Wnt信号调控骨髓源巨噬细胞参与肾脏纤维化的作用及机制

The role and mechanism of Wnt signaling in the regulation of bone marrow-derived macrophages involved in renal fibrosis

目的观察转化生长因子β1(TGF-β1)对骨髓来源巨噬细胞(BMDM)的影响,探讨Wnt信号调控该过程的作用与机制。方法无菌分离获取小鼠BMDM,以集落刺激因子(M-CSF)作为刺激因素,于37℃、5%CO_2培养箱中培养7d,倒置相差显微镜观察细胞形态改变,流式细胞仪鉴定BMDM纯度。后以TGF-β1作为干预因素,分别设立对照组(A组:a-MEM高糖培养基+M-CSF)、实验组(B组:a-MEM高糖培养基+M-CSF+TGF-β1),M-CSF浓度为60 ng/ml,TGF-β1浓度为5 ng/ml。以α-SMA作为肌成纤维细胞表面标志物,以F4/80作为巨噬细胞表面标志物,3 d后流式检测α-SMA^+F4/80^+细胞比例、Western blot测定各组Wnt3a、DVL(dishevelled蛋白)、β-catenin蛋白的表达。应用SPSS 19.0统计软件进行数据处理。结果 M-CSF刺激骨髓细胞7 d后流式检测BMDM比例最高,纯度达(94.44±6.11)%。以M-CSF诱导7d为分组时间点,与对照组相比较,3 d后实验组α-SMA^+F4/80^+细胞比例明显升高(t=6.365,P=0.0007),Wnt3a、DVL、β-catenin的蛋白表达也显著增高(t_(Wnt3a)=12.06,P<0.0001;t_(DVL)=8.168,P=0.0002,t_(β-catenin)=6.752,P=0.0005)。结论TGF-β1可刺激BMDM向肌成纤维细胞转化;Wnt信号通路可能参与调控BMDM的纤维化过程;通过降低巨噬细胞Wnt信号通路的活性可能成为延缓纤维化发生的潜在治疗思路。

Objective To observe the impact of transforming growth factor（ TGF-β1） on bone marrow-derived macrophages（ BMDM）,and explore whether Wnt signaling could play a regulative role in the process of fibrosis or not. Methods Mouse BMDM was obtained by aseptic isolation,and colony stimulating factor（ M-CSF） was used as a stimulating factor. The cells were cultured in a 5% CO_2 incubator for 7 days at 37 ℃. The morphology of the cells was observed by inverted phase contrast microscope. The purity of BMDM was identified by flow cytometry. TGF-β1 was used as an intervention factor to establish a control group（ group A： high glucose complete medium ＋ M-CSF） and an experimental group（ group B：high glucose complete medium ＋ M-CSF ＋ TGF-β1）,with the M-CSF concentration of 60 ng/ml and the TGF-β1 concentration of 5 ng/ml. α-SMA was used as a surface marker of myofibroblasts,and F4/80 was used as a surface marker of macrophages. After 3 days,the ratio of α-SMA^＋F4/80^＋cells was detected by flow cytometry, and expression of Wnt3a, disheveled protein（ DVL）, and β-catenin of each group was determined by Western blot. Data processing was performed with SPSS19. 0 statistical software. Results After 7 days of M-CSF stimulation,the ratio of BMDM was the highest by flow cytometry,and the purity was（ 94. 44 ± 6. 11） %. Compared with the control group,after 3 days,the ratio of α-SMA^＋F4/80~＋cells was significantly increased（ t = 6. 365,P = 0. 0007）,and the protein expression of Wnt3a,DVL,and β-catenin was also significantly increased（ t_（Wnt3a）= 12. 06,P〈0. 0001; t_（DVL）= 8. 168,P = 0. 0002,t_（β-catenin）= 6. 752,P = 0. 0005） in the experimental group. Conclusions TGF-β1 could stimulate the transformation of BMDM into myofibroblasts. Wnt signaling pathway might be involved in the regulation of BMDM fibrosis. Decreasing the activity of Wnt signaling pathway in macrophages might be a potential therapeutic approach to delaying fibrosis.