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Title: [Influence of human amniotic mesenchymal stem cells on macrophage phenotypes and inflammatory factors in full-thickness skin wounds of mice].
Author: Shi CS, Wang DL, Sun J, Yang QX, Wei ZR, Deng CL, Xu GC, Huang GT, Xiao SE.
Journal: Zhonghua Shao Shang Za Zhi; 2020 Apr 20; 36(4):288-296. PubMed ID: 32340419.
Abstract:
Objective: To explore the influence of human amniotic mesenchymal stem cells (hAMSCs) on the in vivo and in vitro regulation of macrophage phenotypes and inflammatory factors associated with wound healing of full-thickness skin wounds in mice. Methods: Fresh amniotic membrane discarded from full-term delivery by 5 healthy pregnant women in the Department of Obstetrics and Gynecology of the Affiliated Hospital of Zunyi Medical University was used for the isolation and culture of hAMSCs by enzyme digestion method. The third passage of cells was used for identification of adipogenic and osteogenic differentiation. The fourth passage of cells was used for identification of hAMSCs surface markers. Ten C57BL/6 mice (all male, aged 6 to 8 weeks, the same gender and age below) were selected for extracting mouse peritoneal macrophages by intraperitoneal lavage, and M1-type macrophages were induced by Dulbecco's modified eagle medium (DMEM) medium containing interferon-γ. The M1-type macrophages were divided into hAMSCs+ macrophage group and macrophage alone group. Then 1×10(4) hAMSCs/per well of fourth passage were added to macrophage in hAMSCs+ macrophage group and cultured in 2 mL DMEM medium for routine culture. In macrophage alone group, each well was only added with 2 mL DMEM medium for routine culture. On day 1 and 7 in culture, the content of interleukin-12 (IL-12), arginase 1, and IL-10 in the cell culture supernatant of the 2 groups were detected by enzyme-linked immunosorbent assay with sample number of 6/per group. (2) Full-thickness skin wound model was reproduced in the back of 56 C57BL/6 mice, which were divided into hAMSCs group and phosphate buffer solution (PBS) group using the random number table, with 28 mice in each group. Mice in hAMSCs group were subcutaneously injected with 100 μL of cell suspension containing 1×10(7) hAMSCs per mL in PBS suspension along the wound edge. While mice in PBS group were only subcutaneously injected with 100 μL PBS along the wound edge. On post injection day (PID) 1, 3, 7, and 14, 7 mice in the two groups were sacrificed respectively. Histopathological observation was performed with hematoxylin-eosin staining. The expressions of macrophage surface markers [CD68 and inducible nitric oxide synthase (iNOS) double positive cells and CD68 and arginase 1 double positive] in the wounds were detected by immunofluorescent staining. The mRNA expressions of IL-10, macrophage inflammatory protein 1α (MIP-1α), and MIP-2 in the wounds were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with analysis of variance for factorial design, t test, and Bonferroni correction. Results: (1) On day 1 in culture, the content of IL-12 and arginase 1 in the cell culture supernatant of the two groups were similar (t=0.448, 0.536, P>0.05), and the content of IL-10 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group (t=14.722, P<0.01). On day 7 in culture, the content of IL-12 in the cell culture supernatant of hAMSCs+ macrophage group was significantly lower than that in macrophage alone group (t=13.226, P<0.01), and the content of arginase 1 and IL-10 was significantly higher than that in macrophage alone group (t=30.172, 31.406, P<0.01). (2) On PID 1, a large number of inflammatory cells infiltration were observed in the skin wounds of both groups. On PID 3, the inflammatory cells infiltration in the skin wounds increased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 