【作者】 任贵云；

【导师】 董福生； 石培凯；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2003， 硕士

【摘要】 目的：研制透明质酸外敷膜，通过动物实验观察其对外科手术切口愈合的影响，探讨透明质酸外敷膜抑制瘢痕的作用机制，为临床应用提供实验依据。方法：1.透明质酸外敷膜的制备：透明质酸、羧甲基纤维素钠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羧甲基纤维素钠溶液，待完全溶解后，再放入透明质酸，用玻璃棒搅拌配制成均匀的透明质酸凝胶，然后将凝胶平铺于玻璃板上，置低温干燥箱干燥后，在超净台内切割成长5.5 cm、宽1.0 cm的透明质酸外敷膜。2.48只SD雄性大鼠，随机分为8组，实验采用自身对照设计，左侧为治疗组，右侧为对照组。大鼠在全麻下于背部脊柱两侧旁1㎝处，平行脊柱分别做长约5 cm的切口，切开皮肤、皮下组织，深达肌层，3~0丝线缝合。治疗组伤口表面贴敷透明质酸膜后，无菌纱布包扎；对照组伤口表面贴敷生理盐水纱条，无菌纱布包扎。每日换药一次，治疗组用药至术后第14天停止用药。分别于术后1、3、5、7、9、14、21、28天处死一组动物，留取标本，光镜、电镜观察组织形态学变化及透明质酸含量测定。结果：1.大体观察：术后1、3日；治疗组：伤口较清洁，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。对照组：部分伤口有结痂，部分伤口渗液较多，不易清洁；创周红肿。术后5、7日，对照组炎症<WP=4>减轻，但缝线反应较重；治疗组仅有轻微缝线反应。术后9、14、21、28日，对照组伤口愈合后可见明显的愈合线，而治疗组愈合线不明显。2. HE染色观察：术后1日，对照组，表面见血凝块及炎性渗出物，表皮不连续，真皮切口两侧见较多炎细胞浸润；治疗组，表面见透明质酸和炎性渗出物组成的嗜伊红物，真皮切口两侧炎细胞浸润较少。术后3日，对照组棘细胞层增生明显、肥大，排列较紊乱，炎细胞浸润明显；治疗组棘细胞层增生不及对照组明显，层次清楚。术后5日，两组表皮均已愈合，对照组角化层形成，真皮浅层已愈合，深层近皮下组织处裂隙较宽；治疗组角化程度较轻，真皮愈合较慢，浅层至深层裂隙较一致，炎细胞浸润较明显。术后7日，两组皮肤全层已愈合，对照组角化程度较重，真皮深层炎细胞呈灶状聚集；治疗组基底层细胞排列较整齐，真皮深层炎细胞浸润较对照组为轻。术后9日，对照组成纤维细胞胞体较大，胞核椭圆形，成层壮聚集排列；治疗组成纤维细胞胞体较小，胞核细长，疏松排列。术后14、21、28日对照组角化较重，胶原排列成束、致密，与切口线垂直；治疗组角化较轻，表皮层厚度基本正常、平坦，胶原束较小，排列相对疏松。3.masson染色观察：术后5、7天，治疗组切口两侧可见新生胶原纤维量较少，胶原束较小，排列相对疏松；对照组切口两侧可见量较多的新生胶原纤维，胶原束较粗大，胶原束与切口垂直排列，相对较致密。4.透射电镜：术后3日时，对照组为功能活跃的成纤维细胞；治疗组成纤维细胞功能活跃，但可见轻度褪形性变。术后7日，对照组成纤维细胞周围可见大量胶原纤维分布；治疗<WP=5>组成纤维细胞发生褪形性变，胞核圆形或卵圆形，核染色质丰富、边集呈高电子密度团块，胞浆内可见大小不等圆形高电子密度斑块，细胞周围胶原纤维较少，可见中等电子密度均匀基质样物质。术后14天，对照组表皮细胞胞体小，细胞连接紧密，细胞间桥多见、排列混乱；治疗组可见正常成纤维细胞及有褪形性变的成纤维细胞，周围有大量胶原纤维，从横断面见，纤维较细小、排列疏松；表皮细胞胞体较大，细胞间桥较少、连接较疏松。5.成纤维细胞计数：术后1、3、5天时，治疗组成纤维细胞数量较对照组多(P<0.05)；术后7、9、14天时治疗组和对照组成纤维细胞计数无明显差异(P>0.05)。炎细胞计数：术后1、3、5日时，对照组伤口浸润的炎细胞数目较治疗组多 (P<0.05)，浸润的炎细胞在术后1日时以中性粒细胞为主，术后3、5日时以淋巴细胞为主。术后7、9、14天时，对照组慢性炎细胞较治疗组多(P<0.05)。6.HA含量分析：两组间同一时间点比较，术后1天，两组伤口局部透明质酸含量均较高，无明显差异(P>0.05)；术后3、5天时，治疗组局部透明质酸含量高于对照组(P<0.05), 术后7、9、14天治疗组和对照组伤口局部透明质酸含量，无明显差异（P>0.05）。各组内不同时间点比较，治疗组术后1、3天局部透明质酸含量较高，无明显差异(P>0.05)，但明显高于术后5、7、9、14天时伤口局部透明质酸含量(P<0.05)；而对照组术后1天伤口局部透明质酸含量明显高于术后3天时局部透明质酸含量(P<0.05)，术后5、7、9、14天的透明质酸含量与术后3天时无明显差异(P>0.05)。结论：1.初步研究制备了高分子量的HA外敷膜。2.<WP=6>高分子量HA外敷膜使HA渗透进入皮肤，抑制瘢痕形成。3.HA外敷膜在创伤愈合早期有较强的透皮作用；随着表皮层愈合，HA透皮作用减弱。4.HA外敷膜对外科手术切口具有一定的抑制炎症反应的作用。5.HA外敷膜在伤口愈合过程中，通过对成纤维细胞的作用，从而影响胶原的合成和沉积。6.HA外敷膜可促使表皮层愈合并延缓角化层形成。更多还原

