整合素β_1介导血小板源生长因子诱导的血管平滑肌细胞与单核细胞间黏附作用

Adhesion between monocytes and vascular smooth muscle cells induced by integrin β_1-mediated platelet-derived growth factor

目的探讨血小板源生长因子(PDGF)对血管平滑肌细胞(VSMC)与人单核细胞系(THP-1)间黏附作用的影响以及可能的分子机制。方法培养的大鼠主动脉血管平滑肌细胞分别用2.5、5、10和20ng/ml PDGF 刺激不同时间后,在无或含有整合素β_1单克隆抗体培养液中,加入经PKH26标记的 THP-1细胞与 VSMC 共培养,测定黏附的 THP-1细胞数量。采用 Western 免疫印迹法检测 PDGF 对 VSMC 表达整合素β_1的影响。结果 PDGF 刺激 SMC 促进 THP-1与 VSMC 的黏附的作用随着浓度的增加而增强,THP-1与血管平滑肌细胞黏附的数量是对照组的1.1、2.1、3.1和4.4倍;在10 ng/ml PDGF 刺激下,黏附的 THP-1细胞数量是对照组的3.1倍(135±47个细胞/视野 vs 43±14个细胞/视野,P<0.001);整合素β_1单克隆抗体能显著抑制 PDGF 刺激的 THP-1细胞与 VSMC的黏附作用,黏附的细胞数量(54±13个细胞/视野,P<0.01);PDGF 促进 THP-1与 VSMC 黏附的作用在 PDGF 刺激后8h 达到高峰。免疫印迹显示 PDGF 能诱导 VSMC 表达整合素β_1,在8h 左右达高峰。结论血小板源生长因子能够呈剂量和时间依赖性地促进单核细胞与血管平滑肌细胞的黏附,该作用部分是通过整合素β_1介导的,这一效应在动脉粥样硬化发病过程中可能具有重要意义。

Objective To investigate the effects of platelet-derived growth factor （PDGF）-BB on the vascular smooth muscle cell （VSMC）-monocyte interaction and the mechanism thereof. Methods Rat aortic VSMC were pretreated with PDGF-BB of the concentrations of 0, 2.5, 5, 10, and 20 ng/ml for 8 hours and then co-incubated with human monocytes of the line THP-1 labeled with PKH26, a cellular membrane fluorescent marker, i.e. , to be subjected to binding assay to observe the dose-effect relationship. Other VSMC were co-cultured with PDGF of the concentration of 10 ng/ml for 1, 2, 4, 12, and 24 hours respectively and then interacted with PKH26-abeled THP-1 cells so as to observe the time-response relationship. Monoclonal antibody against integrin β1 was co-cultured with VSMC for 30 min, and then PKH26-1abeled THP-1 cells were added to block the effect of PDGF. After the incubation of THP-1 cells and VSMC, the amount of bound cells was counted using fluorescent phase-contrast microscopy. The effect of PDGF on the expression of integrin β1 was detected by Western blotting. Results The amounts of THP-1 cells bound to VSMC pretreated with PDGF of the concentrations of 2.5, 5, 10, and 20 ng/ml respectively were 1.1, 2.1,3.1, and 4.4 times that of the untreated cells. Incubated with 10 ng/ml PDGF for 0-18 h, the adhesion rate between the VSMC and THP-1 cells increased time-ldependently. After the blocking by the antibody against integrin β1, the adhesion rate between the VSMC and THP-1 cells decreased from （25.4 ± 11.4） % to （9.2 ± 4.1 ） % （ P 〈 0.01 ）. Western blotting showed that the level of integrin ~1 of the VSMC treated by 10 ng/ml PDGF increased since 4 h after the treatment, peaked 8 h later, and then decreased gradually. Conclusion PDGF-BB can induce the binding of monocytes to VSMC via the integrin-β1 signaling pathway. The effect may therefore facilitate the progression of atherosclerosis by augmenting the VSMC-monocyte adhesive interaction.