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目的EGF对创面修复具有积极作用,EGF受体(EGF receptor,EGFR)可调节皮肤创面愈合,通过建立糖尿病大鼠烫伤创面模型,观察局部应用重组人EGF(recombinant human EGF,rhEGF)联合血糖控制对创面EGFR及其mRNA的影响,探讨rhEGF促进创面愈合的机制。方法136只清洁级雄性Wistar大鼠,体重(188.57±6.59)g,随机分为A、B、C、D组(n=34)。A、B、C组大鼠腹腔内一次性注射链脲佐菌素(60mg/kg)制备糖尿病模型,D组于对应时间点注射等量柠檬酸缓冲液,作为正常对照组。糖尿病模型成功制备后8周,取4组大鼠背部去毛后置于80℃热水6s,制备背部深Ⅱ度烫伤模型。A组于烫伤模型制备前1周开始控制血糖至D组水平;模型制备24h内创面清创后喷洒150U/cm2rhEGF,含1%磺胺嘧啶银霜剂辅料缝扎固定;B组不控制血糖,伤后处理同A组;C组同A组方法控制血糖,模型制备后24h内采用含1%磺胺嘧啶银霜剂辅料缝扎固定;D组伤后处理同A组。烫伤后3、7、11、15、21d测定各组大鼠创面愈合率;并于烫伤后1、3、5、7、11、15、21d,取创面皮肤组织采用mRNA原位杂交法检测EGFR mRNA表达、免疫组织化学染色及Western blot检测EGFR蛋白表达情况结果各组大鼠均存活至实验完成。烫伤后3d各组创面愈合率比较,差异无统计学意义(P>0.05);7、11、15、21d,A、D组高于B、C组(P<0.05);A、D组间及B、C组间比较,差异均无统计学意义(P>0.05)。mRNA原位杂交观察显示,烫伤后各组均有EGFR mRNA表达,主要分布于真皮的成纤维细胞、毛细血管内皮细胞及创缘残存的皮肤附属器上皮中;A、B、C、D组分别于烫伤后5、7、7、11d表达达峰值,且A、D组峰值较B、C组高,差异有统计学意义(P<0.05);A、D组间及B、C组间差异无统计学意义(P>0.05)。免疫组织化学染色及Western blot检测均可见各组EGFR蛋白表达,并逐渐增强,A、B、C、D组分别于烫伤后7、7、11、11d达峰值,A、D组间及B、C组间比较差异无统计学意义(P>0.05),B、C组与A、D组比较,差异有统计学意义(P<0.05)。结论联合血糖控制以及局部应用rhEGF可促进糖尿病大鼠烫伤创面愈合,并能有效刺激EGFRmRNA及EGFR蛋白表达。
Objective EGF has a positive effect on wound healing. EGF receptor (EGFR) regulates skin wound healing. By establishing a scald wound model in diabetic rats, we observed the effect of topical application of recombinant human EGF (rhEGF) combined with glycemic control Wound surface EGFR and its mRNA, to explore the mechanism of rhEGF to promote wound healing. Methods Thirty - six male Wistar rats of clean grade were randomly divided into A, B, C and D groups (n = 34), weighing 188.57 ± 6.59 g. Rats in group A, B and C were given a single intraperitoneal injection of streptozotocin (60mg / kg) to prepare diabetic model. Group D received the same amount of citrate buffer at the corresponding time points as control group. Eight weeks after the successful preparation of the diabetic model, four groups of rats were removed from the back and placed in hot water at 80 ° C for 6 seconds to prepare a deep second-degree scald burn model. Group A received blood glucose control to level D one week before preparation of scald model. After wounding, the wounds were sprayed with 150U / cm2rhEGF after 1 year and wounds fixed with 1% sulfadiazine cream. The rats in group B did not control blood sugar and injury After treatment with the A group; C group with A group method to control blood glucose, 24h after model preparation with 1% sulfadiazine silver cream accessories suture fixation; D group after injury treatment with the A group. At 3, 7, 11, 15 and 21 days after scald, the wound healing rate of each group was measured. At 1, 3, 5, 7, 11, 15 and 21 days after scald, the skin tissues of wounds were detected by in situ hybridization The mRNA expression, immunohistochemical staining and Western blot were used to detect the expression of EGFR protein. Results All rats survived until the experiment was completed. There was no significant difference in wound healing rate between the three groups after burn for 3 days (P> 0.05), while the levels in groups A and D were higher than those in groups B and C at 7, 11, 15 and 21 days (P <0.05) There was no significant difference between B and C groups (P> 0.05). The results of in situ hybridization showed that EGFR mRNA was expressed in all groups after scald, which mainly distributed in dermal fibroblasts, capillary endothelial cells and epithelial remnant epithelium of wounds. Groups A, B, C and D The peak value at 5, 7, 7 and 11 days after scald was reached, and the peak values in group A and group D were higher than those in group B and C (P <0.05). There was no significant difference between group A and group D No statistical significance (P> 0.05). Immunohistochemical staining and Western blot showed that the expression of EGFR protein in each group was gradually increased. The peak values of A, B, C and D reached the peak at 7, 7, 11, There was no significant difference between C and C groups (P> 0.05). There was significant difference between B and C groups and A and D groups (P <0.05). Conclusion Combined blood glucose control and topical application of rhEGF can promote wound healing of diabetic rats and can effectively stimulate the expression of EGFR mRNA and EGFR protein.