Study On The Relationship Of Ox-LDL And Vascular Local RAS And Their Impact On Vascular Smooth Muscle Cell Cytokines Expression

We studied the relationship between ox-LDL and local RAS of artery wall and the effect of the two on smooth muscle cells cytokines expression with methods of imrminohistochemistry, cell biology and molecular biology. The whole study includes three parts.Part I To Observe the Relationship between Ox-LDL and Vascular Local RAS with Experimental MethodsObjective: Former researches have showed that ox-LDL and local RAS both play vital rules in the process of atherosclerosis. LOX-1 is one of the important receptors of ox-LDL, and ang II can not play without AT1R or AT2R. The aim of this study is to observe the correlation between ox-LDL and local RAS, and to observe the receptor pathway of ox-LDL and RAS with LOX-1, AT1R and AT2R blockers polyinosinic acid, losartan and PD123319.Methods: (1) The expression of ox-LDL, LOX-1 and ACE in rabbit atherosclerotic tissue: Get rabbit atherosclerotic model by high lipid diet, and rabbits received normal diet served as control group, amounting to 5 respectively. Then make artery section from the middle part of aorta. Do HE staining and immunohistochemistry staining for ox-LDL, LOX-1 and ACE. (2) The impact of ox-LDL on the expression of LOX-1, ACE, AT1R, AT2R mRNA of vascular smooth muscle cells: Primary generation culture of human umbilical artery smooth muscle cell, and take the 5th to 7th generation cells for experiment. The experiment was divided into 4 groups. The first group co-cultured with 20 g/ml ox-LDL for 1, 6, 12 and 24h; The second with 1, 10,100 ug/ml ox-LDL for 24h; The third with 20 ug/ml ox-LDL + 250 ug/ml polyinosinic acid for 24h; The last was the control group, with 0.1 % FBS Ml99 for 24h. Then detect the mRNA with RT-PCR. (3) The impact of ox-LDL on the activity of ACE of vascular smooth muscle cell: Primary generation culture of humanumbilical artery smooth muscle cell. The experiment included two large groups. One was divided into two groups further, for control group with 0.1 % FBS Ml 99 and experimental group with 20 ug/ml ox-LDL for 24h, amounting to 10 respectively, and detected ACE activity with ultraviolet kit. The other was divided into three groups. The first group co-cultured with 20 ug/mL ox-LDL +0.1umol/mL ang I for 24h, and the second with 20 ug/mL ox-LDL + O.lumol/mL ang I + 10umol/L captopril for 24h, and the last was control group with 0.1 mol/mL ang I for 24h, amounting to 3 of them respectively, and detected ang II level with radio-immunity kit. (4) The impact of ang II on the expression of LOX-1 -. ACE^ AT1R, AT2R mRNA of vascular smooth muscle cells: Primary generation culture of human umbilical artery smooth muscle cell. The experiment included four groups. One with O.lumol/L ang II for 24h. One with O.lumol/L ang II+lumol/L losartan for 24h. One with O.lumol/L ang 11+ lumol/L PD123319 for 24h, the last with 0.1 % FBS M199 for control group. Then detected mRNA.Results: ?There is no expression of ox-LDL, LOX-1 and ACE in rabbit aortic artery tissue of control group, with evidently enhanced expression in atherosclerotic tissue of rabbit aortic artery after high lipid diet. Ox-LDL mainly exists at the areas with abundant foam cells in atheroma, and mainly in cytoplasm. LOX-1 mostly expressed at the endothelial cell area, and some of them also expressed at the area of macrophage and smooth muscle cells near inflammation. ACE mostly expressed at the area with active inflammation, such as the shoulder part of the plaque and the broken area of intima. Endothelial cell area has a relative higher expression of ACE, and the area of macrophage and smooth muscle cells near inflammation also does. The distributing characters of LOX-1 and ACE are very similar. (2) Ox-LDL can augment LOX-1 mRNA expression of VSMC in low concentration(< lOug/ml), and LOX-1 mRNA expression increases with ox-LDL concentration increases. Ox-LDL can enhance ACE mRNA expression continuously in high concentration (> lOug/ml). Ox-LDL can enhance AT1R mRNA expression in low concentration, and AT.1R mRNA expression increases with ox-LDL concentration augments. lOug/m...