S75 Cyclical stretch induces gaq/11 mediated tgfß activation in lung fibroblasts
S75 Cyclical stretch induces gaq/11 mediated tgfß activation in lung fibroblasts
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S75 循环拉伸诱导肺成纤维细胞中 gaq/11 介导的 tgfä 激活
DOI：
10.1136/thoraxjnl-2017-210983.81
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Goodwin A
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文献类型：
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作者：
Goodwin A
方法使用Flexcell系统对尿样胚胎成纤维细胞（mef）和人肺成纤维细胞（hlf）进行周期性拉伸，拉伸方案设计为模拟相关生物的潮汐呼吸（除非另有说明，拉伸率为15%，mef为1 Hz， hlf为0.3 Hz）。western blot检测磷酸化Smad2 （pSmad2）、总Smad2和α -平滑肌肌动蛋白（αSMA）蛋白的表达。利用密度测定中pSmad2与总Smad2的比值来测定tgf - β的活化。我们比较了野生型（WT）、G α12/13-和G αq/11缺陷MEFs之间以及有和没有IPF患者的HLFs之间拉伸介导的TGFβ激活。结果循环拉伸48 h后mef和hlf中pSmad2表达显著升高（p< 0.05）。拉伸对两种细胞α - sma总蛋白表达均无影响。拉伸诱导的pSmad2在WT MEFs（拉伸率为5%、10%、15%和20%）中的表达没有“剂量-反应”关系。缺乏G αq/11信号而不缺乏G α12/13信号的MEFs在周期性拉伸时激活的tgf - β显著低于WT MEFs （p< 0.05）。来自IPF和非IPF供者的hlf在48小时的周期性拉伸后都激活了TGFβ，但IPF成纤维细胞比来自非IPF患者的成纤维细胞激活了更多的TGFβ（6个IPF细胞系和6个非IPF细胞系；p< 0.05）。结论周期性拉伸是G αq/11介导的肺成纤维细胞TGFβ活化的生理相关刺激。IPF成纤维细胞增强了拉伸介导的TGFβ激活，表明呼吸相关拉伸信号的失调是IPF进展的潜在机制。对这些途径的更深入了解可能会确定新疗法的靶点，从而阻止IPF的进展。
MethodsMurine embryonic fibroblasts (MEFs) and human lung fibroblasts (HLFs) were subject to cyclical stretch using the Flexcell system, and stretch regimens designed to mimic tidal breathing in the relevant organism (15% elongation unless otherwise stated, frequency: 1 Hz in MEFs, 0.3 Hz in HLFs). Phosphorylated Smad2 (pSmad2), total Smad2, and alpha smooth muscle actin (αSMA) protein expression were assessed by western blot. TGFβ activation was determined using the ratio of pSmad2 to total Smad2 on densitometry. Comparisons of stretch-mediated TGFβ activation were made between wild-type (WT), G α12/13-, and G αq/11-deficient MEFs, and between HLFs from patients with and without IPF.ResultsCyclical stretch induced a significant increase in pSmad2 expression after 48 hours in both MEFs and HLFs (p< 0.05). However, stretch did not affect total αSMA protein expression in either cell type. There was no'dose-response'relationship in stretch-induced pSmad2 expression in WT MEFs (5%, 10%, 15% and 20% elongation). MEFs deficient in G αq/11 signalling, but not G α12/13 signalling, activated significantly less TGFβ than WT MEFs in response to cyclical stretch (p< 0.05). HLFs from both IPF and non-IPF donors activated TGFβ after 48 hours of cyclical stretch, but IPF fibroblasts activated significantly more TGFβ than fibroblasts from patients without IPF (6 IPF and 6 non-IPF cell lines; p< 0.05).ConclusionCyclical stretch is a physiologically relevant stimulus that drives G αq/11-mediated TGFβ activation in lung fibroblasts. IPF fibroblasts have enhanced stretch-mediated TGFβ activation, indicating that dysregulation of breathing-related stretch signalling is a potential mechanism of IPF progression. A greater understanding of these pathways may identify targets for new therapies that could halt the progression of IPF.