脱细胞真皮基质对M1型巨噬细胞向M2型巨噬细胞转变的影响

Effect of acellular dermal matrix on transformation of M1-type macrophages into M2-type macrophages

目的观察脱细胞真皮基质(ADM)对巨噬细胞特征性标志物表达的影响。方法实验分组:A组,巨噬细胞组;B组,ADM(5μg/μl)与巨噬细胞共培养组;C组,脂多糖(LPS,100 ng/ml)及γ-干扰素(IFN-γ)(10 ng/ml)联合诱导巨噬细胞组;D组,白细胞介素-4(IL-4,10 ng/ml)诱导巨噬细胞组;E组,LPS(100 ng/ml)及IFN-γ(10 ng/ml)联合诱导巨噬细胞后再与ADM(5μg/μl)共培养组。采用实时反转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western blot)检测不同组巨噬细胞一氧化氮合成酶(iNOS)、分化抗原86(CD86)、甘露糖受体(CD206)和精氨酸酶-1(Arg-1)mRNA和蛋白的相对表达水平,组间比较采用t检验,多组间采用单因素方差分析。结果RT-PCR检测结果显示,M1型巨噬细胞标志物iNOS、CD86的表达在C组中最高(2.82±0.07、3.07±0.24,FiNOS=210.3,FCD86=85.6,P<0.01)。M2型巨噬细胞标志物CD206、Arg-1的表达在D组中最高(3.30±0.26、3.23±0.32,FCD206=115.4,FArg-1=71.2,P<0.01)。B组与A组比较,M1型和M2型巨噬细胞标志物的表达差异无统计学意义(t=1.706,P>0.05)。E组与C组比较,M1型巨噬细胞标志物iNOS、CD86的表达量下降,分别由C组的2.82±0.07、3.07±0.24下降到E组的2.21±0.10、1.96±0.04;而M2型巨噬细胞标志物CD206、Arg-1的表达量增高,分别由C组的1.01±0.16、0.97±0.16上升到E组的1.87±0.14、1.91±0.14;两组差异有统计学意义(tiNOS=8.523,tCD86=7.702,tCD206=7.028,tArg-1=7.904,P<0.05)。Western blot检测结果与RT-PCR的检测结果趋势一致。结论ADM有诱导巨噬细胞从M1型向M2型极化的作用。

Objective To observe the effect of acellular dermal matrix(ADM)on the expression of characteristic markers of macrophages.Methods Experimental groups were set up:group A,macrophage group;group B,ADM(5μg/μL)and macrophage co-culture group;group C,lipopolysaccharide(LPS,100 ng/ml)and interferon-γ(IFN-γ,10 ng/ml)combined with macrophage induction group;group D,interleukin-4(IL-4,10 ng/ml)induced macrophage group;group E,macrophages induced by LPS(100 ng/ml)and IFN-γ(10 ng/ml)and co-cultured with ADM(5μg/μL).Real-time reverse transcription-polymer-chain reaction(RT-PCR)and Western blotting were used to detect the mRNA and protein expression levels of inducible nitric oxide synthase(iNOS),CD86,CD206 and Arg-1 in macrophages.GraphPad 7.0 statistical software was used.Experimental data were expressed as±s,the t test was used for comparison between the two groups,and one-way ANOVA was used for multiple groups.Results The RT-PCR revealed the similar results of mRNA and protein quantification.The expression of iNOS and CD86,markers of M1 macrophage,was the highest in group C(2.82±0.07,3.07±0.24,FiNOS=210.3,FCD86=85.6,P<0.01).The expression of M2-type macrophage markers CD206 and Arg-1 was the highest in group D(3.30±0.26,3.23±0.32,FCD206=115.4,FArg-1=71.2,P<0.01).There was no significant difference in the expression of M1-type and M2-type macrophage markers between group A and group B(t=1.706,P>0.05).Compared with group C,the expression levels of iNOS and CD86 in M1-type macrophages decreased from 2.82±0.07 and 3.07±0.24 in group C to 2.21±0.10 and 1.96±0.04 in group E,respectively.The expression levels of CD206 and Arg-1 increased from 1.01±0.16 and 0.97±0.16 in group C to 1.87±0.14 and 1.91±0.14 in group E,respectively.The difference between the two groups was statistically significant(tiNOS=8.523,tCD86=7.702,tCD206=7.028,tArg-1=7.904,P<0.05).Western blotting results were consistent with the RT-PCR results.Conclusion ADM can induce the polarization trend of macrophages from M1-type to M2-type.