Experimental Study On The Effects Of Drugs And Cytokines On Type Ⅰ Collagen Promoter And Matrix Metalloproteinases Activity

The formation of atherosclerosis is a very complicated process, including endothelium impairment, inflammatory reaction, lipid deposition and so on. Among them the most significant pathology change is the increasing of extracellular matrix secreted by VSMCs, especially the increasing of collagen. The research work on collagen, therefore might provide an insight in understanding the mechanisms of AS and provide a basis on clinical therapy. Once the atheromatous plaque formated, however, the collagen is a important protein for keeping plaque stable. Reducing the decomposion of collagen could increase the stability of plaque, decrease the onset of acute coronary event. Some inflamatory factors (such as OX-LDL,T cell)facilitate the secretion of matrix metalloproteinases(MMPs) from mono-macrophage and VSMC,which attenuate the fibrous cap by decomposing the collagen within it. Hence the effective treatment for AS should partly aim at inhibiting inflamatory reactive and reducing the secretion and activiation of MMPs. The previous studies about collagen were mainly concentrated on the organ fibrosis and revealed less about the vessels. We designed a series of experiments, hopefully to reveal some molecular mechanisms about AS and clarificate the effects of some drugs and cytokines on collagen on transcription level and on the activity of MMPs. The focal point of our experiments is about the effects of Pravastatin--one of the HMG-COA reductase inhibitors on collagen in order to comprehend the other effects of Pravastatin besides lowering lipid. The contents and results are as follows:1. Changes of the content and component of collagen in arteries of different agesThoracic aorta were taken from the body of accidental death, whose ages ranged from 25~62. After taken from dead bodies, aorta were immersed into formaldehydeand embedded by paraffin. The sections were stained with HE and collagen-elastic fibers and immunohistochemistry experiments were performed with collagen type Ⅰ and Ⅲ. The pathology results indicated that with the increasing of ages, the content of collagen in artery wall was increased and most of it was collagen type Ⅰ.2. The effects of drugs on VSMCs proliferation and the cell cycleThe smooth muscle cells obtained from male Sprague-Dawley rat's thoracic aorta were primary cultured in vitro in DMEM supplemented with 10% NCS . Pravastatin(10-6~10-2M),OX-LDL(50ug~200ug),Pravastatin(10-3M)+OX-LDL(50ug~200ug)were added into the plate to co-incubated with the cells respectively. MTT method was used to detect the cell proliferation after 24, 48 and 72 hours. After different concentrations of pravastatin were incubated with VSMCs for 24 hours, the cell cycle was detected by flow cytometer to observe the effect of Pravastatin on cell apoptosis. The results displayed that different concentrations of Pravastatin had no significant effect on proliferation of VSMCs ,but OX-LDL could accelerate the proliferation of VSMCs with concentration and time-dependent manner. Pravastatin could inhibit the proliferation effect induced by OX-LDL. The results of flow cytometer indicated that Pravastatin could make the apoptosis peak higher and have dose-dependent.3. Construction of pCOLH plasmids and analysis of their activityFibroblasts from human skin were primary and passage cultured. Using the genome DNA from healthy bodies as the template, five fragments with the same 3' end were obtained by PCR method. The six promoter fragments of 0.2kb,0.4kb,0.7kb,1.6kb and 2.4kb were ligated to pCAT3-Enhancer(without promoter, but contains SV40 enhancer and chloramphenicol acetyltransferase reporter gene) respectively. The five recombinants obtained were named as pCOLH20.2,pCOLH20.4,pCOLH 20.7,pCOLH 21.6 and pCOLH22.4.The promoter of them were correspondent to the sequences of -129～+58,-339～+58bp,-616～+58bp,-1476～+58bp,-2292～+58bp respectively in the upstream of the human α2(Ⅰ) collagen gene. The constructions were tansiently transfected using FuGENE 6 and the plasmidpSVβ...