混合谱系激酶结构域样蛋白对小鼠巨噬细胞和成纤维细胞间相互作用的影响

doi:10.16431/j.cnki.1671-7236.2025.12.001

摘要/Abstract

摘要： 【目的】探究混合谱系激酶结构域样蛋白(mixed lineage kinase domain-like protein，MLKL)在小鼠皮肤伤口愈合过程中对巨噬细胞和成纤维细胞的影响及其调控机制。【方法】建立C57BL/6J小鼠和MLKL基因敲除(MLKL^-/-)小鼠皮肤创伤模型。采用免疫组化法检测小鼠皮肤损伤后MLKL的动态表达及巨噬细胞和成纤维细胞的募集情况。通过体外试验，建立成纤维细胞与M1/M2型巨噬细胞条件培养基(M1/M2 macrophages conditioned medium，M1/M2ø CM)共培养体系。通过Western blotting检测成纤维细胞中转化生长因子-β(transforming growth factor-β，TGF-β)、成纤维细胞生长因子-2(fibroblast growth factor 2，FGF-2)和基质金属蛋白酶9(matrix metalloproteinase-9，MMP-9)的蛋白表达情况。将M1/M2型巨噬细胞与成纤维细胞条件培养基(fibroblast conditioned medium，FBCM)共培养，通过ELISA检测M1型巨噬细胞中白细胞介素-6(interleukin-6，IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)分泌情况。通过ELISA和Western blotting分别检测M2型巨噬细胞中IL-10分泌量及精氨酸酶(Arginase)蛋白表达情况。【结果】免疫组化染色结果显示，C57BL/6J小鼠皮肤伤口愈合第3、5和7天均检测到MLKL阳性信号，MLKL表达水平随伤口愈合时间的延长而升高，且MLKL^-/-小鼠伤口周围CD86呈阳性的M1型巨噬细胞、CD163呈阳性的M2型巨噬细胞和Vimentin呈阳性的成纤维细胞的细胞数量少于C57BL/6J小鼠。在共培养体系中，与未处理组相比，C57BL/6J来源的M1ø CM和M2ø CM处理显著提高了成纤维细胞中TGF-β、FGF-2和MMP-9蛋白表达水平(P＜0.05)，而MLKL^-/-来源的M1ø CM和M2ø CM处理后，成纤维细胞中FGF-2和MMP-9蛋白表达量无显著变化(P＞0.05)，TGF-β蛋白表达量显著降低(P＜0.05)。C57BL/6J FBCM处理巨噬细胞后，M1型巨噬细胞中IL-6和TNF-α含量及M2型巨噬细胞中IL-10含量和精氨酸酶蛋白表达量均显著升高(P＜0.05)；MLKL^-/- FBCM处理巨噬细胞后，相关细胞因子含量及蛋白表达量均低于C57BL/6J FBCM处理组(P＜0.05)。【结论】MLKL可通过调控巨噬细胞和成纤维细胞的相互作用，影响巨噬细胞的募集和成纤维细胞的增殖能力，从而促进皮肤伤口愈合。

关键词: 成纤维细胞; 巨噬细胞; MLKL; 伤口愈合

Abstract: 【Objective】 This experiment aimed to explore the effects of mixed lineage kinase domain-like protein (MLKL) on macrophages and fibroblasts during the process of skin wound healing in mice and its regulatory mechanism. 【Method】 The back trauma models of C57BL/6J mice and MLKL gene knockout (MLKL^-/-) mice were established.Immunohistochemistry was used to detect the dynamic expression of MLKL,as well as the recruitment of macrophages and fibroblasts after skin injury in mice.Through in vitro experiments,a co-culture system of fibroblasts and M1/M2 macrophages conditioned medium (M1/M2ø CM) was established.Western blotting was performed to evaluate the protein expression of transforming growth factor-β (TGF-β),fibroblast growth factor-2 (FGF-2),and matrix metalloproteinase-9 (MMP-9) in fibroblasts.M1/M2 macrophages were co-cultured with fibroblast-conditioned medium (FBCM).The secretion of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in M1 macrophages was measured by ELISA.The secretion of IL-10 and the expression of Arginase protein in M2 macrophages were determined by ELISA and Western blotting,respectively. 【Result】 The immunohistochemical staining results showed that positive MLKL signals were detected on the 3rd,5th and 7th days of skin wound healing in C57BL/6J mice.The expression level of MLKL increased with the prolongation of wound healing time.Moreover，the number of CD86-positive M1 macrophages,CD163-positive M2 macrophages and Vimentin-positive fibroblasts around the wound in MLKL^-/- mice was less than that in C57BL/6J mice.In co-culture experiments,compared with the untreated group,the treatment of M1ø CM and M2ø CM derived from C57BL/6J significantly increased the protein expression levels of TGF-β,FGF-2 and MMP-9 in fibroblasts (P＜0.05),whereas after treatment with M1ø CM and M2ø CM derived from MLKL^-/-，there was no significant difference in the protein expression of FGF-2 and MMP-9 in fibroblasts (P＞0.05),while the expression level of TGF-β protein was significantly decreased (P＜0.05).After macrophages were treated with C57BL/6J FBCM,the contents of IL-6 and TNF-α in M1 macrophages,the content of IL-10 and the expression level of Arginase protein in M2 macrophages were significantly increased (P＜0.05).After macrophages were treated with MLKL^-/- FBCM,the contents of related cytokines and protein expression levels were both lower than those in C57BL/6J FBCM treatment group (P＜0.05). 【Conclusion】 MLKL could promote skin wound healing by regulating the interaction between macrophages and fibroblasts,influencing the recruitment of macrophages，thereby promoting the healing of skin wounds.

Key words: fibroblast; macrophages; MLKL; wound healing

中图分类号:

S858.31

董妍沁, 赵佳敏, 包文慧, 巩志国, 任佩佩, 王奕心, 代佳山, 赵非凡, 张双翼, 刘博. 混合谱系激酶结构域样蛋白对小鼠巨噬细胞和成纤维细胞间相互作用的影响[J]. 中国畜牧兽医, 2025, 52(12): 5529-5538.

DONG Yanqin, ZHAO Jiamin, BAO Wenhui, GONG Zhiguo, REN Peipei, WANG Yixin, DAI Jiashan, ZHAO Feifan, ZHANG Shuangyi, LIU Bo. Effect of MLKL on the Interaction Between Macrophages and Fibroblasts in Mice[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 5529-5538.

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