低分子量透明质酸寡糖片段介导内皮细胞增殖的信号通路

Signaling of Hayluronan Oligosaccharide-mediated Proliferation in Endthelial Cells

为研究低分子量透明质酸寡糖片段(hyaluronan oligosaccharides,o-HA)对血管内皮细胞生长与迁移的影响,及透明质酸(hyaluronan,HA)受体(CD44与RHAMM)在此过程中的作用,首先通过细胞计数、MTT实验、细胞周期分布及单层细胞损伤模型修复实验,观察o-HA对血管内皮细胞(猪髂总动脉内皮细胞,porcine vascular endothelial cell line,PIEC)增殖及创伤愈合的影响.结果显示,o-HA明显促进血管内皮细胞生长,并且能够促进内皮细胞向创伤区迁移.蛋白质免疫印迹分析证明,o-HA作用于PIECs后,细胞Src激酶、ERK-1/2的磷酸化程度增强,c-Myc蛋白、周期蛋白D1表达水平增高.Src激酶特异性的化学抑制剂PP2可轻度抑制ERK-1/2磷酸化;进而通过抗-CD44与抗-RHAMM抗体分别预先封闭细胞表面相应的特异性受体位点后,再用o-HA刺激细胞,探讨HA受体在o-HA介导PIECs信号传导过程中的作用.结果显示,抗CD44抗体不能抑制o-HA介导的ERK-1/2磷酸化;而抗RHAMM抗体可轻度抑制o-HA介导的ERK-1/2磷酸化.结果提示,o-HA具有促进血管内皮细胞增殖及创伤愈合的作用,其机制可能是通过血管内皮细胞表面受体RHAMM实现的.该作用可能通过激活Src激酶及细胞内MAPK(ERK-1/2)信号通路,启动早期反应基因转录,诱使c-Myc蛋白高表达,从而促进血管内皮细胞生长.该作用也可能与上调细胞周期蛋白D1的表达有关.

To investigate the effects of hyaluronan oligosaccharides （o-HA） on the proliferation and the migration of endothelial cells （EC）, as well as the mechanisms of its involvement in angiogenesis, porcine vascular endothelial cells （PIECs） were treated with o-HA and the cell proliferation was determined by cell counting and flow cytometry. The cell viability and motility were assessed by MTr and wound recovery assays, respectively. The results indicated that o-HA caused a significant increase of PIEC proliferation and migration after o-HA treatments. An increased expression of c-Myc and cyclin D1 induced by o-HA was observed by Westem blotting. The levels of phosphorylated Sre kinase and ERK-1/2 were also upregulated after o-HA treatments. To further distinguish which of the two known HA receptors, namely CD44 and RHAMM, is responsible for the downstream tyrosine phosphorylation, o-HA treated PIECs were pre-blocked with the anti- RHAMM antibody or the anti-CD44 antibody, then assayed for ERK-1/2 phosphorylation by immunoblotting with an anti-phospho-ERK-1/2 antibody. We found that only the anti-RHAMM antibody slightly inhibited o- HA-induced tyrosine phosphorylation of ERK-1/2 . The results suggested that in vitro pro-proliferation effect of o-HA in ECs, was mediated by RHAMM receptors, resulted in the activation of the Src kinase and MAPK （ERK-1/2） signal pathway and thus promoted the cell proliferation with the involvement of cyclin D1 expression.