7, the inflammatory cells infiltration in the wounds decreased in both groups, and the inflammatory cells infiltration in hAMSCs group was less than that in the PBS group. On PID 14, no obvious inflammatory cells infiltration was observed in the wounds in the two groups. (3) On PID 1 and 14, the percentages of CD68 and iNOS double positive cells and CD68 and arginase 1 double positive cells in the wounds were similar in the two groups (t(1 d)=0.134, 0.693, t(14 d)=1.146, 2.585, P>0.05). On PID 3 and 7, the percentages of CD68 and iNOS double positive cells in the wounds in hAMSCs group were significantly lower than those of PBS group (t=6.396, 4.787, P<0.01), while the percentages of CD68 and arginase 1 double positive cells were significantly higher than those of PBS group (t=3.928, 4.473, P<0.01). (4) On PID 1, the mRNA expressions of IL-10 in the wounds of mice in the two groups were similar (t=2.005, P>0.05). On PID 3, 7, and 14, the mRNA expressions of IL-10 in the wounds of mice in hAMSCs group were significantly higher than those of PBS group (t=7.758, 124.355, 80.823, P<0.01). On PID 1, 3, 7, and 14, the mRNA expressions of MIP-1α and MIP-2 in the wounds of mice in hAMSCs group (0.341±0.212, 0.648±0.004, 0.611±0.106, 0.763±0.049, 1.377±0.099, 1.841±0.042, 1.181±0.035, 0.553±0.028) were significantly lower than those of PBS group (3.853±0.035, 6.914±0.163, 3.648±0.113, 2.250±0.046, 11.119±0.495, 8.634±0.092, 5.722±0.021, 4.862±0.036, t=43.198, 101.904, 51.845, 58.231, 51.074, 177.501, 291.752, 251.614, P<0.01). Conclusions: hAMSCs demonstrates biological effects of promoting the transformation of M1-type macrophages into M2-type macrophages in full-thickness skin wounds of mice. They can up-regulate the expression of anti-inflammatory and anti-fibrotic factor IL-10, and down-regulate the expression of important inflammation mediated factors MIP-1α and MIP-2. 目的： 探讨人羊膜间充质干细胞(hAMSC)在体内外对小鼠全层皮肤缺损创面巨噬细胞表型的调控及对创面愈合相关炎症因子的影响。 方法： (1)取遵义医科大学附属医院妇产科5名足月分娩健康孕妇产后弃用的新鲜羊膜，酶消化法分离纯化培养hAMSC，取第3代细胞，经成骨、成脂诱导分化鉴定；取第4代细胞，经hAMSC表面标志物鉴定。取10只C57BL/6小鼠(均为雄性，6～8周龄，性别、鼠龄下同)，腹腔灌洗法提取小鼠腹腔巨噬细胞，用含γ干扰素的DMEM培养基培养诱导制备M1型巨噬细胞。将M1型巨噬细胞分为hAMSC＋巨噬细胞组及单纯巨噬细胞组。hAMSC＋巨噬细胞组每孔巨噬细胞加入1×10(4)个第4代hAMSC后加入DMEM培养基2 mL，常规培养；单纯巨噬细胞组每孔巨噬细胞仅加入2 mL DMEM培养基常规培养。于培养1、7 d，酶联免疫吸附测定法检测2组细胞培养上清液中白细胞介素12(IL－12)、精氨酸酶1及IL－10含量(样本数为6)。(2)取56只C57BL/6小鼠，建立背部全层皮肤缺损创面模型，按随机数字表法分为hAMSC组和磷酸盐缓冲液(PBS)组，每组28只。hAMSC组小鼠于创周皮下注射100 μL采用PBS悬浮的含1×10(7)个/mL hAMSC的细胞悬液，PBS组小鼠创周仅注射100 μL PBS。于注射后1、3、7、14 d，2组分别取7只小鼠，处死后行苏木精－伊红染色组织病理学观察，免疫荧光染色检测创面巨噬细胞表面标志物[CD68与诱导型一氧化氮合酶(iNOS)双阳性、CD68与精氨酸酶1双阳性]表达，实时荧光定量反转录PCR检测创面IL－10、巨噬细胞炎症蛋白1α(MIP－1α)、MIP－2的mRNA表达。对数据行析因设计方差分析、t检验、Bonferroni校正。 结果： (1)培养1 d，2组细胞培养上清液中IL－12、精氨酸酶1含量相近(t＝0.448、0.536，P>0.05)，hAMSC＋巨噬细胞组细胞培养上清液中IL－10含量明显低于单纯巨噬细胞组(t＝14.722，P<0.01)。培养7 d，hAMSC＋巨噬细胞组细胞培养上清液中IL－12含量明显低于单纯巨噬细胞组(t＝13.226，P<0.01)，精氨酸酶1和IL－10含量明显高于单纯巨噬细胞组(t＝30.172、31.406，P<0.01)。(2)注射后1 d，2组小鼠创面均有大量炎性细胞浸润；注射后3 d，2组小鼠创面炎性细胞浸润均增多，hAMSC组炎性细胞浸润较PBS组少；注射后7 d，2组小鼠创面炎性细胞浸润均减少，hAMSC组炎性细胞浸润较PBS组少；注射后14 d，2组小鼠创面均未见明显炎性细胞浸润。(3)注射后1、14 d，2组小鼠创面CD68与iNOS双阳性细胞百分比、CD68与精氨酸酶1双阳性细胞百分比相近(t(1 d)＝0.134、0.693，t(14 d)＝1.146、2.585，P>0.05)；注射后3、7 d，hAMSC组小鼠创面CD68与iNOS双阳性细胞百分比明显低于PBS组(t＝6.396、4.787，P<0.01)，CD68与精氨酸酶1双阳性细胞百分比明显高于PBS组(t＝3.928、4.473，P<0.01)。(4)注射后1 d，2组小鼠创面IL－10的mRNA表达相近(t＝2.005，P>0.05)；注射后3、7、14 d，hAMSC组小鼠创面IL－10的mRNA表达明显高于PBS组(t＝7.758、124.355、80.823，P<0.01)。注射后1、3、7、14 d，hAMSC组小鼠创面MIP－1α和MIP－2的mRNA表达(0.341±0.212、0.648±0.004、0.611±0.106、0.763±0.049，1.377±0.099、1.841±0.042、1.181±0.035、0.553±0.028)均明显低于PBS组(3.853±0.035、6.914±0.163、3.648±0.113、2.250±0.046，11.119±0.495、8.634±0.092、5.722±0.021、4.862±0.036，t＝43.198、101.904、51.845、58.231，51.074、177.501、291.752、251.614，P<0.01)。 结论： hAMSC具有促进小鼠全层皮肤缺损创面M1型巨噬细胞向M2型巨噬细胞转化的生物学效应；可以上调抗炎及抗纤维化因子IL－10的表达，下调重要的炎症反应介导因子MIP－1α和MIP－2的表达。.

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