P311在小鼠浅Ⅱ度烧伤及体外细胞创伤模型中对表皮干细胞迁移的影响  

Effects of P311 on the migration of epidermal stem cells in mice with superficial partial-thickness burn and injured cell model in vitro

作　　者：孙薇[1] 姚志慧[1] 詹日兴[1] 张小容[1] 崔艳艳[1] 谭江琳[1] 杨思思[1] 胡晓红[1] 周俊峄[1] 吴军[1] 罗高兴[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市疾病蛋白质组学重点实验室,重庆400038

出　　处：《中华烧伤杂志》2012年第3期213-218,共6页Chinese Journal of Burns

基　　金：国家自然科学基金面上项目（30973116、81171809）

摘　　要：目的观察P311在小鼠浅Ⅱ发烧伤技体外细胞创伤模型中对表皮干细胞（ESC）辽移的作用并探讨其机制。方法（1）取18只C57BL／6小鼠，其中15只行背部浅Ⅱ度烧伤，于伤后6、12、24、48、72h取创面及创周皮肤组织，各时相点3只；余下3只小鼠作为正常对照，同法取止常皮肤标本。采用生物素-链霉亲和素-过氧化物酶（SP）染色法观察P311在上述皮肤组织中的表达。（2）取6只新生的C57BL／6小鼠，连续3d腹腔注射50μg／g溴脱氧尿苷（BrdU，2次／（1）建立ESC示踪模型。7周后于其背部制作浅Ⅱ度烧伤创面。伤后72h取创面皮肤组织制作连续切片，SP法免疫组织化学染色，观察P311阳性和BrdU阳性细胞空间共定位情况。（3）构建腺病毒空载体pAdEasy-增强型绿色荧光蛋h（EGFP）及P311腺病毒表达载体pAdEasy-EGFP-P311，并进行包装。应用Ⅳ型胶原快速黏附法分离培养人ESC,按照随机数字表法将其分为P311高表达组和EGFP对照组，每组3孔，分别以pAdEasy-EGFP-P311及pAdEasy-EGFP腺病毒感染ESC。2组ESC经丝裂毒素C处理2h后行划痕实验。划痕后0（即刻）、24、48、72h测量地固定范围内划痕剩余面积，计算划痕愈合面积百分比。对数据进行独立样本t检验。结果（1）浅Ⅱ度烧伤后不同时相点，P311在创面不同部位的表达量各不相同。伤后创面毛囊、新生表皮巾P311表达量随时间延长逐渐上升；创用表皮及毛囊的P311表达量于伤后12h达高峰，72h回落至正常。（2）应用BrdU标记的小鼠浅Ⅱ度烧伤创Ⅲ新生表皮层可见P311阳性细胞和BrdU阳性细胞共定位。（3）划痕后48、72h，P311高表达组ESC划痕愈合面积百分比分别为（69±31）％、（89±26）％，明显高于EGFP刘照组[（35±12）％、（46±31）％，t值分别为-2．336、-2，611，P值均小于0．05]。结论P311可明显促进小鼠浅Ⅱ度烧伤创面及体外细胞创伤模型中的ES[ Abstract] Objective To study effects of P311 on tile migration of epidermal stern cells （ESCs） in mice with superficial partial-thickness burn and injured cell model in vitro and to explore the mechanism. Methods （ 1 ） Eighteen male C57 BL/6 mice were used. Fifteen of them were inflicted with superficial par- tial-thickness burn on the back. In three injured mice wound tissue and skin of wound edge were obtained atpost burn hour （PBH） 6, 12, 24, 48, 72 respe, etively. The rest three mice were used as normal control, and samples were harvested with the same method as above. The expressions of P311 in harvested samples were assessed with biotin-streptavidin-peroxidase （SP） staining. （2） Six newly born C57BL/6 mice were in- traperitoneally injeeled with 50 μg/g BrdU （two times a day） for three clays fur ESCs-lahelling. Seven weekslater, the miee were inflicted with superficial partial-thickness burn on the baek. Serial slices of burn woundtissue were prepared at PBH 72 and immunohistochemically stained with SP for observation of the co-localiza- tion of BrdU-positive ESCs and P311-positive cells. （3） The empty vector pAdEasy-enhanced green fluores- cence protein （EGFP） and the adenovirus P311-expressing vector named pAdEasy-EGFP-P311 were con- structed and packed. Human ESCs were isolated by the method of rapid adhesion to collagen IV. After being divided into P311 high-expressing group （ n = 3） and EGFP control group （ n = 3）, the ESCs in two groups were respectively infected by pAdEasy-EGFP-P311 and pAdEasy-EGFP. Scratching assay was per- formed on ESCs in both groups after they were treated by mitomycin C for 2 hours. The remaining area within the fixed range was measured at post scratching hour （PSH） 0, 24, 48, and 72, and the wound-area healing rate was calculated. Data were processed with independent samples t test. Results （ 1 ） Expression a- mount of P311 was different in different parts of wound at different time points after burn. Expression amount of P311

关 键 词：烧伤 创伤和损伤 细胞运动 P311 表皮干细胞 创面愈合 

分 类 号：R644[医药卫生—外科学]