生长停滞特异性蛋白6调控巨噬细胞极化在小鼠创面修复中的作用

Role of growth arrest specifi c protein 6 in regulating macrophage polarization in wound healing in mice

目的探讨生长停滞特异性蛋白6(growth arrest-specifi c protein 6,Gas6)调控巨噬细胞极化在创面修复中的作用。方法采用清洁级雄性B6小鼠随机(随机数字法)分为正常组、皮肤缺损组、皮肤缺损组+生理盐水组、皮肤缺损+Gas6(1μg)组、皮肤缺损+Gas6(5μg)组和皮肤缺损+Gas6(10μg)组,每组16只,其中10只用来观察各组小鼠皮肤创面愈合情况,剩余6只于造模后第5天分离创面组织巨噬细胞,酶联免疫吸附法(ELISA)检测IL-6,IL-10水平,RTPCR检测精氨酸酶-1(Arg-1)、诱导性一氧化氮合酶(iNOS)的mRNA表达水平,流式细胞术检测巨噬细胞M1型标记物CD197和M2型标记物CD163、F4/80的表达,HE染色检测皮肤创面病理改变,Masson染色分析创面肉芽组织及胶原沉积情况。结果皮肤缺损造模后第3天伤口表面开始结痂。Gas6治疗组伤口面积小于PBS组,伤口愈合情况优于PBS组。与正常组相比,皮肤缺损组小鼠第5天巨噬细胞CD197的比例升高明显(P=0.0049)、CD163和F4/80双阳性的比例明显降低(P=0.0086)、IL-6水平明显升高(P=0.0013)、IL-10水平明显升高(P=0.0014)、iNOS mRNA水平明显升高(P=0.008)、Arg-1 mRNA水平明显降低(P=0.0121)、组织炎症浸润加重。与PBS对照组相比,Gas6治疗组CD197的比例明显下降(P=0.000)、CD163和F4/80双阳性比例明显升高(P=0.000)、IL-6水平明显降低(P=0.000)、IL-10水平明显升高(P=0.0003)、iNOS mRNA水平明显降低(P=0.0018)、Arg-1 mRNA水平明显升高(P=0.001)、组织炎性细胞减少,胶原纤维增多。结论Gas6可促使皮肤缺损小鼠巨噬细胞由M1向M2转化,加快缺损皮肤创面愈合。

Objective To investigate the role of growth arrest specific protein 6(Gas6)in regulating macrophage polarization in wound healing.Methods Clean male B6 mice were randomly(random number)divided into the normal group,skin defect group,skin defect group+normal saline group(PBS group),skin defect+Gas6(1μg)group,skin defect+Gas6(5μg)group,and skin defect+Gas6(10μg)group.Ten mice in each group were used to observe the healing of skin wounds.Macrophages were isolated from the wound tissues of the remaining 6 mice on the fi fth day after modeling.The levels of IL-6 and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA),the mRNA expression levels of arginase-1(Arg-1)and inducible nitric oxide synthase(iNOS)were detected by RTPCR,and fl ow cytometry was used to detect the expression of M1 marker CD197,M2 marker CD163 and F4/80.HE staining was used to detect the pathological changes of skin wounds.Masson staining was used to analyze the granulation tissue and collagen deposition.Results Scab began to form on the surface of the wound on the third day after the skin defect model was established.The wound area of the Gas6 treatment group was smaller than that of the PBS group,and the wound healing was better than that of the PBS group.Compared with the normal group,the proportion of CD197 in macrophages of the skin defect group was signifi cantly increased(P=0.0049),the proportion of CD163 and F4/80 double positive was signifi cantly decreased(P=0.0086),the level of IL-6 was signifi cantly increased(P=0.0013),the level of IL-10 was signifi cantly increased(P=0.0014),the level of iNOS mRNA was signifi cantly increased(P=0.008),and Arg-1 was signifi cantly increased in the skin defect group The mRNA level was signifi cantly decreased(P=0.0121),and the infl ammatory infi ltration was aggravated.Compared with the PBS group,the proportion of CD197 in the Gas6 treatment group was signifi cantly decreased(P=0.000),the double positive rates of CD163 and F4/80 were signifi cantly increased(P=0.000),the level of IL-6 was signifi cantly decreased(P=0.000),the level of IL-10 was signifi cantly increased(P=0.0003),the level of iNOS mRNA was signifi cantly decreased(P=0.0018),the level of Arg-1 mRNA was signifi cantly increased(P=0.001),and the number of inflammatory cells and the number of collagen fibers were increased.Conclusions Gas6 can promote the transformation of macrophages from M1 to M2 in mice with skin defect,which is benefi cial to the wound healing of skin defect.