【Abstract】 Objective: To develop the hyaluronic acid (HA) film, to discuss the mechanism and to observe the effect of the external using film on the wound healing of rats. To provide an experimental basis for clinical application. Methods: 1.Preparation of the external using film made from HA: Under the asepsis condition, the external using film of HA was completed. 2.Forty-eight male SD rats were randomly divided into 8 groups. Meanwhile, the experiment was designed self-contrast, the left of rat was used as treatment group and the right of rat was used as control group. 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 cut of treatment group was applied with HA film and then bound up with sterile gauze; and the control applied with NS gauze and then bound up also with sterile gauze. On the 1st, 3rd, 5th, 7th, 9th, 14th, 21st and 28th days after the surgery, 6 rats were killed to be collected to specimens. Under light microscope (LM) and transmission election microscope (TEM), the histologic change was observed and the content of HA was determined.<WP=8>Results: 1.General observation: After surgery, in the treatment group, the wound was clean, with little seepage on the surface of the wound, slight inflammation, no obvious red and swollen around the cut, and the healing line in the treat group 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 the control group, there was few inflammatory cells infiltrated on both sides of the 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. 3.Observation under TEM: After surgery, compared with the control group, there was a slight degeneration in the FBs of the treatment group, with round or ovate nucleus, and rich chromatin gathered high electron density clots. The epidermic cells were big, with few loosely linked intercellular bridges. 4.The numbers of FBs: On the 3rd and 5th day, there were more FBs in the treatment group than those in the control group (P<0.05), and there was no significant difference between the two groups at different time (P>0.05). The numbers of inflammatory cells: There were more inflammatory cells in<WP=9>the control group than those in the treatment group (P<0.05). 5.HA content: On the 1st day after the surgery the content of HA in both groups was no statistical difference (P>0.05). On the 3rd, 5th, the HA content was higher in treatment group than that in the control group (P<0.05), and there was no significant difference between the two groups at different time(P>0.05). In the treatment group there was no significant difference of HA content on the 1st and 3rd(P>0.05), on the 3rd day, the content of HA in control group began to drop down (P<0.05).Conclusions: 1.The external using film of HA was made in this study. 2. The external using film made from high molecular weight HA could repress the formation of scar, for HA could infiltrate into dermis. 3.HA external using film had powerful infiltrating activity at early stage of wound healing. With the healing of the epidermis layer, the infiltrating activity of HA began to decrease.4.The inflammation after wound could be inhibited by HA film. 5.During the healing, the HA film could influence the synthesis and deposition of collagen by affect FB. 6.HA could accelerate the healing of epidermis while delayed the formation of keratinization layer.更多还原