Application of hnrnpa2b1 in cardiac fibroblasts
A fibroblast and heart technology, applied in the field of cell engineering and genetic engineering, can solve the problems of unclear specific mechanism of action and less research, and achieve good application value and the effect of promoting proliferation
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Embodiment 1
[0051] 1. Pretreatment.
[0052] Experimental object: Cardiac fibroblasts from suckling rats of Wistar rats born 2-3 days old, provided by Beijing Weitong Lihua Company, and the medium used is DMEM high-glucose medium from Gibco, USA.
[0053] (1) Preparation before the experiment: ultra-clean bench ultraviolet sterilization for 30 minutes.
[0054](2) Take cardiac fibroblasts from suckling mice, digest them with type II collagenase, and perform differential adhesion in culture flasks to remove non-fibroblasts.
[0055] (3) discard the supernatant, re-add the culture medium, at 37°C, 5% CO 2 Incubate in the incubator for 24 hours.
[0056] 2. Inoculation and transfection of cardiac fibroblasts.
[0057] (1) When the confluence of cardiac fibroblasts reaches 90% under microscope, the medium is discarded, and the cells are washed twice with preheated PBS containing 1% double antibodies (penicillin and streptomycin).
[0058] (2) Add preheated 0.25% trypsin to the culture bot...
Embodiment 2
[0079] qRT-PCR experiments.
[0080] (1) Wash the adherent cells 2-3 times with cold PBS, tilt the cell culture dish, aspirate the PBS, add 1 mL of Trizol, let stand for 5 min, until the cells are completely lysed under the microscope, blow evenly with a pipette tip and remove Transfer to a 1.5 mL RNase-free centrifuge tube.
[0081] (2) Add 200 μL of precooling solution (Trizol interstitial fluid: chloroform ratio: 5:1) to each tube, shake vigorously for 15-30 s, place on ice for 5 min, and centrifuge at 12,000 rpm at 4°C for 15 min. The liquid is divided into three layers: the upper layer is colorless RNA, the middle layer is a white aqueous phase containing phenol-chloroform, and the lower layer is light red. Carefully pipette off the upper colorless liquid phase (avoid touching the middle phase). Transfer the upper liquid phase (about 300 μL) into a new 1.5 mL centrifuge tube, add an equal amount of isopropanol solution, gently shake up and down 10 times, and let stand o...
Embodiment 3
[0117] Drug Affinity Reaction Target Stability Test (DARTS for short).
[0118] The specific operation steps are as follows:
[0119] (1) Preparation of M-PER lysate (1mL).
[0120]Mix 10 μL of protease inhibitor cocktail, 10 μL of 200 mM phosphatase inhibitor, 50 μL of 1 M sodium fluoride, 100 μL of 100 mM β-glycerophosphate disodium, 100 μL of 50 mM sodium pyrophosphate, and 730 μL of M-PER to obtain M-PER lysate.
[0121] (2) Wash the cells twice with pre-cooled PBS, aspirate the PBS, add 500 μL of M-PER lysate to each large dish (2 / 3 dishes can be combined into one according to the cell density), scrape the cells with a cell scraper and move them to a 1.5 mL centrifuge tube , and incubated for 30 min at 4°C on a rotary shaker.
[0122] (3) Centrifuge at 13,000 rpm at 4°C for 10 min, take the supernatant, and measure the concentration. Store at -80°C or proceed to the next step.
[0123] (4) Two 1.5mL centrifuge tubes were each filled with 225μL protein, one tube was ad...
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