The Study Of The Mechanisms Of HSP60and Vascular Smooth Muscle Cell Proliferation

Objective: Atherosclerosis (AS) is a common diseases and frequently-occurringdisease, which severely threaten human health and often leads to fatal cardiovascularand cerebrovascular diseases. There are a variety of factors involved in the chronicinflammatory processes. And inn atherosclerotic plaques biological characteristics,endothelial cells, vascular smooth muscle cells and inflammatory are significantassociation.Phenotypic transformation of vascular smooth muscle cells (VSMCs) andproliferation are important factors in atherosclerosis incidence. VSMC could keepappropriate blood pressure by diastole and contraction. Smooth muscle cell state is divided into"synthesis" and "expansion", which show the shift between these two states. When thecells exhibited a "synthetic" state, the "expansion" state of the gene in the inhibitionstate, and the proliferation and migration of some genes related to the high expression.This kind of "synthetic" state of the cells can secrete the relevant matrixmetalloproteinase and elastin, which eventually led to the repair and remodeling ofdamaged blood vessels. When VSMCs were subjected to outsiders injury, the cells willreduce its specificity the telescopic protein expression, and thus can increase cellproliferation and migration, at the same time, the matrix metalloproteinase synthesiswill be a corresponding increase, this process can promote the repair of vascular injury,If this repair process is abnormal, it will lead to some diseases, such as hypertension,atherosclerosis and vascular stenosis and other vascular diseases.Heat shock protein60(HSP60) is belong to exogenous molecules which couldinduce autoimmune reactions, and is closely related to the development of the AS.Studies have shown that endogenous of HSP60can be detected within the cells in thehuman atherosclerotic area, and the extent of disease and the amount of the expressionof HSP60in a positive correlation. Also some research found that HSP60as a pro-inflammatory factor, show different expression levels in patients with heart diseaseand different degrees of coronary artery disease in patients with serum. And higherexpression of HSP60serum level was positively correlated with the severity of thepatient’s condition. Serum HSP60expression level is higher, the possibility ofoccurrence of cardiovascular disease is greater.Although many studies have shown that VSMC may play important roles in AS,however, the mechanism is not verified. This study will focus on the role of HSP60inVSMC proliferation, and the mechanism of HSP-60and AS formation.Methods:(1) A7r5cell culture: A7r5cells were cultured in DMEM with10%FBS in37℃、CO2incubator;(2) The MTT method to observe the changes of cell proliferation.By different concentrations of HSP60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml)stimulation of A7r5cells for24h, To observe the effect on the regulation of cellproliferation, to determine the optimum concentration of inducing cell proliferation;(3)By using10ng/ml HSP60stimulation of VSMC for24h, the changes of cell cycle wasdetected by flow cytometry;(4) the effect of HSP60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml) on the expression of CDK4were detected by Western blot;(5) theeffect of HSP60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml) on the expression ofcyclin1were detected by Western blot;(6) the effect of HSP60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml) on the expression of PCNA were detected by Westernblot.Results:(1) the MTT method to observe the cell proliferation results showed: byusing different concentrations of HSP-60(1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml) after stimulating, the cells proliferation in20ng/ml and50ng/ml HSP60didnot change significantly. However, with1ng/ml or10ng/ml HSP60stimulation, cellproliferation was increased significantly (P <0.05);(2) Cells stimulated with10ng/mlHSP60, significantly increased the cell cycle was detected by flow cytometry in the Sstage;(3) of different concentrations (1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/mlHSP60) after VSMC stimulation, the expression of CDK4was increased by Westernblot detection, and in which100ng/ml HSP60cell proliferation was more obvious;(4)of different concentrations (1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml HSP60) afterVSMC stimulation, the expression of cyclin1was increased by Western blot detection,and in which50ng/ml HSP60cell proliferation was more obvious;(5) of differentconcentrations (1ng/ml,10ng/ml,20ng/ml,50ng/ml,100ng/ml HSP60) after VSMC stimulation, the expression of PCNA was increased by Western blot detection, and inwhich10ng/ml HSP60cell proliferation was more obvious.Conclusions:(1) HSP60can induce the proliferation of vascular smooth muscle cells,and are positively with concentration. The cell proliferation is more obvious in the10ng/ml HSP60;(2) HSP-60promote the proliferation of vascular smooth muscle cellsis related to cell cycle;(3) Proliferation of vascular smooth muscle cells by theregulation of HSP60are interacted with CDK4/cyclin D1pathway and PCNA.