| Objective: Atherosclerosis(AS) is an inflammatory disease of cardiovascular system, and inflammatory response is present in the whole pathological process of atherosclerosis. Osteopontin(OPN) is one of adhesive proteins in extracellular matrix, which can be synthesized and secreted by proliferating vascular smooth muscle cells (VSMCs). OPN can induce cell adhesion and migration. In addition, it has the function of cytokine-like, which involves in inflammatory response. OPN plays an important role in atherosclerosis development. In the present study, we used anti-osteopontin antibody as an inhibitor, which was given intravenously, to block the function of osteopontin in vascular lesion sites, and investigated preventive effect of the inhibitor on AS and it's mechanism.Methods:1. Establishment of experimental AS model in rat: 12 SD rats were randomly divided into two groups (n=6), model group and prevention group. Each of model group was fed by orogastric intubation with Vitamin D3 (500000 IU/kg) within 3 days, and then fed with a high-fat diet for 21 days. For prevention group, with being fed with Vitamin D3 and a high-fat diet, each animal was administrated anti-osteopontinmonoclonal antibody(0.5ml) by tail vein injection (The titer was 1:1000). Antibody was delivered at two days intervals for seven times. The weight of rats was measured before experiment and at the end of experiment. The anus temperature of each rat was measured at 0, 2, 4, 6, 8, 10, 12 and 24 hours respectively after administration. During the experiment, general conditions of rats were observed. Blood samples were taken from all rats at 0, 10, 17 and 24 days after experiment respectively, and serum was separated for detection of levels of Ca2+, TNF-αand CRP. The aortas were collected for section preparation, SM-αactin immunohistochemistry, and OPN Western blotting analysis at the end of experiment.2. SM-αactin immunohistochemical staining: The sections were incubated with special SM-αactin primary monoclonal antibody at 4℃ overnight (The negative control sections were treated by PBS instead of primary antibody), and in turn polyvalent biotinylated antibody (1:1000 dilution) and the streptavidin-peroxidase at 37℃ for 15 min. 3,3-diaminobenzidine(DAB) was added as a chromogen and stained.3. Western blotting analysis of OPN expression: The extracts from arteries of two groups rats were separated on 8% SDS-PAGE, and then blotted onto nitrocellulose membrane. The membrane was immunologically reacted with anti-OPN antibody.4. Serum and tissue Ca2+ levels assay: We performed as kitdescription with fully automatic biochemical analyzer.5. Determination of the levels of serum inflammatory factors: Serum CRP and TNF-αwere measured respectively by immunonephelometric and radioimmunoprecipitation assays.Result:1. The change of general conditions: The general conditions of two groups were fine. Rats' weight hadn't changed during the whole experiment. After antibody were administrated by tail vein injection the first four times, the body temperature of rats in prevention group went up to the highest point within 8 hours, which was 38.4±0.14℃(P<0.05), and then declined after 8 hours, and maintained at the normal range. There was no change in body temperature of rats after antibody was given the last three times. During the whole experiment, the body temperature of rats in model group fluctuated at the normal range.2. The morphological changes: The morphological observation of the aorta section showed that, in model group, space in subendothelial layer area widened, and there presented many small vacuoles and even large vacuoles in some areas. The smooth muscle cells proliferated, abnormally arranged or protruded, and elastic membranes abnormally arranged and broke. The significant calcifications in arterial medium layer were observed. Furthermore, foam-like cells were presented around the calcific regions. In prevention group, all of the morphological changes were slight, compared with those of...
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