猪复方壳多糖组织工程皮肤替代物的构建  

Construction of tissue engineered dermal substitute of pig composite Chitosan

作　　者：吴国选[1] 伍津津[1] 朱堂友[1] 鲁元刚[1] 毕建军[1] 雷霞[1] 

机构地区：[1]解放军第三军医大学大坪医院皮肤科,重庆市400042

出　　处：《中国临床康复》2006年第13期67-69,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家八六三计划资助项目(2001AA216041;2003AA205020)~~

摘　　要：目的:采用组织工程方法,在鼠尾胶原中加入壳多糖及培养的贵州小型香猪成纤维细胞与角质形成细胞,进行猪复合壳多糖组织工程皮肤替代物的构建。方法:实验于2001-09/2002-12在解放军第三军医大学大坪医院完成。①实验所需皮肤标本取自3月龄雌性贵州小型香猪4只,取新鲜全层皮肤于D-Hanks平衡盐液浸泡,将标本剪成0.3cm×2.0cm皮条,4℃消化14～16h后备用。②消化后的标本分离表皮和真皮,分别用于角质形成细胞和成纤维细胞的分离培养。以传代培养的猪角质形成细胞和成纤维细胞作为种子细胞,以加入壳多糖、糖胺聚糖等天然细胞外基质的鼠尾胶原作为真皮基质,于钢丝网上进行气液界面培养,苏木精-伊红染色观察培养物的结构。结果:①角质形成细胞的培养情况:接种的猪角质形成细胞于培养第2天可见细胞贴壁生长,形成细胞集落,15d左右融合成片,呈“铺路石样”。②真皮成纤维细胞的培养情况:消化法接种的猪成纤维细胞培养两三天后即见细胞贴壁生长,10d左右基本融合,呈旋涡状生长。③猪真皮基质浸没培养的变化情况:刚制备的真皮基质呈红色,倒置显微镜下凝胶各层面均可见尚未恢复形态的成纤维细胞。3d后随着凝胶的收缩,人工真皮逐渐与培养板壁和底分离,透光度下降,不易见到凝胶中的细胞。真皮基质的收缩发生于第3天,第6～12天真皮基质的直径最大收缩到45%左右,以后逐渐减慢。④猪的复合皮肤替代物的培养情况:培养第3天在真皮基质上加入传代后的猪角质形成细胞,其上可见薄的角质形成细胞层。浸没培养6d后角质形成细胞层色素逐渐加深。气液界面培养14d后的人工皮肤有一定的弹性、韧性以及抗拉力和抗挤压力。⑤复方壳多糖组织工程皮肤的光镜观察结果:镜下表皮可明显区分基底层、棘层和角化不全的角质层,共有7～12层细胞。真、表AIM： Fibroblast and keratinocyte of cultured Guizhou miniature pigs and chitin are added into murine tail collagen to construct composite Chitosan tissue engineering dermal substitute with tissue engineering method. METHODS： This experiment was conducted in Daping Hospital, Third Military Medical University of Chinese PLA from September 2001 to December 2002. ① Dermal samples of the experiment were chosen from four 3-month-old female Guizhou miniature pigs .Fresh full-thickness skin was dipped in D-Hanks balance salt solution, then the samples were cut into 0.3 cm×2.0 cm pieces and digested at g ℃ for lg to 16 hours for use. ② Epidermis and dermis of the sample were isolated after digestion, then they were used for the isolation and culture of keratinocyte and fibroblast, respectively. Pig kerafinocyte and fibroblast after passage culture were used as seed cells, and murine tail collagen with chitin and glycosaminoglycan and other natural extracellular matrix as dermal matrix, then they were put on the steel wire screen for air-liquid interface culture. Then, the structure of culture was observed with hematoxylin-eosin staining. RESULTS： ① Culture of keratinocyte： On the 2^nd day of the culture of inoculated pig keratinocyte, cells grew adherently and formed cell colony, and converged into pieces on the 15^th day, presenting ＂slabstone-like＂. ② Culture of dermal fibroblast： pig fibroblasts inoculated with digestion method were cultured for 2 to 3 days, and cells grew adherently and converged basically on about 10 days, presenting whirlpool shape. ③ Change of submergence of pig dermal matrix： Dermal matrix which was prepared immediately presented red. Unrecorvered fibroblasts in morphology were found in each gel layer under an inverted microscope. 3 days later, with the shrinkage of gel, synthetic derma gradually isolated from the wall and bottom of the culture plate, transparency was decreased and the cells in the gel were not easy to be found. Shrinkage of dermal matrix occurred

关 键 词：生物医学工程 皮肤 人工 真皮 细胞 培养的 壳多糖 猪 

分 类 号：R318[医药卫生—生物医学工程]