SDF-1对创缘表皮干细胞定向趋化及促创面愈合效应的研究_表皮干细胞

　　[摘要]目的:观察干细胞趋化因子SDF-1在创面愈合过程中调控表皮干细胞定向迁移促进创面愈合的过程。方法:制作大鼠背部全层皮肤缺损模型,随机分为三个实验组:①SDF-1组;②AMD3100(CXCR4受体的拮抗剂)组;③空白对照组。分别于致伤后1天、3天、5天、7天和12天照相计算伤后不同时间创面占初始创面的百分比并取材。运用HE染色和免疫组化技术观察创缘表皮干细胞定向迁移分化的特点,寻找其规律。结果:与其他两个处理组相比,SDF-1处理组愈合时间缩短,其创缘皮表皮基底层细胞分裂增殖旺盛,创缘表皮干细胞数目明显多于其他两组。结论:在创面愈合过程中,SDF-1可以调控表皮干细胞向创缘迁移,加速创面上皮化。
　　[关键词]基质细胞衍生因子-1;表皮干细胞;创面愈合
　　[中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455(2010)08-1156-05
　　
　　SDF-1 accelerates wound healing via recruitment of epidermal stem cells
　　ZHANG Yi1,GONG Xi-yuan1,SUN Xue-wu1,QIU Xiao-dong1,GUO Dan-feng1,ZHANG Guan-jun1,HAN Ji-qin1,
　　CAO Chuan2
　　(1.Department of Plastic Surgery,Jinan Central Hospital of Shandong University,Jinan 200013,China; 2.Department of Plastic Surgery,Southwest Hospital,Third Military Medical University,Chongqing 400038,China)
　　
　　Abstract:ObjectiveTo investigate the stem cell chemokine SDF-1 in the regulation of wound healing migration of epidermal stem cells to promote wound healing process.MethodsRat dorsal full-thickness skin defects were randomly divided into three experimental groups: ①SDF-1 group; ②AMD3100 (CXCR4 receptor antagonist) group;③control group. 1 day,3 days,5 days,7 days and 12 days after injury camera calculates the wound at different times,and total percentage of the initial wound drawn. Using HE staining and immunohistochemical observation of a margin of epidermal migration and differentiation of stem cell characteristics,to find its own rules. ResultsCompared with the other two treatment groups,SDF-1 treatment to shorten healing time,the margin of skin epidermal basal cell division and proliferative margin of the number of epidermal stem cells was more than the other two groups.ConclusionIn the wound healing process,SDF-1 can regulate epidermal stem cells migrate to the wound edge,accelerate wound epithelization.
　　Key words:SDF-1; epidermal stem cells; wound healing
　　
　　位于表皮基底层的表皮干细胞是皮肤组织发生、创面修复与改建的关键细胞[1-2]。β1整合素和角蛋白19(Keratin-19 K19)染色呈阳性为其鉴别手段[3-4]。研究发现,创面修复过程中表皮干细胞可向创缘定向迁移[5]。理论上讲,这种迁移趋势是由趋化因子与其靶细胞膜上特异性受体结合,从而调控这些细胞的迁移、增殖和分化来发挥促修复作用。基质细胞衍生因子-1(stromal cellderived factor-1 SDF-1)和其受体CXCR4广泛表达于多种组织细胞,对一些组织器官发育和损伤后修复再生起重要调节作用。我们前期工作发现,细胞免疫荧光染色显示表皮干细胞表面CXCR4受体表达呈阳性,应用流式细胞仪检测表皮干细胞表面CXCR4抗原阳性率为43%,且创面在愈合过程中的创缘组织匀浆内的SDF-1表达量明显增多[6],由此我们猜测在创面愈合过程中,SDF-1/CXCR4轴有可能参与调控创缘表皮干细胞向创面内迁移从而促进创面修复过程。
　　
　　1材料和方法
　　1.1 主要设备及试剂:石蜡切片机,德国LEICA公司生产;实验摄像显微镜,日本Olympus生产。
　　AMD3100由美国Sigma公司提供;SDF-1因子由美国peprotech公司提供;K19抗体(Keratin19 Ab-1)由NeoMarker公司提供(小鼠来源);β1整合素抗体(β1 Integrin Ab-1)由Chemicon International公司提供(小鼠来源);过氧化物酶标记的链霉卵白素(Streptavidin/Peroxidase)试剂盒和DAB染色试剂盒由北京中杉生物技术有限公司提供;戊巴比妥钠由上海行知化工工厂提供。
　　1.2 动物模型的制备:出生50天的健康清洁级SD大鼠90只,雌雄不限,由第三军医大学附属大坪医院动物所提供。随机分为3组:SDF-1组、AMD3100组、空白对照组。1%戊巴比妥钠溶液腹腔注射后,鼠背部脱毛,用直径为6mm的打孔器制作2个全层皮肤缺损的圆形创面。三个治疗组于致伤后即刻、1天、2天、3天、4天分别在创缘注射SDF-1因子1μg,AMD3100 125μg,生理盐水0.1ml,1次/天,随后观察创面愈合情况。
　　1.3 致伤后处理及取材:所有实验动物均行肉眼观察,并记录创面愈合情况(创面愈合以创面完全封闭且完全上皮化为判定标准)。三组在致伤后1天、3天、5天、7天和12天随机处死5只大鼠,取背部2个创面的创缘组织(附带部分正常皮肤组织),置于10%甲醛液内固定。常规石蜡包埋,系列切片,进行HE染色、β1整合素和K19染色。免疫组化检测采用链亲和素-过氧化物(Streptavidin/Peroxidase,SP)法。小鼠抗大鼠β1整合素多克隆抗体(工作浓度1:1000),小鼠抗大鼠K19单克隆抗体(工作浓度1:1000)。实验操作严格按试剂盒说明说进行,DAB显色,苏木精复染。另外以PBS代替β1整合素多克隆抗体、K19单克隆抗体作为空白对照。
　　1.4 观察指标
　　1.4.1 创面面积计算:用数码相机拍摄鼠背部创面及创面旁的标尺,以标尺校准创面面积大小,应用Image J软件分析测量创面面积。
　　1.4.2 免疫组织化学染色阳性细胞的判定:结果以细胞膜(β1整合素)、细胞浆(K19)呈棕黄色着色为强阳性,黄色着色为阳性,淡黄色为弱阳性。
　　1.4.3 统计学处理:统计学分析采用SPSS10.0统计软件作单因素方差分析,结果以x±s表示。以P�0.05作为统计学差异显著,P 　　[5]李建福,付小兵,盛志勇,等. 创面愈合过程中创缘表皮干细胞的再分布[J].中华医学杂志,2003,83(3):228-230.
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　　[收稿日期]2010-06-10[修回日期]2010-07-12
　　编辑/张惠娟