外源性阻断RAGE效应对糖尿病小鼠创面中巨噬细胞浸润的影响

Effect of blocking RAGE on macrophage infiltration of diabetic wound healing

目的·通过外源性阻断糖基化终末产物受体(RAGE)后,观察RAGE效应对糖尿病小鼠创面愈合过程中巨噬细胞浸润的影响。方法·96只雄性8周龄C57BL/6J小鼠随机分成糖尿病组(n=72)和正常对照组(n=24)。多次小剂量链脲佐菌素腹腔注射诱导糖尿病小鼠模型,并用灭菌9 mm直径环钻于小鼠背部制造全层皮肤缺损创面模型。伤后正常对照组创面局部用0.9%氯化钠溶液(N组);糖尿病组再随机分成3组,糖尿病空白对照组(C组)创面局部外用0.9%氯化钠溶液,糖尿病Ig G对照组(I组)创面局部外用兔Ig G,糖尿病RAGE干预组(R组)创面局部外用RAGE抗体。再于伤后第3日和第7日,给予相同处理。通过大体评估创面愈合情况;于伤后第1、3、7日切取创缘皮肤样本,免疫组织化学染色标记并计数创面中巨噬细胞数量,透射电子显微镜下观察巨噬细胞超微结构。结果·(1)伤后第14日,R组创面愈合面积百分比显著高于C组和I组(均P=0.000)。(2)伤后第1日,N组和R组CD68+巨噬细胞数均显著多于C组(均P=0.000);伤后第14日,N组和R组巨噬细胞数均显著少于C组(均P=0.000)。(3)电子显微镜下对比创面组织的巨噬细胞形态及结构,存在明显不同。结论·糖尿病创面环境中,RAGE效应与创面巨噬细胞浸润数量及形态的异常密切相关,从而参与了糖尿病创面的难愈。

Objective·To investigate the influence of blocking receptor for advanced glycation end products （RAGE） on macrophages infiltration in diabetic wound healing. Methods·Ninety-six male C57BL/6J mice （8-week-old） were divided into diabetic group （n=72） and normal group （n=24） randomly. Diabetic mice were induced by streptozocin multiple intraperitoneal injection. One full-thickness excisional wound （diameter of 9 mm） was created by a sterilized punch. Diabetic mice were divided into 3 groups in which different topical treatments were applied to the wounds. Anti-RAGE antibody were applied in group R, rabbit IgG applied in group I, normal saline applied in group C. Normal mice were applied with saline topically （group N）. All treatments were repeated on day 3 and day 7 after wounded. The wound healing conditions were observed. The wound and surrounding tissues from animals in each group were excised on day 1, 3, and 7 after wounded. Immunohistochemistry was utilized to investigate the changes of macrophages infiltration in quantity. Macrophages were also analyzed with respect to morphology by transmission electron microscopy （TEM）. Results·① The wound closure ratio of group R was higher than those of group C and group I on day 14 after being wounded （P=0.000）. ② On day 1, the numbers of macrophages in group R and group N were both bigger than those of group C and group I, but smaller on day 14 （P=0.000）. ③ The morphological characters of macrophages also existed great differences under TEM. Conclusion·Number and morphology of macrophages are both abnormal in diabetic wound in a RAGE pathway depending manner. Based on macrophages, it suggests that impaired healing of diabetic wound is closely related to RAGE pathway.