Effects of thrombopoietin on TGFβ1-induced myofibroblast transdifferentiation in human lung fibroblasts
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摘要:
目的 探讨血小板生成素处理对TGFβ1介导的肺成纤维细胞向肌成纤维细胞转化的影响。 方法 CCK8法测定血小板生成素及TGFβ1组合处理后细胞生长的抑制率，采用荧光实时定量PCR及免疫荧光染色分别检测不同处理组细胞αSMA、COL1A2基因及蛋白的表达水平，应用免疫荧光染色检测细胞增殖相关蛋白Ki-67的表达水平，应用荧光实时定量PCR检测HO-1基因的表达水平。 结果 TGFβ1处理能够诱导肺成纤维细胞分化为肌成纤维细胞，细胞形态发生显著变化且增殖速度提高，肌成纤维细胞标志基因/蛋白αSMA及COL1A2的表达水平随诱导时间延长显著提高（P < 0.01）。在TGFβ1处理同时添加血小板生成素（TPO），αSMA、COL1A2基因和蛋白的表达水平均显著下调（P < 0.01）。此外，TPO显著抑制TGFβ1诱导的细胞增殖，且增殖相关蛋白Ki- 67的表达水平较TGFβ1处理组显著降低（P < 0.05）。此外，发现TGFβ1处理导致HO-1的表达水平下降，而添加TPO能够挽救该基因的表达水平（P < 0.05）。 结论 血小板生成素能够抑制TGFβ1诱导的肺成纤维细胞向肌成纤维细胞的转化，这一发现对肺纤维化的对症治疗具有潜在的应用价值。 Abstract:Objective To investigate the effects of thrombopoietin (TPO) on proliferation and collagen synthesis in pulmonary fibroblasts induced by TGFβ1. Methods Cultured human embryonic lung fibroblasts (HFLs) were treated with recombinant human TGF-β1 to induce myofibroblast differentiation. Different concentrations of recombinant human TPO were applied individually or in combination. Cell proliferation rate was determined using the CCK8 assay. Q-PCR and immunofluorescence assay were employed to examine the mRNA and protein expression of α-smooth muscle actin (αSMA) and type I collagen (COL1)A2. Results TGFβ1 treatment induced HFL transdifferentiation to myofibroblasts was determined by the expression of αSMA, a myofibroblast-specific marker. Cell proliferation increased during the induction. COL1 gene and protein expression were upregulated by TGFβ1 induction (P < 0.05). The TGFβ1-induced mRNA and protein expression of αSMA and COL1A2 was decreased by TPO treatment (P < 0.05), as determined by reverse transcription quantitative polymerase chain reaction and immunofluorescence analysis, respectively. The inhibitory rate showed a dose dependent effect within a certain TPO concentration range. The CCK8 assay demonstrated that TPO downregulated the TGFβ1-induced proliferation (P < 0.05). Furthermore, the expression of heme oxygenase- 1 (HO- 1) was downregulated in TGFβ1- induced lung fibroblasts, and these effects were attenuated by TPO administration (P < 0.05). Conclusions TPO can inhibit the TGFβ1-induced proliferation and differentiation of human lung fibroblasts. These effects may be mediated in part by HO-1-related signaling pathways. -
Key words:
- thrombopoietin /
- TGFβ1 /
- lung cancer /
- pulmonary fibroblast /
- collagen synthesis
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图 1 TGFβ1诱导HFL细胞向肌成纤维细胞转化
A：TGFβ1处理不同时间后HFL细胞培养形态观察（上图比例尺200 μm；下图比例尺100 μm）；B：TGFβ1处理不同时间后HFL细胞内αSMA和COL1A2基因表达水平变化；相对表达水平与处理前样本比较，GAPDH为内参基因**P<0.01；C：TGFβ1处理前后细胞内αSMA和COL1蛋白的免疫荧光检测
图 2 TPO抑制TGFβ1介导的肺成纤维细胞向肌成纤维细胞的转化
A：TGFβ1处理前后细胞c-MPL基因表达水平分析（相对表达水平与处理前样本比较，GAPDH为内参基因）；B：TGFβ1联合不同浓度TPO处理后HFL细胞培养形态观察（上图比例尺200 μm；下图比例尺100 μm）；C：TGFβ1联合不同浓度TPO处理24 h及48 h后αSMA和COL1A2基因相对表达水平检测（相对表达水平与TGFβ1单独处理样本比较，GAPDH为内参基因）*P<0.05；**P<0.01
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