胎鼠皮肤无瘢痕愈合培养液优化及皮肤细胞增殖活跃区研究  

Optimization of culture medium for scarless healing of fetal mouse wounds and analysis of regions with active cell proliferation

作　　者：王海涛[1] 姜仁军[1] 李树伟[1] Wang Haitao Jiang Renjun Li Shuwei(Xinjiang Production ＆ Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, College of Life Sciences, Tarim University, Alaer 843300, Xinfiang, Chin)

机构地区：[1]塔里木盆地生物资源保护利用兵团重点实验室 塔里木大学生命科学学院,新疆阿拉尔843300

出　　处：《中华皮肤科杂志》2017年第2期99-104,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（31560685）;塔里木大学研究生科研创新项目（TDGR1201521）

摘　　要：目的 优化昆明远交系（KM）胎鼠皮肤无瘢痕愈合模型的培养液，定位并分析皮肤创伤愈合中细胞增殖活跃区。方法 取60只胎龄15 d小鼠背部皮片180块，用无菌微创器在皮片上统一造创，随机等分到6组培养液中培养3 d，包括高糖改良Eagle培养基（DMEM）组、DMEM ＋ 5%胎牛血清（FBS）组、DMEM ＋ 10% FBS组、低糖Eagle培养基（MEM）组、MEM ＋ 5% FBS组、MEM＋ 10% FBS组。HE染色后观察各组皮肤创口愈合情况，筛选最适培养条件。在最适培养条件下采用EdU细胞标记物定位皮肤创口愈合处细胞增殖活跃区，并用免疫荧光检测其中AP？1蛋白表达。结果 胎鼠微创皮片培养3 d后，DMEM组、DMEM ＋ 5% FBS组、DMEM ＋ 10% FBS组、MEM组、MEM ＋ 5% FBS组、MEM ＋ 10% FBS组皮片愈合率分别为0、3.33%、6.67%、3.33%、46.67%、26.67%，6组皮片愈合率差异有统计学意义（χ^2 = 41.39，P 〈 0.05）。两两比较显示，MEM ＋ 5% FBS组皮片愈合率显著高于除MEM ＋ 10% FBS组外的其他4组（均P 〈 0.01）。logistic回归分析显示，MEM（OR = 11.717，95% CI 3.274 ~ 41.934，P 〈 0.001）及FBS浓度（5% FBS：OR = 24.625，95% CI 3.027 ~ 200.299，P = 0.003；10% FBS：OR = 13.449，95% CI 1.618 ~ 111.813，P = 0.016）均是促进胎鼠微创皮片愈合的因素。在MEM ＋ 5% FBS培养条件下，真皮和表皮愈合形态最好，且创面处表皮无增厚现象。EdU细胞增殖标记分析显示，胎鼠皮肤的真皮乳头层和表皮基底层是创面愈合过程中两个主要的细胞增殖活跃区，同时AP？1蛋白在这两个细胞增殖活跃区也有大量表达。结论 MEM ＋ 5% FBS为体外培养胎鼠皮肤创伤愈合的最适培养液。胎鼠皮肤的真皮乳头层和表皮基底层是皮肤创伤愈合中两个主要的细胞增殖活跃区，在创面愈合过程中起到重要作用。Objective To optimize the culture medium for scarless wound healing in outbred fetal Kunming （KM） mice, and to locate and preliminarily analyze the region with active cell proliferation. Methods After 6 pregnant mice were sacrificed at the gestational age of 15 days, a total of 180 skin grafts were obtained from the back of 60 fetal mice, and wounds on the skin grafts were made uniformly with minimally invasive sterile device. Then, these skin grafts with wounds were randomly and equally divided into 6 groups to be treated with high-glucose Dulbecco′s modified Eagle′s medium （DMEM）, DMEM ＋ 5% fetal bovine serum （FBS）, DMEM ＋ 10% FBS, low-glucose Eagle′s minimum essential medium （MEM）, MEM ＋ 5% FBS, and MEM ＋ 10% FBS for 3 days, respectively. Then, these skin grafts were embedded in optimal cutting temperature （OCT） compound, and subjected to hematoxylin and eosin （HE） staining to select the optimal culture condition for wound healing. Under the optimal culture condition, the region with active cell proliferation was located by labeling and tracking cutaneous stem cells with 5-ethynyl-2-deoxyuridine （EdU）, and activator protein-1 （AP-1） was detected by immunofluorescent staining. Results After 3-day cultivation, wound healing rates significantly differed among the DMEM group, DMEM ＋ 5% FBS group, DMEM ＋ 10% FBS group, MEM group, MEM ＋ 5% FBS group and MEM ＋ 10% FBS group （0, 3.33%, 6.67%, 3.33%, 46.67% and 26.67%, respectively, χ2 = 41.39, P 〈 0.05）. Additionally, the MEM ＋ 5% FBS group showed a significantly higher wound healing rate than the other groups except the MEM ＋ 10% FBS group （all P 〈 0.01）. Logistic regression analysis showed that the type of basal medium （MEM： OR = 11.717, 95% CI： 3.274 - 41.934, P 〈 0.001） and FBS concentrations （5% FBS： OR = 24.625, 95% CI： 3.027 - 200.299, P = 0.003; 10% FBS： OR = 13.449, 95% CI： 1.618 - 111.813, P = 0.016） were factors influencing fetal wound healing. Under the culture con

关 键 词：伤口愈合 瘢痕 转录因子AP-1 无瘢痕愈合 胎鼠 

分 类 号：R751[医药卫生—皮肤病学与性病学]