Functions and application of activin receptor-like kinase 4 in treatment of cardiac hypertrophy
A technology for cardiac hypertrophy and cardiac function, applied to the function and application of activin receptor-like kinase 4 in the treatment of cardiac hypertrophy, the application of drugs for alleviating and/or treating cardiac hypertrophy, and the field of preparation and prevention, which can solve the problems that have not yet been seen in ALK4 To achieve the effect of inhibiting myocardial hypertrophy and improving heart function
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Embodiment 1
[0055] [Example 1] Effect of ALK4 interference (AdshALK4) and overexpression (AdALK4) adenovirus on Ang II-stimulated primary cardiomyocyte hypertrophy
[0056] 1. Primary neonatal SD rat cardiomyocyte culture
[0057] (1) Eight newborn Sprague-Dawley suckling mice were disinfected with 75% alcohol below the neck, and the heart was removed with ophthalmic scissors and micro forceps, and placed in a glass plate filled with 10mL DMEM / F12 solution. Take another one and repeat the above process.
[0058] (2) Wash the heart with DMEM / F12 medium, and cut the heart into 1-2mm 3 fragments. Transfer to a serum bottle with a rotor, suck off DMEM / F12, and add trypsin digestion solution. Rotate at 120r / min, digest for 15min, rest for a few seconds, and discard the supernatant.
[0059] (3) Add trypsin digestion solution, the rotation speed is 120r / min, and digest for 15min. Stand still for a few seconds, draw the supernatant, terminate the digestion with DMEM / F12 medium with 20% calf...
Embodiment 2
[0076] [Example 2] Construction of heart-specific ALK4 knockout mice and ALK4 transgenic mice:
[0077] (1) Construction of cardiac-specific ALK4 gene knockout mice (see figure 2 A):
[0078] According to gene information, using CRISPR Design (URL: http: / / crispr.mit.edu / ) to design a CRISPR targeting site in intron 1 and 2 respectively. The target sequences are:
[0079] ALK4-sgRNA 1: ggTCCATCCAAATGGGTCCTGC TGG
[0080] ALK4-sgRNA2: ggCTCAGGGCATGAAGCGTTTT TGG
[0081] In addition, a donor plasmid (Donor Vector) for homology repair was designed, which includes homology arms on both sides, exons 2 and 3 in the middle, and two loxp sequences in the same direction.
[0082] ①Construction of targeting vector: The two primers corresponding to sgRNA1 and sgRNA2 were respectively fused into double-stranded DNA, and then ligated into pUC57-sgRNA vector treated with restriction endonuclease BsaI with T4 DNA ligase. There is a T7 promoter upstream of the vector, which can be use...
Embodiment 3
[0103] [Example 3] Myocardial hypertrophy model acquisition
[0104] 1. Grouping of experimental animals: male background C57BL / 6 cardiac-specific Cre mice (α-MHC-Cre (WT), cardiac-specific ALK4 knockout mice (ALK4-KO) and cardiac-specific ALK4 transgenic mice ( TG) and non-transgenic mice (NTG), through aortic constriction (AB) to establish myocardial hypertrophy model. Randomly divided into 8 groups, grouped as follows: C57BL / 6 background wild-type mice sham operation group (WT Sham) and AB operation group (WT AB), ALK4 knockout mouse sham operation group (ALK4-KOSham) and AB operation group (ALK4-KO AB), non-transgenic mouse sham operation group (NTG Sham) and AB operation group (NTG AB), heart-specific ALK4 transgenic mouse sham operation group (TG Sham) and AB operation group (TG AB).
[0105] 2. The myocardial hypertrophy model adopts aortic arch coarctation (AB) surgery, and the operation process of the model is as follows:
[0106] 2.1 Preoperative preparation
[01...
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