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Effects of ROCKⅠon Migration of Vascular Smooth Muscle Cells Induced by PDGF

Author: LinHaiShuang
Tutor: GaoYing
School: Dalian Medical University
Course: Biochemistry and Molecular Biology
Keywords: ROCKⅠ VSMCs Migration MAPK PDGF
CLC: R363
Type: Master's thesis
Year: 2011
Downloads: 10
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Abstract


Objectives:Atherosclerosis is one of the major diseases that threatens to human health. The pathogenesis of AS has been a hot research of cardiovascular disease. VSMCs proliferate and migrate from the medium to the intima layer of the vessel and lead to abnormal proliferation is an important part of the development of atherosclerosis and other cardiovascular diseases. As a potent chemotactic agents in vitro, PDGF can induce proliferation and migration of VSMC. Rho-kinase, a target effector of the small G protein Rho has two isoformsⅠ, ROCKⅠand ROCKⅡ, which involved in cell migration, cell proliferation, cell adhesion, cytoskeletal reorganization and other cellular behaviors. These functions also participated in the development of many cardiovascular diseases. But the molecular mechanisms of ROCK involved in cell migration are not clear. As reported, MAPK family members are involved in cell proliferation and migration, such as p38 MAPK, ERK (extracellular signal-regulated kinase). In addition, in the process of cell migration, matrix metalloproteinases (MMPs) can degrade a wide range ECM, clearing the way for the VSMC migration, promoting VSMC migration. This experimental study aims to clarify the molecular mechanism of migration in vascular smooth muscle cell.Methods: (1) The overexpression level of transient and stable transfection ROCKⅠwas detected by Western blot and RT-PCR. (2) The migration of VSMC was detected by Boyden chamber and Wound healing after the overexpression of ROCKⅠ. (3) The p-ERK and p-p38 expression levels of VSMC induced by PDGF were detected by Western blot at different times. (4) The expression levels of p-ERK and p-p38 were detected by Western blot in different concentrations of ERK and p38 inhibitors. (5)The cell migration was detected by Boyden chamber in different concentrations of U0126 and SB203580 treatments. (6) The activity and expression of MMP-2 were assessed by Gelatin Zymognaphy and Western blot. (7) The remodeling of the actin cytoskeleton and cell morphology were detected by immunofluorescence and cell photography.Results: (1) The cell migration was significantly increased with overexpression of ROCKⅠby transient transfection. (2) PDGF stimulated a transient increase in the phosphorylation of ERK and p38 respectively. (3) U0126 and SB203580 (the inhibitor of ERK and p38) caused dose-dependent inhibition of ERK and p38 phosphorylation to the basal level. (4) With different concentration of U0126 and SB203580 treatments, the expression of p-ERK and p-p38 were decreased with the concentration dependence. (5) The protein expression and activity of MMP2 were decreased with U0126 and SB203580 treatments. (6) The reduction of cell filopodia and change of cell morphological were observed after U0126 and SB203580 treatments.Conclusions: (1) We further confirmed that ROCKⅠplays a leading role in the migration of vascular smooth muscle cells. (2) PDGF-mediated cell migration, probably performs through Rho/ROCK/MAPK pathway, and through the degradation of extracellular matrix.

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