旁分泌TGF-β1促进缺氧诱导的肉鸡肺血管平滑肌细胞增殖与迁移

Paracrine TGF-β1 promotes hypoxia-induced proliferation and migration of pulmonary vascular smooth muscle cells in broiler chicken

制备禽转化生长因子-β1(transforming growth factor-β1,TGF-β1)重组蛋白(chTGF-β1)和禽特异性TGF-β1抗体,采用免疫组化法检测肺动脉高压(pulmonary arterial hypertension,PAH)肉鸡和正常肉鸡肺血管中TGF-β1的表达。分离培养肉鸡肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)和内皮祖细胞(endothelial progenitor cells,EPCs),构建低氧细胞模型,采用Western blot检测TGF-β1表达;将常氧(21%O_(2))和缺氧(5%O_(2))培养的PASMCs用chTGF-β1(20μg/L)处理24 h,采用CCK-8试验和细胞划痕试验分别检测细胞增殖和迁移。将肺动脉节段置于常氧和缺氧条件下培养48 h,采用免疫组化法检测肺动脉中膜Ⅰ型TGF-β受体(TGFβR-1)的表达。结果显示,TGF-β1在正常肉鸡肺小动脉中膜中仅有微弱表达,在PAH肉鸡肺小动脉中膜中表达增强;chTGF-β1可促进低氧诱导的PASMC增殖及迁移;低氧对PASMCs中TGF-β1表达无显著性影响,但可显著促进EPCs表达TGF-β1;在低氧条件下,离体培养的肺动脉中膜Ⅰ型TGF-β受体(TGFβR-1)表达升高。结果表明,在PAH发病过程中,TGF-β1可能通过旁分泌方式作用于PASMCs,促进肺血管中膜肥厚。

This study was conducted to investigate the effect of transforming growth factor(TGF)-β1 on the proliferation and migration of broiler pulmonary vascular smooth muscle cells(PAMSCs)induced by hypoxia,aiming to reveal the role of TGF-β1 in pulmonary vascular remodeling in broilers with pulmonary arterial hypertension(PAH).Chicken TGF-β1 recombinant protein(chTGF-β1)was prepared and used to produce anti-chicken TGF-β1 antibody.The expression of TGF-β1 in lung tissue of PAH broilers and normal broilers was determined by immunohistochemistry method.Broiler PASMCs and endothelial progenitor cells(EPCs)were isolated and used to establish hypoxic cell model,and were subjected to Western blot analysis for TGF-β1.A CCK-8 experiment and wound healing experiment were conducted to detect proliferation and migration of PASMCs treated with chTGF-β1(20μg/L)under normoxia(21%O_(2))and hypo xia(5%O_(2)).Pulmonary artery was cultured under normoxia or hypoxia,and the expression of typeⅠTGF-βreceptor(TGFβR-1)was determined by immunohistochemistry.The results demonstrated a weak expression of TGF-β1 in the media of pulmonary arterioles in normal broilers.In contrast,PAH broilers had increased TGF-β1 expression in pulmonary arterioles.Hypoxia exposure induced TGF-β1 expression in EPCs but not in PASMCs.However,TGF-β1 treatment enhanced hypoxia-induced proliferation and migration of cultured PASMCs.Hypoxia exposure resulted in an increase of TGFβR-1 expression in the media of ex vivo pulmonary arteries.The results suggest that TGF-β1 acts on PASMCs in a paracrine manner to promote media hypertrophy in PAH broilers.