负压伤口治疗对糖尿病足创面肉芽组织血小板源生长因子表达的影响

目的探讨负压伤口治疗技术(NPWT)的应用对糖尿病足肉芽组织血小板源生长因子(PDGF)表达的影响。 方法选取解放军白求恩国际和平医院2010年1月至2014年12月收治的40例糖尿病足患者，采用单纯随机抽样法将患者随机分为NPWT组(20例)和对照组(20例)，NPWT组创面采用负压伤口治疗技术治疗，对照组采用传统纱布换药治疗。两组分别于治疗前及治疗第12天取创面肉芽组织2份，一份置于4%多聚甲醛溶液中保存，行苏木精－伊红(HE)染色和免疫组织化学染色，HE染色观察肉芽组织形成情况，免疫组织化学染色观察肉芽组织PDGF的表达情况，通过Motic Medical 6.0数码医学图像分析系统检测吸光度值对PDGF蛋白行定量分析。同时，另一份肉芽组织标本－80℃冰箱中保存，采用Western blot法检测PDGF蛋白表达量。数据应用SPSS 13.0软件进行分析，PDGF吸光度值及PDGF蛋白表达量比较采用两独立样本t检验。 结果两组治疗前HE染色可见炎症细胞多，成纤维细胞、新生血管形成较少；两组治疗第12天肉芽组织HE染色均可见新鲜肉芽组织的形成，表现为新生血管、成纤维细胞增多，但与对照组相比，NPWT组新生肉芽组织增多较为明显。免疫组织化学分析显示，两组治疗后均可见PDGF蛋白表达上调；NPWT组治疗第12天PDGF吸光度值(0.41±0.05)与治疗前(0.29±0.04)比较，差异有统计学意义(t＝8.63，P<0.05)；对照组治疗第12天PDGF吸光度值(0.33±0.04)与治疗前(0.28±0.04)比较，差异也有统计学意义(t＝3.62，P<0.05)；但是，NPWT组治疗前后PDGF吸光度变化值为0.13±0.05，与对照组治疗前后PDGF吸光度变化值(0.05±0.03)比较，差异有统计学意义(t＝5.89，P<0.05)。Western blot分析显示，NPWT组治疗第12天PDGF蛋白表达量为0.24 ±0.04，与治疗前PDGF蛋白表达量(0.11±0.03)比较，差异有统计学意义(t＝11.01，P<0.05)；对照组治疗第12天PDGF蛋白表达量为0.17±0.03，与治疗前PDGF蛋白表达量(0.12 ±0.03)比较，差异亦有统计学意义(t＝6.06，P<0.05)；但是，NPWT组治疗前后PDGF蛋白表达量的变化值为0.12 ±0.03，与对照组治疗前后PDGF蛋白表达量的变化值(0.05 ±0.02)比较，差异有统计学意义(t＝9.06，P<0.05)。 结论应用NPWT可明显促进创面肉芽组织PDGF表达上调，从而促进创面的愈合。

Effect of negative pressure wound therapy on the expression of platelet derived growth factor in granulation tissue of diabetic foot wounds

ObjectiveTo assess the endogenous regulation of platelet derived growth factor (PDGF) in the foot wound healing response induced by negative pressure wound therapy (NPWT) in human diabetic patients in vivo. MethodsFrom January 2010 to December 2014, 40 patients with diabetic foot wounds fitting the inclusion criteria were found and enrolled in the clinical trial. The subjects were randomly assigned for NPWT group or control group with simple random sampling method. Twenty patients were treated with NPWT, while the same number of patients in the control group received conventional gauze therapy. Granulated tissue biopsies were collected before(0 day) and after(12 days) treatment in both groups. All biopsies were subdivided into two parts. One part was preserved in 4% paraformaldehyde for hematoxylin eosin staining and immunocytochemical staining of PDGF, the other part was stored at -80℃ for Western blot analysis of PDGF expression. Experimental data were analyzed using SPSS 13.0 software and differences of absorbance value and protein expression of PDGF between the two groups were analyzed using two-sample t test. ResultsThe HE staining showed that both NPWT group and control group wounds developed characteristic granulated tissue after treatment, which presented intense fibroblasts and neoformation of vessels. However, NPWT group wounds developed much more characteristic granulated tissue than control group wounds. The immunohistochemical data revealed that PDGF expression was up-regulated in NPWT group and control group after treatment. The absorbance value of PDGF was higher after treatment than that before treatment in NPWT group, the difference was statistically significant (0.41±0.05 vs 0.29±0.04, t=8.63, P<0.05). Also, the absorbance value of PDGF was higher after treatment than that before treatment in control group, the difference was statistically significant (0.33±0.04 vs 0.28±0.04, t=3.62, P<0.05). However, the change value (difference between before treatment and after treatment) of absorbance value of PDGF was higher in NPWT group than that in control group, the difference was statistically significant (0.13±0.05 vs 0.05±0.03, t=5.89, P<0.05). The Western blot analysis of PDGF was consistent with that of immunohistochemical analysis. The protein levels of PDGF was higher after treatment than that before treatment in NPWT group, the difference was statistically significant (0.24 ±0.04 vs 0.11±0.03, t=11.01, P<0.05). Also, the protein level of PDGF was higher after treatment than that before treatment in control group, the difference was statistically significant (0.17±0.03 vs 0.12±0.03, t=6.06, P<0.05). But, the change value ( difference between before treatment and after treatment) of protein level of PDGF was higher in NPWT group than that in control group, the difference was statistically significant (0.12 ±0.03 vs 0.05 ±0.02, t=9.06, P<0.05). ConclusionNPWT obviously enhances PDGF expression of granulation tissue in diabetic foot wound and subsequently promotes wound healing.