色素上皮衍生因子在创面中的表达变化及对人真皮微血管内皮细胞的作用与机制  

Expression of pigment epithelium-derived factor in wound healing and its effect and mechanism on human dermal microvascular endothelial cells

作　　者：舒斌[1] 朱君佑 祁少海[1] Shu Bin;Zhu Junyou;Qi Shaohai(Department of Burn Surgery, First Affiliated Hospital, Sun Yat- sen University, Guangzhou 510080, China)

机构地区：[1]中山大学附属第一医院烧伤外科,广州510080

出　　处：《中华损伤与修复杂志（电子版）》2021年第4期301-309,共9页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金面上项目(81971833);广东省自然科学基金面上项目(2021A1515011806)。

摘　　要：目的探讨色素上皮衍生因子(PEDF)在创面愈合过程中的表达变化情况以及对人真皮微血管内皮细胞(hDMEC)的作用和潜在机制。方法运用生物信息学方法,通过NCBI GEO数据库行基因芯片分析,明确PEDF在创面愈合过程中表达变化情况;选择3只SD大鼠,制作创面模型,收集皮肤组织样本进行实时荧光定量PCR及蛋白质印迹法检测,验证PEDF在创面愈合过程中表达变化情况;细胞划痕试验探究PEDF对hDMEC迁移能力的影响,细胞计数试剂盒(CCK)-8试验探究PEDF对hDMEC增殖能力的影响,成管实验探究PEDF对hDMEC血管形成能力的影响;STRING预测PEDF-蛋白间相互作用关系,最终通过蛋白质印迹法检测PEDF与hDMEC Wnt/β-catenin通路间关系。数据比较采用单因素方差分析和LSD-t检验。结果(1)R语言分析基因芯片GSE28914人皮肤正常创面愈合过程中PEDF变化情况,发现人正常皮肤组织中PEDF表达量为-0.04±0.03,而人皮肤正常创面愈合过程中,PEDF在凝血期(创后即刻)一过性升高(0.84±0.12),比较差异有统计学意义(差异倍数=0.89,P=0.002),炎症期(创后第3天)迅速下降(-0.60±0.09),比较差异有统计学意义(差异倍数=-0.55,P=0.010),最终逐渐恢复到正常皮肤水平(0.03±0.06),比较差异有统计学意义(差异倍数=-0.07,P=0.602);(2)SD大鼠皮肤组织PEDF表达量高于创后第3天,SD大鼠皮肤中PEDF的RNA相对表达量为1.15±0.13,创后第3天SD大鼠创面组织中PEDF的RNA相对表达量为0.45±0.04,比较差异有统计学意义(t=9.21,P<0.001)。SD大鼠皮肤组织中相对PEDF蛋白表达水平为1.32±0.15,创后第3天SD大鼠创面组织中PDEF蛋白表达水平为0.83±0.08,比较差异有统计学意义(t=5.11,P<0.001);(3)PEDF抑制hDMEC迁移能力,siPEDF组、rPEDF组、对照组的内皮细胞迁移率分别是(92.01±3.04)%、(19.51±3.93)%、(57.03±1.065)%,3组比较差异有统计学意义(F=459.30,P<0.001)。siPEDF组的迁移率明显高于对照组,差异有统计学意Objective To investigate the expression of pigment epithelium-derived factor(PEDF)in wound healing and its effect and mechanism on human dermal microvascular endothelial cells(hDMEC).Methods The expression of PEDF during wound healing was determined by bioinformatics and gene chip analysis in NCBI GEO database;3 SD rats were selected to make wound models,and skin tissue samples were collected for real-time fluorescence quantitative PCR and western blotting to verify the expression changes of PEDF in the process of wound healing.The cell scratch test was conducted to explore the effect of PEDF on the migration ability of hDMEC,the cell counting kit(CCK)-8 test was conduccted to explore the effect of PEDF on the proliferation of hDMEC,and the tube formation experiment was conducted to explore the effect of PEDF on the angiogenesis of hDMEC.STRING was used to predicte the PEDF-protein interaction relationship,and finally passed western blotting was used to detect the relationship between PEDF and hDMEC Wnt/β-catenin pathway.Data were compared with one-way ANOVA and LSD-t test.Results(1)Bioinformatics analysis of gene chip GSE28914 by R showed that PEDF expression in human normal skin tissue was-0.04±0.03,and PEDF increased transiently during the coagulation phase(immediately after trauma)(0.84±0.12),the difference was statistically significant(Fold change=0.89,P=0.002),decreased rapidly on the third day after trauma(-0.60±0.09),the difference was statistically significant(Fold change=-0.55,P=0.010),and finally returned to normal skin level(0.03±0.06),the difference was statistically significant(Fold change=-0.07,P=0.602).(2)The expression of PEDF in skin tissue of SD rats was higher than that in wound tissue on the third day after trauma.The relative expression level of PEDF RNA in skin of SD rats was 1.15±0.13,and that in wound tissue of SD rats on the third day after trauma was 0.45±0.04,the difference was statistically significant(t=9.21,P<0.001).The relative expression level of PEDF protein in skin tissue

关 键 词：伤口愈合 细胞迁移分析 细胞增殖 色素上皮衍生因子 人真皮微血管内皮细胞 血管生成 

分 类 号：R73[医药卫生—肿瘤]