Zhang Zhi, Chu Guang, Wu Hong-Xian, Zou Ni, Sun Bao-Gui, Dai Qiu-Yan
Shanghai First People’s Hospital, Shanghai Jiao Tong University School of Medicine, PR China.
Clin Invest Med. 2011 Jun 1;34(3):E138-46. doi: 10.25011/cim.v34i3.15186.
The goal of this study was to investigate the crosstalk between vascular endothelial cells (ECs) and smooth muscle cells (SMCs) using a three-dimensional (3-D) co-culture model. In addition, the role of IL-8 in this crosstalk was investigated.
A 3-D co-culture model was constructed using a Transwell chamber system and type I collagen gel. Human umbilical artery smooth muscle cells (HUASMCs) were suspended in the gel and added to the upper compartment of the Transwell. Human umbilical vein endothelial cells (HUVECs) were then grown on the surface of the gel. The growth of HUASMCs was tested with a CFDA SE cell proliferation kit. IL-8 and other bioactive substances were investigated by ELISA and real-time PCR. The alteration of p-ERK expression related to the change in IL-8 levels was also examined by Western blot analysis.
The proliferation rate of HUASMCs in the 3-D co-culture model was 0.679 ± 0.057. Secretion and transcription of VEGF, t-PA, NO and VCAM-1 in the 3-D co-culture model were different than in single (2-D) culture. When 3-D co-cultured, IL-8 released by HUVECs was significantly increased (2.35 ± 0.16 fold) (P﹤0.05) and the expression of VCAM-1 from HUASMCs was reduced accordingly (0.55±0.09 fold). In addition, increasing or decreasing the level of IL-8 changed the level of p-ERK and VCAM-1 expression. The reduction of VCAM-1, resulting from increased IL-8, could be blocked by the MEK inhibitor, PD98059.
Crosstalk between HUVECs and HUASMCs occurred and was probably mediated by IL-8 in this 3-D co-culture model.
本研究旨在利用三维（3-D）共培养模型研究血管内皮细胞（ECs）和平滑肌细胞（SMCs）之间的相互作用。此外，还研究了白细胞介素-8（IL-8）在这种相互作用中的作用。
使用Transwell小室系统和I型胶原凝胶构建三维共培养模型。将人脐动脉平滑肌细胞（HUASMCs）悬浮在凝胶中并添加到Transwell的上室。然后将人脐静脉内皮细胞（HUVECs）接种在凝胶表面。使用CFDA SE细胞增殖试剂盒检测HUASMCs的生长情况。通过酶联免疫吸附测定（ELISA）和实时聚合酶链反应（PCR）研究IL-8和其他生物活性物质。还通过蛋白质免疫印迹分析检测与IL-8水平变化相关的磷酸化细胞外信号调节激酶（p-ERK）表达的改变。
三维共培养模型中HUASMCs的增殖率为0.679±0.057。三维共培养模型中血管内皮生长因子（VEGF）、组织型纤溶酶原激活剂（t-PA）、一氧化氮（NO）和血管细胞黏附分子-1（VCAM-1）的分泌和转录与单层（二维）培养不同。三维共培养时，HUVECs释放的IL-8显著增加（2.35±0.16倍）（P﹤0.05），HUASMCs中VCAM-1的表达相应降低（0.55±0.09倍）。此外，增加或降低IL-8水平会改变p-ERK和VCAM-1的表达水平。IL-8增加导致的VCAM-1减少可被丝裂原活化蛋白激酶激酶（MEK）抑制剂PD98059阻断。
在这种三维共培养模型中，HUVECs和HUASMCs之间发生了相互作用，可能由IL-8介导。
