炎症与修复相关细胞因子基因在临床慢性难愈性创面中差异表达的分析

Analysis of differential gene expressions of inflammatory and repair-related factors in chronic refractory wounds in clinic

目的比较压疮和糖尿病足等慢性难愈性创面组织形态及炎症与修复相关细胞因子基因表达差异。方法收集2016年8月—2017年9月,南昌大学第一附属医院收治的10例泌尿外科行包皮环切术患者[均为男性,年龄(38±4)岁]10个包皮样本为正常皮肤组,9例热力或电烧伤患者[男6例、女3例,年龄(51±8)岁]、13例压疮患者[男9例、女4例,年龄(51±14)岁]、10例糖尿病足患者[男8例、女2例,年龄(61±10)岁]手术过程中切除的有血运创缘组织样本,分别为烧伤创面组(9个样本)、压疮组(13个样本)、糖尿病足组(10个样本)。取压疮组和糖尿病足组各10张切片,各取5张切片采用免疫荧光法观察组织形态及创面Ki67和CD31表达情况。取正常皮肤组10个样本、烧伤创面组9个样本、压疮组13个样本、糖尿病足组10个样本行实时荧光定量反转录PCR分析血管内皮生长因子192(VEGF192)、转化生长因子β(TGF-β)、血管细胞黏附分子1(VCAM-1)、细胞间黏附分子1(ICAM-1)、白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)的mRNA表达。对数据行Mann-WhitneyU检验、Kruskal-Wallis秩和检验。结果(1)糖尿病足组创面Ki67表达水平(390±100)高于压疮组创面(182±14,Z=-2.611,P<0.01)。(2)糖尿病足组创面虽然有大量血管内皮细胞(CD31阳性细胞)存在,但其分布无序,未形成完整管腔。压疮组创面血管内皮细胞数量远少于糖尿病足组创面,但形成完整管腔。(3)烧伤创面组、压疮组、糖尿病足组创面的VEGF192的mRNA表达水平均低于正常皮肤组(H=13.72、30.50、15.20,P<0.05或P<0.01),尤以压疮组表达水平最低;压疮组创面VEGF192的mRNA表达水平明显低于糖尿病足组(H=15.30,P<0.01)。与正常皮肤组比较,烧伤创面组创面TGF-β的mRNA表达水平无明显变化(H=-9.50,P>0.05),而压疮组、糖尿病足组创面TGF-β的mRNA表达水平明显降低(H=18.04、14.50,P<0.01);压疮组创面TGF-β的mRNA表达水平与糖尿病足组相近(H=3.54,P>0.05)。(4)与正常皮肤组比较,烧伤创面组和压疮组创面VCAM-1的mRNA表达水平均明显升高(H=-22.50、-11.50,P<0.05或P<0.01),糖尿病足组创面VCAM-1的mRNA表达水平无明显变化(H=10.00,P>0.05);烧伤创面组创面ICAM-1的mRNA表达水平无明显变化(H=-9.50,P>0.05),压疮组和糖尿病足组创面ICAM-1的mRNA表达水平明显降低(H=16.50、16.50,P<0.01)。压疮组创面VCAM-1的mRNA表达水平明显高于糖尿病足组(H=-21.50,P<0.01),ICAM-1的mRNA表达水平与糖尿病足组相近(H=0,P>0.05)。(5)与正常皮肤组比较,糖尿病足组创面IL-1β的mRNA表达水平无明显变化(H=-10.00,P>0.05),烧伤创面组和压疮组创面IL-1β的mRNA表达水平均明显升高(H=-32.50、-21.50,P<0.01);烧伤创面组、压疮组、糖尿病足组创面IL-6的mRNA表达水平均明显升高(H=-17.50、-30.50、-11.80,P<0.05或P<0.01);烧伤创面组创面TNF-α的mRNA表达水平无明显变化(H=-9.50,P>0.05),压疮组和糖尿病足组创面TNF-α的mRNA表达水平均明显降低(H=18.04、14.50,P<0.01)。压疮组创面IL-1β和TNF-α的mRNA表达水平均明显低于烧伤创面组(H=11.00、27.54,P<0.05或P<0.01),IL-6的mRNA表达水平明显高于烧伤创面组(H=-13.00,P<0.05)。糖尿病足组创面IL-1β和TNF-α的mRNA表达水平均明显低于烧伤创面组(H=22.50、24.00,P<0.01),IL-6的mRNA表达水平与烧伤创面组相近(H=5.70,P>0.05)。结论糖尿病足和压疮创面从增殖细胞核抗原水平、血管形成能力到生长因子、细胞黏附因子和炎性细胞因子基因表达水平均表现出明显差异。

