Function and application of interferon regulatory factor 4 (IRF4) in scaffold and endarterectomy restenosis
A technology for restenosis and vascular stenosis, which is applied in the direction of medical preparations containing active ingredients, peptide/protein components, drug combinations, etc., can solve the problems of high incidence and affect the treatment effect, and achieve the effect of inhibiting restenosis
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Embodiment 1
[0029] Example 1 Mouse Vascular Injury Model (VI) Obtained
[0030] 1. Experimental animal grouping: WT and IRF4-KO mice aged 8-10 weeks and weighing 24-27g were used and divided into four groups: WT vascular injury group; WT sham operation group; IRF4-KO vascular injury group; IRF4 - KO sham operation group, 60 mice in each group. Twenty mice in each group were sacrificed 7 days, 14 days, and 28 days after the operation, and blood vessels in the injured segment were collected for analysis.
[0031] 2. Operation procedure of mouse vascular injury model:
[0032] 1) Accurately weigh the body weight (g) of the mouse in dynamic mode with an electronic balance, accurately prepare 3% pentobarbital sodium solution with double distilled water, shake gently to dissolve it fully, and use 80mg / kg body weight dose to calculate Accurately extract the corresponding volume of pentobarbital sodium solution with a 1mL syringe, and intraperitoneally inject the anesthetized mouse. After the m...
Embodiment 2
[0037] Example 2 Determination of Intimal Neogenesis in Vascular Injury Model (VI) Mice
[0038] 1. Mice Harvesting
[0039] 1) Anesthetize the mouse, cut the heart and let the blood out.
[0040] 2) Cut the carotid artery from the proximal bifurcation of the carotid artery, take 0.5-0.6cm long, and keep the external carotid artery knot.
[0041] 3) Put the carotid artery in PBS, and gently drain the residual blood in the lumen with micro forceps.
[0042] 4) Put the blood vessel into a 1.5mL EP tube filled with 1mL 4% paraformaldehyde for fixation.
[0043] 2. Pathological detection
[0044] 2.1 Preparation of paraffin specimen slices
[0045] Paraffin specimen sections are prepared by professional pathological staff in the laboratory. The main operating procedures include trimming the heart → processing the embedding frame → washing with running water → dehydrating → transparent → dipping in wax → embedding → sectioning (3 μm) → spreading → drying or baking Backup.
[...
Embodiment 3
[0053] Example 3 Detection of proliferation level of blood vessel wall cells
[0054] The expressions of proliferating cell nuclear antigen (PCNA) and cell cycle protein (Cyclin D1) were detected by immunofluorescence staining. Required primary antibody information: PCNA (#2586; 1:100; mouse; Cell Signaling Technology), cyclin D1 (#2978; 1:25; rabbit; Cell Signaling Technology); required secondary antibody information: Alexa Fluor 568-conjugated goat anti-rabbit IgG (A11011; Invitrogen, Carlsbad, CA), Alexa Fluor 568-conjugated goat anti-mouse IgG (A11004; Invitrogen, Carlsbad, 150 d, CA).
[0055] The main steps are:
[0056] 1) Baked slices: put the paraffin slices in the oven for more than 30 minutes.
[0057] 2) Dewaxing: Xylene 5min×3.
[0058] 3) Hydration: 100% ethanol 5min×2; 95% ethanol 5min; 70% ethanol 5min; ddH 2 O dipping for 5min×2.
[0059] 4) Citrate tissue antigen repair (high pressure repair): Take a certain amount of pH6.0 citrate antigen repair working...
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