Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators.
Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators.
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DOI：
10.1111/j.1524-475x.2010.00638.x
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发表时间：
2010-11
期刊：
影响因子：
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通讯作者：
Saadeh PB
中科院分区：
文献类型：
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作者：
Nguyen PD;Tutela JP;Thanik VD;Knobel D;Allen RJ Jr;Chang CC;Levine JP;Warren SM;Saadeh PB
糖尿病的特点是一些鲜为人知的现象，包括伤口愈合功能障碍和血管生成受损。P53是一种主要的细胞周期调节因子，在糖尿病伤口中表达上调，最近被证明在血管生成途径中发挥调节作用。我们之前已经描述了一种用siRNA局部沉默伤口床中的靶基因的新方法。我们假设沉默p53导致改善糖尿病伤口愈合和血管生成介质的增加。在糖尿病db/db小鼠上建立成对的4毫米支架伤口。局部应用p53 siRNA，均匀分布在琼脂糖基质中，于创面后第1天和第7天应用于创面（单独使用基质和无活性siRNA作为对照）。分别于伤后第10天和第24天处死。用光度法评估伤口愈合时间，并采集伤口进行组织学、免疫组织化学和免疫荧光分析。通过western blot、RT-PCR和ELISA检测血管生成细胞因子的表达。采用方差分析/t检验检验显著性（p< 0.05）。局部p53沉默导致创面愈合更快，治疗组创面愈合时间为18±1.3d，对照组为28±1.0d。治疗组在每个时间点显示伤口结构改善，同时显示局部p53几乎完全敲除。此外，治疗过的伤口显示CD31内皮细胞染色比对照组增加1.92倍。Western blot分析证实在治疗过的伤口中p53几乎完全敲除。第10天，VEGF分泌（ELISA）较对照组（33.0±3.8 pg/ml）显著增加（109.3±13.9 pg/ml），而RT-PCR显示，治疗创面中SDF-1表达较对照组增加1.86倍。这种情况在伤口愈合后和对照组关闭前（第24天）发生逆转。增强血管生成细胞因子谱和内皮细胞标记物与局部p53 siRNA基因治疗改善糖尿病伤口愈合有关。
Diabetes is characterized by several poorly understood phenomena including dysfunctional wound healing and impaired vasculogenesis. P53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with siRNA. We hypothesized that silencing p53 results in improved diabetic wound healing and augmentation of vasculogenic mediators. Paired 4-mm stented wounds were created on diabetic db/db mice. Topically applied p53 siRNA, evenly distributed in an agarose matrix, was applied to wounds at post-wound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at post-wound days 10 and 24. Wound time to closure was photometrically assessed, and wounds were harvested for histology, immunohistochemistry, and immunofluorescence. Vasculogenic cytokine expression was evaluated via western blot, RT-PCR, and ELISA. ANOVA/t-test was used to determine significance (p<=0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18 ± 1.3d in the treated group versus 28 ± 1.0d in controls. The treated group demonstrated improved wound architecture at each time point while demonstrating near complete local p53 knockdown. Moreover, treated wounds showed a 1.92 fold increase in CD31 endothelial cell staining over controls. Western blot analysis confirmed near complete p53 knockdown in treated wounds. At day 10, VEGF secretion (ELISA) was significantly increased in treated wounds (109.3 ± 13.9 pg/ml) versus controls (33.0 ± 3.8 pg/ml) while RT-PCR demonstrated a 1.86 fold increase in SDF-1 expression in treated wounds versus controls. This profile was reversed after treated wounds healed and prior to closure of controls (day 24). Augmented vasculogenic cytokine profile and endothelial cell markers are associated with improved diabetic wound healing in topical gene therapy with p53 siRNA.