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目的探讨局部应用NGF联合胰岛素对糖尿病大鼠烫伤创面血管中凋亡相关因子Bcl-2、Bax表达的影响及创面愈合的机制。方法取75只清洁级雄性Wistar大鼠,体重200～220 g,随机分为正常对照组(A组)、糖尿病对照组(B组)、胰岛素治疗组(C组)、NGF治疗组(D组)、NGF联合胰岛素治疗组(E组),每组15只。B、C、D、E组大鼠采用两步给药法腹腔注射链脲佐菌素(streptozotocin,STZ)建立糖尿病模型,STZ剂量分别为第1天10 mg/kg,第3天50 mg/kg;A组给予相同剂量柠檬酸缓冲液。模型制备后1个月,采用水蒸气烫伤法于各组大鼠背部制备2个深Ⅱ度烫伤创面。烫伤模型制备后,A、B组创面外敷3层生理盐水纱布;C组创面外敷3层浸润5 U胰岛素诺和灵30R的纱布,并每日腹部皮下注射诺和灵30R 4～6 U/kg;D组创面外敷3层浸润5 mL NGF溶液(25 U/mL)的纱布;E组联合C、D组方法处理。观察大鼠一般情况,伤后7、11、15、21 d大体观察各组创面愈合情况并计算创面愈合率,伤后3、7、11、15、21 d取创面组织行组织学及免疫组织化学染色观察,检测创面Bcl-2、Bax、CD34的表达并计算微血管密度。结果各组大鼠均存活至实验完成。随时间延长,各组创面逐渐缩小,其中E组创面愈合速度、皮肤角化、毛发生长及肉芽组织和胶原纤维生长均优于其余各组。伤后各时间点E组创面愈合率均高于其余各组,差异有统计学意义(P<0.05)。伤后随时间延长,各组CD34、Bcl-2表达逐渐增强,至15 d达高峰,21 d表达减弱;E组各时间点表达均强于其他各组(P<0.05)。伤后3 d各组均未见Bax表达,7 d后开始见新生血管内皮细胞Bax表达,且随时间延长表达逐渐增强,其中E组表达强度均低于其余各组(P<0.05)。结论局部联合应用NGF和胰岛素可通过抑制创面血管内皮细胞凋亡、增加创面血管生成,促进糖尿病大鼠创面愈合。
Objective To investigate the effect of topical application of NGF combined with insulin on the expression of Bcl-2 and Bax in blood vessels of scalded wounds in diabetic rats and the mechanism of wound healing. Methods Totally 75 male Wistar rats of clean grade weighing 200-220 g were randomly divided into three groups: normal control group (group A), diabetic control group (group B), insulin treatment group (group C), NGF treatment group (group D) ), NGF combined with insulin treatment group (E group), 15 rats in each group. The rats in groups B, C, D and E were given diabetes mellitus by two-step intraperitoneal injection of streptozotocin (STZ). The STZ doses were 10 mg / kg on day 1, 50 mg / kg; Group A was given the same dose of citrate buffer. One month after the model preparation, 2 deep Ⅱ degree scald wounds were prepared on the back of rats in each group by steam scalding method. After the scald model was prepared, three layers of saline gauze were applied on the wounds of group A and group B. The wounds of group C were infiltrated with 5U insulin and noradren 30R gauze on the wound surface of group C, and noradren 30R 4 ~ 6 U / kg ; Group D wound surface with three layers of 5ml NGF solution (25 U / mL) gauze; Group E combined C, D group method. The general condition of the rats was observed. The healing status of the wounds in each group was observed on the 7th, 11th, 15th and 21st day after injury. The wound healing rate was calculated. At 3, 7, 11, 15 and 21 days after wounding, the histology and immunohistochemistry The expression of Bcl-2, Bax, CD34 in wounds was detected by chemical staining and the microvessel density was calculated. Results All rats survived until the experiment was completed. With the extension of time, wounds in all groups gradually decreased. The wound healing rate, skin keratosis, hair growth, granulation tissue and collagen fiber growth in group E were better than those in other groups. The wound healing rate of group E at each time point after wounding was higher than that of the other groups, the difference was statistically significant (P <0.05). Over time, the expressions of CD34 and Bcl-2 in each group increased gradually and peaked on day 15 and decreased on day 21. The expression of CD34 and Bcl-2 in each group was stronger than other groups at each time point (P <0.05). No expression of Bax was observed in each group 3 days after injury, and Bax expression was detected in neovascular endothelial cells 7 days later. The expression of Bax gradually increased with time, and the expression intensity of group E was lower than that of the other groups (P <0.05). Conclusion Local combination of NGF and insulin can inhibit wound vascular endothelial cell apoptosis, increase wound angiogenesis and promote wound healing in diabetic rats.