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目的制备NGF-胰岛素复合凝胶并观察其对大鼠深Ⅱ度烫伤创面愈合的影响。方法以卡波姆980为基质,分别加入NGF 4 000 U、胰岛素800 U,制备胰岛素凝胶、NGF凝胶、NGF-胰岛素复合凝胶。观察NGF-胰岛素复合凝胶性状,并行体外药物释放检测。75只SPF级雄性Wistar大鼠,体重200～250 g,随机分为正常对照组(A组)、糖尿病对照组(B组)、局部胰岛素凝胶治疗组(C组)、局部NGF凝胶治疗组(D组)、局部NGF-胰岛素复合凝胶治疗组(E组),每组15只。B、C、D、E组大鼠采用一次性腹腔注射链脲佐菌素(55 mg/kg)制备1型糖尿病模型,A组注射相同剂量柠檬酸钠缓冲液。造模成功后,采用恒温水浴箱法于各组大鼠背部制备深Ⅱ度烫伤模型。A、B组创面外敷空白凝胶基质,C、D、E组对应外敷胰岛素凝胶、NGF凝胶、NGF-胰岛素复合凝胶,每天换药1次。烫伤后观察各组大鼠存活情况,于3、7、11、15、21 d观察创面愈合情况并计算创面愈合率,各时间点每组处死3只大鼠取皮肤全层标本行组织学及免疫组织化学染色观察。结果制备的NGF-胰岛素复合凝胶清亮透明,保湿性及黏附性良好,易于涂抹及清洗。体外释放检测示NGF-胰岛素复合凝胶释药时间可达24 h以上,且30 d内稳定性良好。各组大鼠均存活至实验完成。烫伤后3 d各组创面无缩小,7、11、15、21 d时E组创面愈合率最高,B组最低,与其余各组比较以及E、B组间比较,差异均有统计学意义(P<0.05)。组织学观察示各时间点E组肉芽组织和胶原纤维生长均优于其余各组。免疫组织化学染色示各组CD34、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)均于3 d开始表达,且随时间延长阳性细胞逐渐增多;各时间点E组微血管密度及PCNA最高,B组最低,与其余各组比较以及E、B组间比较差异均有统计学意义(P<0.05)。结论局部应用NGF-胰岛素复合凝胶可显著促进大鼠深Ⅱ度烫伤创面愈合。
Objective To prepare NGF-insulin complex gel and observe its effect on deep second degree scald wound healing in rats. Methods Carbopol 980 was used as matrix to add 4 000 U of NGF and 800 U of insulin respectively to prepare insulin gel, NGF gel and NGF-insulin composite gel. To observe the properties of NGF-insulin complex gel and to test in vitro drug release. Seventy-five SPF male Wistar rats weighing 200-250 g were randomly divided into normal control group (group A), diabetic control group (group B), local insulin gel treatment group (group C) and local NGF gel treatment Group (group D), local NGF-insulin gel group (group E), 15 rats in each group. Rats in groups B, C, D and E were given type 1 diabetes mellitus by intraperitoneal injection of streptozotocin (55 mg / kg), and group A was injected with the same dosage of sodium citrate buffer. After successful modeling, a deep Ⅱ degree scald model was prepared on the back of rats in each group by a constant temperature water bath method. A and B groups were treated with blank gel matrix. Groups C, D and E were given external insulin gel, NGF gel and NGF-insulin composite gel once a day. Survival of rats in each group was observed after scald. Wound healing was observed at 3, 7, 11, 15, and 21 days. Wound healing rate was calculated. Three groups of rats were killed at each time point. Immunohistochemical staining. Results The prepared NGF-insulin complex gel was clear and bright, moisturizing and good adhesion, easy to apply and clean. In vitro release assay showed that the release time of NGF-insulin composite gel was up to 24 h, and the stability was good within 30 d. Rats in each group survived until the experiment was completed. The wound healing rate of group E was the highest on the 3rd, 7th, 15th, 21st and 21st day after burn, the lowest was in group B, the difference was statistically significant compared with the other groups (P <0.05) P <0.05). Histological observation showed that the growth of granulation tissue and collagen fibers in group E were better than the other groups at all time points. Immunohistochemical staining showed that the expression of CD34 and proliferating cell nuclear antigen (PCNA) in each group began to be expressed on the 3rd day, and the number of positive cells gradually increased with time; the microvessel density and PCNA in group E were the highest at each time point, while the lowest in group B , Compared with the rest of the groups and the differences between the E and B groups were statistically significant (P <0.05). Conclusion Topical application of NGF-insulin composite gel can significantly promote deep Ⅱ degree scald wound healing in rats.