA复合膜治疗组。2.HE染色观察：术后3天，聚乙烯醇膜对照组，表面见血凝块和炎性渗出物，表皮不连续，真皮切口未愈，裂隙较大，切口两侧可见成纤维细胞和炎细胞浸润；复合膜组，表皮层愈合，向真皮层轻微凹陷，棘细胞层增生，细胞<WP=5>层次清楚，真皮层部分愈合，成纤维细胞较多；EGF膜治疗组，表皮层愈合，棘细胞层增生，真皮层少部分愈合，大量成纤维细胞，少量炎细胞浸润；HA膜治疗组，表皮未完全愈合，真皮愈合优于EGF膜治疗组和聚乙烯醇膜对照组，次于复合膜组。术后5天，聚乙烯醇膜对照组，表皮层愈合，角化层形成，可见角质颗粒，棘细胞层增生明显，真皮少部分愈合，仍可见裂隙，裂隙上可见增生的成纤维细胞，炎细胞浸润；复合膜组，表皮愈合，角化程度较聚乙烯醇膜对照组轻，角质颗粒少，真皮层基本愈合，可见较多增生的成纤维细胞；EGF膜治疗组，表皮愈合，较平整，真皮未完全愈合，大量成纤维细胞和炎细胞浸润；HA膜治疗组，表皮层愈合，真皮愈合优于EGF膜治疗组和聚乙烯醇膜对照组，次于复合膜组，可见较多成纤维细胞。术后7天，四组表皮层均愈合，聚乙烯醇膜对照组，真皮未完全愈合，真皮成纤维细胞胞体粗大，成层状聚集排列，与愈合线垂直，有炎细胞浸润；复合膜组，真皮层愈合，成纤维细胞胞体较小，胞核细长，疏松排列，较规则；EGF膜治疗组，接近复合膜组，优于聚乙烯醇膜对照组；HA膜治疗组，真皮层未完全愈合，其它情况同复合膜组。术后9天，聚乙烯醇膜对照组，表皮有凹陷，角化较重，胶原排列有明显的方向性，较粗；复合膜组，表皮平坦，胶原束较小，在真皮浅层处排列接近正常组织；EGF膜治疗组、HA膜治疗组表皮基本平坦，胶原束较小，排列相对疏松。术后14、21天，聚乙烯醇膜对照组角化明显，胶原<WP=6>排列成束，致密，与切口线垂直；复合膜组、EGF膜治疗组、HA膜治疗组角化不明显，表皮层厚度基本正常、平坦，胶原束较小，排列相对疏松。3.masson染色：术后7天，聚乙烯醇膜对照组，绿色着色带较宽，切口两侧胶原排列相对致密胶原束粗大，与切口垂直；复合膜组，绿色着色带较窄，胶原束纤细，排列相对疏松，部分切口处胶原排列接近正常组织；EGF膜治疗组、HA膜治疗组，绿色着色带宽度介于复合膜组和聚乙烯醇膜对照组之间，新生胶原纤维量较少，胶原束纤细，排列相对疏松。4.HA免疫组化染色观察：复合膜组，3、5、7天表皮上层染色均为强阳性，第9天为阳性，表皮下层染色由阳性转为弱阳性，真皮层由强阳性转为阳性；EGF膜治疗组， 3天时表皮层强阳性，5、7天弱阳性，9天阴性，到第9天时，真皮层染色也由强阳性转为阳性；HA膜治疗组，表皮层染色除3天为强阳性，其它为阳性，第9天，真皮染色也由强阳性转为阳性；聚乙烯醇膜对照组，除7、9天基底层染色阳性外，表皮层均为阴性，3天真皮染?更多还原

【Abstract】 Objective:To develop the compound group of epidermal growth factor (EGF) and hyaluronic acid (HA),to observe the mechanism and the effect of the compound group on the wound healing of rats.To provides an experimental basis for clinical application.Methods:1.Preparation of the compound group made from EGF and HA:Under the asepsis conditon,the compound group of EGF and HA was completed.In the same condition ,the other three groups, EGF group ,HA group and polyvinyl alcohol group were completed.2.Seventy-two male SD rats were randomly divided into four groups.Under general anesthesia ,two 5cm cuts were made on two sides,parallel to the spine with the deep from epidermis to the muscle layer.Then the cut was sewn with 3~0 suture. The cuts of four groups were applided respectively with compound group of EGF and HA, EGF group, HA group and polyvinyl alcohol group(control group),and then bound up with sterile gauze.On the 3rd,5th,7th,9th,14th and 21st days after the surgery,3 rats in each group were killed to be <WP=10>collected to specimens.The expression of EGF,HA in skin was detected with Streptavidin/Peroxidasa(SP) immunohistichemical methods. Under light microscope (LM),the histologic change was observed and the content of PCIII was determined.Result:1.General observation: After surgery ,in three treatment groups the wound were clean,with little seepage on the surface of the wound,slight inflammation,no obvious red and swollen around the cut,and the healing line was not clear.In the control group ,there were escharosis on the surface of the wound, much more seepage,the wound was hard to clean,red and swollen around the cut and there was an obvious healing line after the wound healing. 2.Observation under LM:After surgery ,compared with cotrol group,in compound group group,there was few inflammatory cell infiltrated on both sides of derma incision;the proliferation of acanthoid cell layer was not obvious;the FBs with a bit small body and slim nucleus spread loosely;quite few new collagen fibers could be seen on both sides of the cut,with comparably small bunches of loosely spread collagen,and they were slight keratinized.EGF group group and HA group group were superior to control group,inferior to group compound group group.3.Distribution of HA in the skin: the upper part of the epidermal and the dermis of compound group group showed HA stain strong positive on the 3rd,5th,7th day,and <WP=11>turned to positive on the 9th day;the below part of the epidermal turned from positive to weak positive. The epidermal of EGF group group showed HA stain strong positive on the 3rd day,and turned to negative on the 9th day,and the dermis turned from strong positive to positive.HA stain of HA group group was similar to compound group group.The below part of the epidermal of control group showed HA stain positive on the 7th,9th day,showed negative on the 3rd,5th day;the dermis showed HA stain weak positive on the 3rd day,turned to negative on the other days. 4.Distribution of EGF in the skin: the upper part of the epidermal of compound group group showed EGFstain strong positive on the 3rd,5th,7th ,9th day; the below part of the epidermal and fibroblast and hair follicle showed EGFstain positive on the 3rd,5th,7th day,and turned to weak positive on the 9th day.The upper part of the epidermal of EGF group group showed EGFstain strong positive, the below part of the epidermal and fibroblast and hair follicle showed EGFstain positive.The epidermal of HA group group showed EGFstain positive. The epidermal of control group showed EGFstain positive on the 3rd day, the hair follicle weak positive; fibroblast and hair follicle showed EGFstain positive on 5th,7th and 9th day.5.PCIII content: the content of compound group group was significant higher than control group on all point time (P<0.05);the content of <WP=12>PCIII in EGF group group and HA group group was obviously higher than control group on all days except the 21st day(P<0.05);the content of PCIII in compound group group was obviously higher than EGF group grou更多还原