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。

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。

(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，差异有统计学意义(t＝25.29，P<0.001)，rPEDF组的迁移率明显低于对照组，差异有统计学意义(t＝15.98，P<0.001)。siPEDF组内皮细胞迁移率为对照组2倍，rPEDF组内皮细胞迁移率为对照组的1/3；(4)PEDF抑制hDMEC增殖能力，siPEDF组、rPEDF组、对照组的内皮细胞增殖率分别是(442.60±58.90)%、(248.90±52.19)%、(333.80±47.70)%，3组比较差异有统计学意义(F＝16.69，P<0.001)。siPEDF组的增殖率明显高于对照组，差异有统计学意义(t＝3.21，P＝0.013)，rPEDF组的增殖率明显低于对照组，差异有统计学意义(t＝2.69，P＝0.028)；(5)PEDF抑制hDMEC成管能力：siPEDF组、rPEDF组、对照组的内皮细胞成管结点数分别是(38.00±4.58)、(11.33±3.51)、(23.67±6.66)个，3组比较差异有统计学意义(F＝20.64，P＝0.002)，siPEDF组的成管结点数明显多于对照组，差异有统计学意义(t＝3.07，P＝0.037)，rPEDF组的成管结点数明显少于对照组，差异有统计学意义(t＝2.84，P＝0.047)；siPEDF组、rPEDF组、对照组的成管长度分别是(1 518.00±250.90)、(365.8±91.52)、(925.50±193.00) ppi，3组比较差异有统计学意义(F＝27.53，P<0.001)，siPEDF组的成管长度明显长于对照组，差异有统计学意义(t＝3.24，P＝0.032)，rPEDF组的成管长度明显短于对照组，差异有统计学意义(t＝4.54，P＝0.011)；(6)PEDF与经典Wnt/β-catenin信号通路关键蛋白存在相互作用，STRING预测蛋白-蛋白之间的相互作用关系，发现PEDF与Wnt/β-catenin信号通路之间存在共同作用蛋白；(7)PEDF抑制Wnt1、β-catenin蛋白表达，siPEDF组、rPEDF组、对照组hDMEC中Wnt1蛋白相对表达水平分别为0.84±0.04、0.12±0.05、0.66±0.08，3组比较差异有统计学意义(F＝114.80，P<0.001)，siPEDF组的Wnt1的蛋白表达水平明显高于对照组，差异有统计学意义(t＝4.95，P＝0.036)，rPEDF组的Wnt1的蛋白表达水平明显低于对照组，差异有统计学意义(t＝13.94，P＝0.005)；siPEDF组、rPEDF组、对照组β-catenin蛋白相对表达水平分别为0.77±0.05、0.50±0.07、0.63±0.06，3组比较差异有统计学意义(F＝15.04，P＝0.005)，siPEDF组的β-catenin的蛋白表达水平明显高于对照组，差异有统计学意义(t＝6.51，P＝0.023)，rPEDF组的β-catenin的蛋白表达水平明显低于对照组，差异有统计学意义(t＝4.56，P＝0.045)。

PEDF随着创面愈合过程，其表达呈动态的变化；PEDF可能通过调节Wnt/β-catenin通路，进而抑制hDMEC的迁移、增殖与成管，最终影响创面愈合。

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).

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.

(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 of SD rats was 1.32±0.15, and the expression level of PDEF protein in wound tissue of SD rats was 0.83±0.08 on the third day after trauma, the difference was statistically significant (t= 5.11, P< 0.001). (3) PEDF inhibited the migration of hDMEC. The migration rates of endothelial cells in siPEDF group, rPEDF group and control group were (92.01±3.04)%, (19.51±3.93)%, (57.03±1.065)%, respectively, and the difference was statistically significant (F=459.30, P< 0.001). The migration rate of siPEDF group was significantly higher than that of control group, the difference was statistically significant (t=25.29, P< 0.001), and the rPEDF group was significantly lower than that of control group, the difference was statistically significant (t=15.98, P< 0.001). The migration rate of endothelial cells in siPEDF group was two times that in control group, and that in rPEDF group was 1/3 of that in control group. (4) PEDF inhibited the proliferation of hDMEC. The proliferation rates of endothelial cells in siPEDF group, rPEDF group and control group were (442.60±58.90)%, (248.90±52.19)%, (333.80±47.70)%, respectively. There was statistically significant difference among the three groups (F= 16.69, P< 0.001). The proliferation rate of the siPEDF group was significantly higher than that of the control group, and the difference was statistically significant (t= 3.21, P= 0.013). The proliferation rate of the rPEDF group was significantly lower than that of the control group, and the difference was statistically significant (t= 2.69, P= 0.028). (5) PEDF inhibited hDMEC tube forming. The number of endothelial tube forming nodes in siPEDF group, rPEDF group and control group were (38.00±4.58), (11.33±3.51), (23.67±6.66) respectively, and the difference was statistically significant (F= 20.64, P= 0.002). The number of endothelial tube forming nodes in siPEDF group was significantly higher than that of control group, the difference was statistically significant (t= 3.07, P= 0.037). The number of nodes in rPEDF group was significantly less than that in control group, the difference was statistically significant (t= 2.84, P= 0.047); the tube length of siPEDF group, rPEDF group and control group were (1 518.00±250.90), (365.8±91.52) and (925.50±193.00) ppi, respectively, and the difference was statistically significant (F= 27.53, P< 0.001). The tube length of siPEDF group was significantly longer than that of control group, and the difference was statistically significant (t= 3.24, P= 0.032). The tube length of rPEDF group was significantly shorter than that of control group, the difference was statistically significant (t= 4.54, P= 0.011). (6) PEDF interacts with key proteins in the classical Wnt/β-catenin signaling pathway. STRING were used to predict the protein-protein interaction relationship and founded that there were co-acting proteins between PEDF and Wnt/β-catenin signaling pathway. (7) PEDF inhibited Wnt1 and β-catenin expression level. The relative expression levels of Wnt1 protein in hDMEC of siPEDF group, rPEDF group and control group were 0.84±0.04, 0.12±0.05 and 0.66±0.08, respectively. The differences among the three groups was statistically significant (F=114.80, P< 0.001). The protein expression level of Wnt1 in siPEDF group was significantly higher than that of control group, the difference was statistically significant (t=4.95, P= 0.036). The protein expression level of Wnt1 in rPEDF group was significantly lower than that of control group, the difference was statistically significant (t=13.94, P=0.005). The relative expression levels of β-catenin protein in siPEDF group, rPEDF group and control group were 0.77±0.05, 0.50±0.07 and 0.63±0.06, respectively, the differences between the three groups was statistically significant(F= 15.04, P= 0.005). The protein expression level of β-catenin in siPEDF group was significantly higher than that of the control group, and the difference was statistically significant (t=6.51, P=0.023). The protein expression level of β-catenin rPEDF group was significantly lower than that in control group, the difference was statistically significant (t=4.56, P=0.045).

The expression of PEDF changes dynamically within the wound healing process; PEDF might inhibit the ability of migration, proliferation, and tube formation in hDMEC by regulating Wnt/β-catenin pathway, and ultimately affect wound healing.