论文部分内容阅读
目的:本研究拟探讨平滑肌蛋白22α(SM22α)对血管平滑肌细胞(VSMC)表面血小板源性生长因子受体β(PDGFR-β)胞吞的影响,进而揭示SM22α对PDGFR-β活性调控的关键步骤——胞吞和泛素化动态平衡的影响及其对血管重构过程的调控作用。方法:PDGF刺激体外培养大鼠VSMC,考察si RNA敲低SM22α蛋白后不同时点的细胞中,激光共聚焦显微镜观察PDGFR-β在细胞膜分布的异同;利用活细胞工作站实时观察SM22α的表达对PDGFR-β在细胞内内吞体和溶酶体分布的异同;SM22α对c-Cbl或TRAF6等E3泛素连接酶活性的影响;si RNA敲低早期内吞体标志蛋白Rab5、再循环内吞体标志蛋白Rab4和Rab11、多泡体(MVB)标志蛋白Rab7,观察SM22α对PDGFR-β亚细胞定位的变化及其与血管重构的关系。结果:研究发现,SM22α表达下调促进细胞表面的PDGFR-β的再循环过程,使PDGFR-β在细胞表面的分子数显著减少,激活信号分子Akt和p42/44磷酸化,促进PDGF诱导的VSMC生长、增殖和迁移过程,SM22α对PDGFR-β再循环的调控与血管重构过程密切相关。结论:PDGFR-β调控异常诱发的生物学行为改变是心血管疾病的主要细胞和分子生物学基础,我们的结果表明,SM22α可能通过影响PDGFR-β胞吞和泛素化动态平衡而影响其活性的调控,进而参与血管重构的病理生理过程,阐明其分子机制可为发掘基于影响PDGFR-β功能的心血管疾病药物设计提供新靶点。
This study was designed to investigate the effect of SM22α on the endocytosis of platelet-derived growth factor receptor β (PDGFR-β) on the surface of vascular smooth muscle cells (VSMCs), and to reveal the key steps of SM22α on the regulation of PDGFR- Effect of endocytosis and ubiquitination on homeostasis and its regulation on vascular remodeling. Methods: PDGF-stimulated rat VSMCs were cultured in vitro. The expression of PDGFR-β in the cell membrane was observed by confocal laser scanning microscope (LSCM) at different time points after si RNA knockdown of SM22α protein. The effect of SM22α expression on PDGFR β in the endocytosis and lysosome distribution of the similarities and differences; SM22α on c-Cbl or TRAF6 and other E3 ubiquitin ligase activity; si RNA knockdown early endosome body marker protein Rab5, recalculated endosomes Rab4 and Rab11, Rab7, the marker of MVB, and observe the changes of subcellular localization of PDGFR-β by SM22α and its relationship with vascular remodeling. Results: It was found that the down-regulation of SM22α promoted the recycling of PDGFR-β on the cell surface, significantly decreased the number of PDGFR-β on the cell surface, activated the phosphorylation of Akt and p42 / 44, and promoted the growth of VSMC induced by PDGF , Proliferation and migration process, SM22α regulation of PDGFR-β recycling and vascular remodeling process are closely related. CONCLUSIONS: The biological behavioral changes induced by the abnormal regulation of PDGFR-β are the major cellular and molecular biological basis of cardiovascular diseases. Our results show that SM22α may affect the activity of PDGFR-β by affecting the dynamic balance of endocytosis and ubiquitination , And then participate in the pathophysiological process of vascular remodeling. The elucidation of its molecular mechanism may provide a new target for the discovery of drug design based on cardiovascular diseases that affect the function of PDGFR-β.