他克莫司抑制兔耳瘢痕增生的作用及其机制研究:他克莫司能修复皮肤吗

　　[摘要]目的：他克莫司是临床广泛应用的一种药物，其对增生性瘢痕是否产生作用并无相关报道，为此提出并证实了他克莫司可以抑制兔耳瘢痕增生。方法：建立兔耳增生性瘢痕模型，选10只新西兰大耳白兔双耳腹侧用打孔器制作直径1cm圆形创面，伤后14天创面上皮化后给药，每只兔左耳为空白对照组涂等剂量凡士林软膏，右耳为他克莫司治疗组。分别在伤后14天、21天、28天、35天和49天采集标本，行HE染色，观察形态学差异；Real-time PCR检测纤维化相关因子TGF-β1、TGF-β2、Collagen-α1等的表达。结果：HE染色可见他克莫司组成纤维细胞数量及胶原纤维较对照组明显减少，PCR结果可见TGF-β1、TGF-β2及Collagen-α1表达较对照组在各时间点均减少。结论：实验组较对照组瘢痕明显减轻，证明他克莫司显著抑制兔耳瘢痕增生，可作为临床上治疗及预防瘢痕增生的全新疗法。
　　[关键词]瘢痕免疫；他克莫司；兔耳模型；增生性瘢痕
　　[中图分类号]R619+.6[文献标识码]A [文章编号]1008-6455（2011）01-0068-03
　　
　　Tacrolimus inhibit scar formation in rabbit ear hypertrophic scar model
　　ZENG Hai-feng,HAN Xiao-xia,LIU Bei,LU Kai-hua,GUO Shu-zhong,XIA Wei
　　(Institute of Plastic Surgery,Xijing Hospital,The Fourth Military Medical University,Xi"an710032,Shaanxi,China)
　　
　　Abstract:ObjectiveTacrolimus was widly used to inhibit rejection of allograft and therapy for various cutaneous diseases; however, there is no report about its effection on hypertrophic scar.Methods10 New Zealand White rabbits were randomly divided into two groups with each ear created four wounds use 1cm diameter punch biopsy at standardised locations on the ventral surface. The right ear wounds were taked by tacrolimus ointment 0.1g per wound twice daily started on day 14 postwounding, the left ears were treated with vaseline at the same time. Experimental and control wounds were harvested on days 14, 21, 28, 35 and 49 post wounding, examined histologically to quantify scar elevation index(SEI). Hematoxylin-eosin staining was performed to reveal differences in scar morphology.Real-time PCR was adopted to measure the variation tendency of the TGF-β1,TGF-β2 and Collagen-α1. ResultsSEI was significantly reduced after tacrolimus treated versus the applied of Vaseline (P 　　1.3.2 标本采集：术后第14、21、28、35及49天每个时间点空气栓塞法随机处死2只兔子，分别沿创面边缘0.5cm处环行切开，切取含创面的兔耳组织，取材后的兔子即退出实验。每个标本沿直径切开，分为两半，其中一半即刻以4%多聚甲醛固定，常规石蜡包埋，垂直于创面瘢痕最凸起处纵行切片，每张切片厚度为5μm，行苏木素-伊红染色（HE）染色；另一半-80℃冰箱保存备用。
　　1.3.3 计算瘢痕指数（Scar elevation index，SEI）：光镜下观察HE染色切片，用测微尺测出瘢痕最高点至耳软骨表面的垂直距离取值为a，瘢痕周围正常皮肤至耳软骨表面的垂直距离取值为b，使用公式SEI= a/b，计算出瘢痕增生指数。
　　1.3.4 Real-time PCR检测：如表1所示设计合成引物，Tm值均设定为60℃。按标本顺序分别切取80mg瘢痕组织，提取总RNA，分光光度计测定RNA浓度，纯化并反转录为cDNA，扩增并检测产物片段大小，-20℃保存。收集所有标本后行Real-time PCR检测纤维化相关因子TGF-β1、TGF-β2及Collagen-α1表达及变化。
　　1.4 统计学分析：实验结果均以均数±标准差（x±s）表示，应用SPSS11.5软件进行t检验处理数据，P