Function and application of centrifugal force and shear stress response gene 1 (recs1) in the treatment of restenosis after vascular injury
A technology for vascular injury and restenosis, which is applied in the functional field of genes, can solve problems affecting the function of vascular VSMC, and achieve the effect of promoting restenosis
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Embodiment 1
[0040] Example 1 Mouse Vascular Injury Model (VI) Obtained
[0041] 1. Grouping of experimental animals: WT and RECS1-KO mice aged 8-10 weeks and weighing 24-27g were used and divided into two groups: WT vascular injury group and RECS1-KO vascular injury group, with 20 mice in each group. mouse. The mice were sacrificed 28 days after the operation, and blood vessels in the injured segment were collected for analysis.
[0042] 2. Operation procedure of mouse vascular injury model:
[0043] 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 mouse is fully anesthetized (about 3 minutes), 8% sodium sulfide is used to ...
Embodiment 2
[0048] Example 2 Determination of Intimal Neogenesis in Vascular Injury Model (VI) Mice
[0049] 1. Mice collection
[0050] 1) Anesthetize the mouse, cut the heart and let the blood out.
[0051] 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.
[0052] 3) Put the carotid artery in PBS, and gently drain the residual blood in the lumen with micro forceps.
[0053] 4) Put the blood vessel into a 1.5mLEP tube filled with 1mL of 4% paraformaldehyde for fixation.
[0054] 2. Pathological detection
[0055] 2.1 Preparation of paraffin specimen slices
[0056] Paraffin specimen slices are prepared by professional pathologists in the laboratory. The main operating procedures include: after overnight fixation in 4% paraformaldehyde, carefully wrap the blood vessel with filter paper, put it into the embedding frame→rinse with running water→dehydration→transparency→wax immersion →embedding...
Embodiment 3
[0064] Example 3 Detection of the proliferation level of blood vessel wall cells
[0065] The expressions of proliferating cell nuclear antigen (PCNA) and cell cycle protein (CyclinD1) were detected by immunofluorescence staining. Required primary antibody information: PCNA (#2586;1:100; mouse; CellSignalingTechnology), cyclinD1 (#2978;1:25; rabbit; CellSignalingTechnology); Required secondary antibody information: AlexaFluor568-conjugatedgoatanti-rabbitIgG (A11011; Invitrogen, Carlsbad, CA), AlexaFluor568-conjugated goatanti-mouse IgG (A11004; Invitrogen, Carlsbad, 150d, CA).
[0066] The main steps are:
[0067] 1) Baked slices: put the paraffin slices in an oven at 55°C for more than 30 minutes.
[0068] 2) Dewaxing: Xylene 5min×3.
[0069] 3) Hydration: 100% ethanol 5min×2; 95% ethanol 5min; 70% ethanol 5min; ddH 2 O dipping for 5min×2.
[0070] 4) Citrate tissue antigen repair (high pressure repair): Take a certain amount of pH6.0 citrate antigen repair working solut...
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