Application of protein in preparation of medicine for preventing and treating atherosclerosis and complications
A technology for atherosclerosis and atherosclerosis, which is applied in the field of biomedicine, can solve the problems of limited research level and no drugs for oxLDL clearance, and achieve the effect of reducing the ratio level.
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[0114] Example 1. sDSS1 protein can interact with oxLDL or LDL.
[0115] 1.1 Experimental materials and methods
[0116] Experimental materials: sDSS1 protein, oxidized low-density lipoprotein (oxLDL) (Solebo, product number: H7980); low-density lipoprotein (LDL) (Solebo, product number: H7960).
[0117] Experimental method: 2μg of oxLDL or LDL and 6μg of sDSS1 protein or 10μg of sDSS1 protein were added to 1.5ml under the condition of 20mM sodium acetate / acetate buffer (pH4.5) or 20mM phosphate buffer (pH7.2), respectively. Mix in EP tubes and incubate at 37 °C for 12 h. The incubation product was added with loading buffer and mixed, and denatured at 100°C for 10 minutes to prepare a loading sample. The prepared samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE). After separation, the SDS-PAGE gel was stained with Coomassie brilliant blue to reveal protein bands.
[0118] 1.2 Experimental results
[0119] In the acetate buffer system (pH 4.5), LDL pro...
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[0122] Example 2. sDSS1 protein can reduce the uptake of oxLDL by vascular endothelial cells.
[0123] 2.1 Experimental materials and methods
[0124]Experimental materials: sDSS1 protein, Dil-oxLDL (Thermo Fisher Scientific, product number: L34358), human umbilical vein endothelial cells (HUVEC) (PromoCell, product number: C-12200)
[0125] Experimental method: HUVEC cells were seeded into 6-well plates according to the cell number of 300,000 per well. After 24 hours of adherence, 1.5ml of 10μg / ml Dil-oxLDL was added, or 1.5ml of 10μg / ml Dil-oxLDL was added at the same time. 2 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml of sDSS1 protein. After continuing to incubate in the incubator for 5 hours, the intracellular fluorescent signal was observed with a fluorescence microscope, and the cells were digested with trypsin to form single cells for flow cytometry to detect the fluorescence intensity.
[0126] 2.2 Experimental results
[0127] After adding Dil-oxLDL to the HUVEC cell cultur...
Example Embodiment
[0128] Example 3. sDSS1 protein inhibits phagocytosis of oxLDL by macrophages.
[0129] 3.1 Experimental materials and methods
[0130] Experimental materials: sDSS1 protein, Dil-oxLDL, phorbol ester (PMA, Sigma-Aldrich, catalog number: P1585), human monocyte THP-1 (Cell Bank of the Chinese Academy of Sciences Type Culture Collection, catalog number: SCSP- 567).
[0131] Experimental method: THP-1 cells were seeded into 6-well plates according to the cell number of 250,000 per well, and the culture medium contained 100 ng / mL PMA. After the cells were activated by PMA for 48 hours, they were replaced with fresh medium and cultured for four days to help the cells to attach. Discard the old medium and add 1.5ml of 10μg / ml Dil-oxLDL, or add 1.5ml of 10μg / ml Dil-oxLDL simultaneously with sDSS1 protein at concentrations of 2μg / ml, 5μg / ml, 10μg / ml, 20μg / ml. After continuing to incubate in the incubator for 5 hours, the intracellular fluorescence signal was observed with a fluoresc...
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