Method For Evaluating Atherosclerotic Lesion, And Kit
a kit and atherosclerotic technology, applied in the field of methods, kits, and atherosclerotic lesions evaluation equipment, can solve the problems of high angiography, and inability to obtain intravascular plaques or the properties of them
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example 1
[0109]ApoE-deficient mice (ApoED; B6.KOR / StmSlc-Apoeshl), which were deficient in the lipid metabolism-related ApoE protein, were used as atherosclerosis models (Matsushima, Y. et al., J. Atheroscler. Thromb. 8: 71-79, 2001; Japan SLC, Inc.). Administration of a high-fat diet accelerates lesion progression in ApoED mice. In addition, lesion progression is accelerated depending on age in weeks. In 12-week-old mice, lesion progression is observed to such an extent that arterial intimal thickening and lipid accumulation are observed (stage 1). In 18-week-old mice, lipid accumulation results in foam cell formation (stage 2). In 25-week-old mice, many forms of unstable atherosclerotic plaque are widely observed in arteries (stage 3), and this corresponds to the most dangerous stage in the atherosclerosis process. In 35-week-old mice, many stable lesions that are highly fibrotic and calcified are observed (stage 4).
[0110]Therefore, for this example, arterial tissue was collected from 12, ...
example 2
[0118]Arterial tissues were excised from five 12-week-old ApoE knockout mice (female) from different lines (ApoEKO; B6.129P2-ApoetmlUnc / J) fed with a normal diet, and then protein fractions (pool) were prepared in a manner similar to that in Example 1. The ApoEKO line differing from the ApoED line described in Example 1 (Piedrahita, J A. et al., Proc. Natl. Acad. Sci. U.S.A., 89: 4471-4475, 1992; Charles River Laboratories, Japan Inc.) were confirmed in advance in a preliminary study to have no lesion formed in arteries (data not shown). The lipid concentrations in blood of a subject were: total cholesterol of 539 mg / dl; triglyceride of 49 mg / dl; LDL cholesterol of 404 mg / dl; and HDL cholesterol of 143 mg / dl. Thus, the progression of arteriosclerosis was suspected. Hence, the ratios of the expression levels of proteins contained in tissue samples were exhaustively analyzed by the method of Example 1 (that had been partially modified) using an arterial tissue obtained from a wild-typ...
example 3
[0120]Ten typical proteins were selected from the candidate marker protein group that exhibited variations in Example 1 (characteristic in ApoE-deficient mice) in the amounts thereof in arterial tissues. The expression of the proteins in atherosclerotic lesion sites was confirmed by immunohistochemical staining of lesion sites (plaque formation sites) in arterial tissues of ApoE-deficient mice at different ages in weeks.
[0121]Each aortic sample subjected to 10% formalin fixation was coronally excised at the aortic arch, and then a paraffin embedded block was prepared. Subsequently, it was sliced to a thickness of 3 μm, and then the slices were subjected to immunohistochemical staining according to a conventional method using hematoxylin.eosin (H&E) staining and antibodies. Primary antibodies used for immunohistochemical staining are as follows.
Anti-properdin antibody (50-fold dilution, sc68367; Santa Cruz),
Anti-CD5 antigen-like protein antibody (100-fold dilution, ab45408; Abcam),
An...
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