Use of aspirin and derivative thereof in preparation of drug for treating airway mucus hypersecretion
A technology of aspirin and its derivatives, which is applied in the field of clinical medicine, can solve problems such as airflow limitation, increased risk of bacterial infection, and progressive decline in lung function, achieving credible experimental results
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Embodiment 1
[0052] Example 1 Aspirin regulates the expression of FOXA2
[0053] 1. Cell level research
[0054] 1.1 Cell culture
[0055] A549 cells were placed in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, at 37°C, 5% CO 2 Subculture in an incubator. The medium was changed every two days, and the cultures were subcultured every 4 days.
[0056] 1.2 Drug handling
[0057] Dexamethasone (Dexamethasone, 10 -6 M, 48h), ambroxol (Ambroxol, 2.1 μg / ml, 48h), tiotropium bromide (tiotropium, 20nM, 48h), aspirin (aspirin, 4mM, 48h) to stimulate A549 cells.
[0058] 1.3 RNA level detection
[0059] 1.3.1 Total RNA extraction
[0060] 1) Wash the cells once with 1ml of phosphate buffer solution pre-cooled in a 4°C refrigerator, add 1ml TRIzol to each well, and act for 2 minutes to fully lyse the cells. Freeze the lysed samples in a -80°C refrigerator. Freezing and thawing can make the cells lyse more fully. ;
[0061] 2) Thaw the TRIzol-t...
Embodiment 2
[0111] Example 2 Study on Lysine Pirin Regulating Airway Mucus Secretion
[0112] 1. Animal handling
[0113] BALB / c female mice (4-6 weeks) were randomly divided into control group (WT), OVA sensitized group (OVA), wild type + lysine-pirin group (WT + L-ASA), OVA sensitized + In the lysinepirin group ((OVA+L-ASA) 4-6 rats in each group), the lysinepirin was administered by nebulization. The left lung was stained with HE and PAS, and the right lung was lavaged with normal saline. The cells in the lavage fluid were counted, sliced, and Swiss-Giemsa stained. The right lung tissue was extracted for RNA and protein detection.
[0114] 1) Preparation of mouse abdominal cavity sensitization mixture
[0115] Configure the corresponding total amount of sensitization mixture according to the number of experimental mice, and prepare according to the following ratio: immune adjuvant Al(OH)3-Mg(OH)2(Imject TM Alum Adjuvant) 0.5ml and 200 micrograms of chicken ovalbumin (dissolved in ph...
Embodiment 3
[0149] Example 3 Regulation of aspirin on the expression of MUC5AC and MUC5B in human primary airway epithelial cells
[0150] 1. Human primary airway epithelial cell culture
[0151]Preparation of Feeder cell: Culture NIH / 3T3 (mouse embryonic fibroblast cell line) in a 15cm culture dish with DMEM (H) medium (containing 10% FBS and 1% PS), and use Mitomycin C (4mg / L) after treating the cells for three hours, freeze them with cell preservation solution for future use.
[0152] 2. Isolation and expansion of primary human airway epithelium
[0153] Resuscitate the prepared Feeder cell (2M) in a 15cm culture dish (treated with PureCol) one day in advance, and replace it with CRCY medium the next day;
[0154] Microdissect a section of human bronchus and cut it longitudinally. Dilute pronase with Accutase to 8-10ml of 0.5mg / ml digestive solution. Add 1ml of FBS to stop the digestion, centrifuge at 3000g for 3min, discard the supernatant, resuspend in CRCY medium, add to the pre-...
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