摘要
目的 研究PPAR-γ、PDGF-βR及FAK在肺动脉平滑肌细胞迁移中的分子调控机制。方法 分离大鼠肺动脉平滑肌细胞,培养至3-7代细胞用于实验。分为4组,CON组:常规培养细胞;PDGF-BB组:细胞同步化后加入PDGF-BB(20 ng/ml),刺激1 h后裂解细胞;Y15组:细胞同步化后加入PDGF-BB(20 ng/ml)预刺激1 h,再加入FAK抑制剂Y15(10 μmol/L)刺激2 h后裂解细胞;ROSI组:细胞同步化后加入罗格列酮(10 μmol/L)刺激1 h,再加入PDGF-BB刺激1 h后裂解细胞。裂解各组处理细胞,Western blot检测各组p-PDGF-βR、p-FAK(Tyr397)水平;划痕实验及Transwell方法比较细胞迁移能力。结果 PDGF-BB组细胞迁移能力较CON组明显增强(77.3%± 4.2% vs 61.3%± 3.5%, P <0.05),p-PDGF-βR、p-FAK(Tyr397)水平提高(2.2 ± 0.12 vs 0.93 ± 0.11;1.07 ± 0.08 vs 0.39 ± 0.02, P <0.05)。Y15组细胞迁移能力较PDGF-BB组明显降低(72.7%± 4.7% vs 77.3%± 4.2%, P <0.05),p-PDGF-βR无明显变化(2.0 ± 0.10 vs 2.2 ± 0.12, P >0.05),p-FAK(Tyr397)水平降低(0.48 ± 0.09 vs 1.07 ± 0.08, P <0.05)。ROSI组细胞迁移能力较Y15组差异无统计学意义(75.3%± 4.04% vs 72.7%± 4.70%, P >0.05),p-PDGF-βR水平降低(1.25 ± 0.10 vs 2.0 ± 0.10, P <0.05);p-FAK(Tyr397)水平提高(0.52 ± 0.07 vs 0.48 ± 0.09, P <0.05);而与PDGF-BB组比较,ROSI组细胞迁移能力(77.3%± 4.2% vs 75.3%± 4.04%)及p-PDGF-βR(2.2 ± 0.12 vs 1.25 ± 0.10)、p-FAK(Tyr397)水平(1.07 ± 0.08 vs 0.52 ± 0.07)均显著降低( P <0.05)。Transwell迁移小室实验结果同划痕实验结果。结论 PPAR-γ/PDGF-βR/FAK(Tyr397)通路,可能是抑制肺动脉平滑肌细胞迁移机制之一。
Objective To investigate the mechanism of PPAR-γ, PDGF-βR and FAK in the regulation of migration of pulmonary artery smooth muscle cells(PASMCs). Methods Rat pulmonary artery smooth muscle cells were isolated and cultured for 3-7 passages for experiments. PASMCs were divided into four groups. In control group, PASMCs were conventionally cultured. In PDGF-BB group, PASMCs were given PDGF-BB(20 ng/ml) for 1 h after synchronization. In Y15 group, PASMCs were pre-stimulated with PDGF-BB(20 ng/ml) for 1 h after synchronization, then given FAK inhibitor Y15(10 μmol/L) for 2 h. In ROSI group, PASMCs were givenrosiglitazone(10 μmol/L) for 1 h after synchronization, and then PDGF-BB for 1 h. The cells in each group were lysed, and the levels of p-PDGF-βR and p-FAK(Tyr397) were detected by Western blot. Migration of PASMCs was determined by Scratch test and Transwell assay. Results The cell migration ability in PDGF-BB group was significantly higher than that in control group(77.3%± 4.2% vs 61.3%± 3.5%, P <0.05), and the levels of p-PDGF-βR and p-FAK (Tyr397) were increased (2.2 ± 0.12 vs 0.93 ± 0.11, 1.07 ± 0.08 vs 0.39 ± 0.02, P <0.05). The migration ability in Y15 group was significantly lower than that in PDGF-BB group(72.7%± 4.7% vs 77.3%± 4.2%, P <0.05), p-FAK (Tyr397) level was also lower(0.48 ± 0.09 vs 1.07 ± 0.08, P <0.05), but there was no significant difference in p-PDGF-βR between two groups(2.0 ± 0.10 vs 2.2 ± 0.12, P >0.05). The cell migration ability was not signi-ficantly different between ROSI group and Y15 group(75.3%± 4.04% vs 72.7%± 4.70%, P >0.05), and the p-PDGF-βR level was lower in ROSI group than in Y15 group(1.25 ± 0.10 vs 2.0 ± 0.10, P <0.05), the level of p-FAK(Tyr397) was higher(0.52 ± 0.07 vs 0.48 ± 0.09, P <0.05). Compared with PDGF-BB group, the cell migration ability was significantly decreased in ROSI group (77.3%± 4.2% vs 75.3%± 4.04%, P <0.05), and p-PDGF-βR(2.2 ± 0.12 vs 1.25 ± 0.10) and p-FAK(Tyr397) level(1.07 ± 0.08 vs 0.52 ± 0.07) were also significa
作者
张德信
胡劲松
李少军
张永红
王珂
李满祥
ZHANG Dexin;HU Jinsong;LI Shaojun;ZHANG Yonghong;WANG Ke;LI Manxiang(Department of Respiratory and Critical Care Medicine,Second Affiliated Hospital of Medical College,Xi’an Jiaotong University,Xi’an 710004,China;Xi’an Jiaotong University Health Science Center;Department of Respiratory and Critical Care Medicine,First Affiliated Hospital,Xi’an Jiaotong University)
出处
《山西医科大学学报》
CAS
2019年第9期1262-1266,共5页
Journal of Shanxi Medical University
基金
陕西省科学技术研究发展计划项目(2016JM8143)
关键词
肺动脉平滑肌细胞
黏着斑激酶
过氧化物酶增生物激活受体
pulmonary artery smooth muscle
focal adhesion kinase
peroxisome proliferator activated receptor-γ