ObjectiveTo compare the tissue morphology and gene expressions of inflammatory and repair-related factors in chronic refractory wound tissue including pressure ulcers and diabetic feet.MethodsDuring August 2016 to September 2017, 10 samples of prepuce were collected after circumcision of 10 urological patients [all male, aged (38±4) years old] admitted in the First Affiliated Hospital of Nanchang University and included in normal skin group, samples of tissue around the edge of wounds with blood supply were collected from 9 heat or electric burn patients [6 male patients, 3 female patients, aged (51±8) years old], 13 pressure ulcer patients [9 male patients, 4 female patients, aged (51±14) years old] and 10 diabetic foot patients [8 male patients, 2 female patients, aged (61±10) years old] during the operations. The samples were divided into burn wound group (9 samples), pressure ulcer group (13 samples), and diabetic foot group (10 samples). Ten slices were taken from pressure ulcer group and diabetic foot group respectively, and 5 slices in each group were used to observe the tissue morphology and expressions of Ki67 and CD31 of wounds respectively with immunofluorescence method. Ten samples from normal skin group, 9 samples from burn wound group, 13 samples from pressure ulcer group, and 10 samples from diabetic foot group were collected for analysis of mRNA expressions of vascular endothelial growth factor 192 (VEGF192), transforming growth factor β (TGF-β), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) , interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) by real time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with Mann-Whitney U test and Kruskal-Wallis rank-sum test.Results(1) The expression level of Ki67 in diabetic foot group (390±100) was higher than that of pressure ulcer group (182±14, Z=-2.611, P<0.01). (2) Although there were a large number of vascular endothelial cells (CD31 positive cells) in wounds of diabetic foot group, their distribution was disordered and failed to form intact lumen. There were less vascular endothelial cells in wounds of pressure ulcer group than those of diabetic foot group, but the complete lumen was formed. (3) The mRNA expression levels of VEGF192 in wounds of burn wound group, pressure ulcer group, and diabetic foot group were significantly lower than the level in normal skin group (H=13.72, 30.50, 15.20, P<0.05 or P<0.01), and the level was the lowest in pressure ulcer group. The mRNA expression level of VEGF192 in wounds of pressure ulcer group was significantly lower than that of diabetic foot group (H=15.30, P<0.01). Compared with that of normal skin group, the mRNA expression level of TGF-β in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), while the mRNA expression levels of TGF-β in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P<0.01). The mRNA expression level of TGF-β in wounds of pressure ulcer group was similar to that of diabetic foot group (H=3.54, P>0.05). (4) Compared with those of normal skin group, the mRNA expression levels of VCAM-1 in wounds of burn wound group and pressure ulcer group were significantly increased (H=-22.50, -11.50, P<0.05 or P<0.01), and there was no significant difference in the mRNA expression level of VCAM-1 in wounds of diabetic foot group (H=10.00, P>0.05);the mRNA expression level of ICAM-1 in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), and the levels of ICAM-1 in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=16.50, 16.50, P<0.01). The mRNA expression level of VCAM-1 in wounds of pressure ulcer group was significantly higher than that of diabetic foot group (H=-21.50, P<0.01), the mRNA expression level of ICAM-1 in wounds of pressure ulcer group was similar to that of diabetic foot group (H=0, P>0.05). (5) Compared with those of normal skin group, except for the mRNA expression level of IL-1β in wounds of diabetic foot group showed no significant difference (H=-10.00, P>0.05), the mRNA expression levels of IL-1β in wounds of burn wound group and pressure ulcer group were significantly increased (H=-32.50, -21.50, P<0.01);the mRNA expression levels of IL-6 were significantly increased in wounds of burn wound group, pressure ulcer group, and diabetic foot group (H=-17.50, -30.50, -11.80, P<0.05 or P<0.01);except for the mRNA expression level of TNF-α in wounds of burn wound group showed no significant difference (H=-9.50, P>0.05), the mRNA expression levels of TNF-α in wounds of pressure ulcer group and diabetic foot group were significantly decreased (H=18.04, 14.50, P<0.01). The mRNA expression levels of IL-1β and TNF-α in wounds of pressure ulcer group were significantly lower than those of burn wound group (H=11.00, 27.54, P<0.05 or P<0.01), while the mRNA expression level of IL-6 was significantly higher (H=-13.00, P<0.05). The mRNA expression levels of IL-1β and TNF-α in wounds of diabetic foot group were significantly lower than those of burn wound group (H=22.50, 24.00, P<0.01), while the mRNA expression level of IL-6 showed no significant difference (H=5.70, P>0.05).ConclusionsThe phenotypes of diabetic foot and pressure ulcer vary from the expressions levels of proliferating cell nuclear antigen and blood vessels forming ability to the expression levels of growth factors, cell adhesion factors, and inflammatory cytokines.