改良保存液处理自体血对糖尿病小鼠伤口成纤维细胞的影响

Effect of modified autologous blood on wound fibroblasts in diabetic mice

目的评价经改良保存液处理的自体血对糖尿病小鼠伤口成纤维细胞的影响,并初步探讨其机制。方法诱导建立糖尿病模型小鼠40只,随机分为献血组和实验组,每组20只。实验组小鼠于伤口模型建立后,剔除死亡小鼠3只,将剩余小鼠随机分为标准组(n=6)、改良组(n=6)和供皮组(n=5),标准组和改良组于术后第1、2和3天分别回输在不同保存液中贮存7 d的自体血,用透明膜示踪法记录并计算伤口愈合面积百分比、RT-qPCR检测伤口组织缺氧诱导因子1α(HIF-1α)mRNA表达量。供皮组于伤后3 d收集创面和边缘的全层皮肤,将皮肤成纤维细胞分离培养后,随机分为四组:细胞标准组(S组)和标准+siRNA-HIF-1α组(S+siRNA组)、细胞改良组(M组)、改良+siRNA-HIF-1α组(M+siRNA组),在细胞灌流培养系统中使用不同方法保存的自体血灌注培养。细胞计数KIT-8(CCK-8)、EdU、Transwell、流式细胞仪评价成纤维细胞细胞存活、增殖和迁移能力及其细胞周期分布;Western blot测定各组成纤维细胞中HIF-1α、血管内皮生长因子(VEGF)和基质金属蛋白酶(MMP)-2蛋白相对含量。结果与标准组比较,改良组小鼠术后伤口愈合面积和HIF-1αmRNA相对表达量明显升高(P<0.05)。与S组比较,M组的细胞存活力增强,增殖细胞及迁移细胞数量明显升高,而S+siRNA组的细胞存活力减弱,增殖细胞及迁移细胞数量明显下降(P<0.05);与M组比较,M+siRNA组细胞存活力、增殖细胞及迁移细胞数量明显降低(P<0.05)。与S组比较,S+siRNA组HIF-1α、VEGF和MMP-2蛋白相对含量明显降低,而M组蛋白相对含量明显增高(P<0.05);与M组比较,M+siRNA组HIF-1α、VEGF和MMP-2蛋白相对含量明显降低(P<0.05)。结论改良处理自体血可增强糖尿病小鼠成纤维细胞的存活、增殖和迁移能力,可能参与伤口的愈合。

Objective To evaluate the effect of autologous blood treated with modified preservation solution on wound fibroblasts in diabetic mice and its related mechanisms.Methods Forty diabetic mice were randomly divided into 2 groups using a random number table:blood donation group(n=20)and experimental group(n=20).After the wound model was established,mice inexperimental group were randomly divided into standard group(n=6),modified group(n=6)and donor group(n=5).Autologous blood stored in different preservation solutions for 7 days was returned to group standard group and modified group respectively on the 1st day,2nd day and 3rd day after operation,the percentage of wound healing area was recorded and calculated by transparent film tracer method,and the expression level of HIF-1αin wound tissue was detected by RT-qPCR.Mice in donor group were fed to the 3rd day after injury to collect the whole skin of the wound and the edge.After the fibroblasts of skin were isolated and cultured,some of them were transfected with HIF-1αsiRNA,the cells were then various groups namely cell modified group(group M),modified-siRNA-HIF-1αgroup(group M+siRNA),cell standard group(group S)and standard-siRNA-HIF-1αgroup(group S+siRNA).Cell-based perfusion assay was applied in order to culture the fibroblasts with different preservation solutions.Fibroblast cell viability,mobility,migration and cell cycle were evaluated by cell count KIT-8(CCK-8)assay,EdU assay,Transwell assay and Flow cytometry,respectively.And the expression of HIF-1α,VEGF and MMP-2 in fibroblasts were determined by Western blot.Results Compared with standard group,the wound healing rate and expression levels of HIF-1αin skin tissue of group modified were significantly increased(P<0.05).Compared with group S,group M exhibited improved cell viability,proliferative cells and migrated cells while the results in group S+siRNAwere reversed(P<0.05).Compared with group M,group M+siRNA exhibited decreased cell viability,proliferative cells and migrated cells(P<0.05).Compared with group S,the protein levels of HIF-1α,VEGF and MMP-2 in group M were significantly increased while they were significantly decreased in group S+siRNA(P<0.05).Compared with group M,the protein levels of HIF-1α,VEGF and MMP-2 in group M+siRNA were significantly decreased(P<0.05).Conclusion Autologous blood treated with modified preservation solution may be involved in wound healing in diabetic mice by improve the survival rate,cell proliferation and migration ability of fibroblast cells.