Application of traf-binding NF-κB activator (tank) and its inhibitors in the treatment of cardiac hypertrophy
A technology for activating factor and myocardial hypertrophy, applied in the field of gene function and application, can solve problems such as limited treatment methods, and achieve the effect of worsening cardiac function, promoting myocardial hypertrophy, and promoting its fibrosis.
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Embodiment 1
[0049] Example 1 Construction of heart-specific TANK knockout mice and TANK transgenic mice
[0050] 1. Construction of heart-specific TANK knockout mice (see the construction strategy in figure 1 )
[0051] Construction of cardiac-specific TANK knockout mice using CRISPR-Cas9 technology. First, use the online CRISPR design tool (http: / / crispr.mit.edu) to design a CRISPR target site in introns 2 and 3 of the mouse TANK gene, respectively. The target sequences are:
[0052] TANKsgRNA1: ggAAAAATAGTGTCAAACTGTTGAC TGG,
[0053] TANK sgRNA2: gGCAGGGTTTCTCTGTTATAGCCC TGG.
[0054] In addition, a donor vector (Donor Vector) for homology repair was designed, which includes homology arms on both sides, exon 3 in the middle and two loxp sequences in the same direction.
[0055] (1) Construction of targeting vector: The two primers corresponding to sgRNA1 and sgRNA2 were respectively fused into double-stranded DNA, and then ligated into the pUC57-sgRNA vector treated with restriction...
Embodiment 2
[0078] Example 2 Acquisition of myocardial hypertrophy model
[0079] 1. Grouping of experimental animals: A model of myocardial hypertrophy was established by coarctation of the aorta (AB). The mice were randomly divided into 10 groups, and the groups were as follows: control group mice sham operation group (α-MHC-MCM Sham, TANK-flox Sham) and control group AB operation group (α-MHC-MCM AB, TANK-flox AB), TANK Gene knockout mice sham operation group (TANK-KO Sham) and AB operation group (TANK-KOAB), non-transgenic mouse sham operation group (NTG Sham) and AB operation group (NTG AB), heart-specific TANK transgenic mice Mouse sham operation group (TG Sham) and AB operation group (TG AB).
[0080] 2. The myocardial hypertrophy model adopts aortic arch coarctation surgery. The model operation process:
[0081] 2.1 Preoperative preparation
[0082] (1) Anesthesia: The mice were weighed first, the amount of anesthetic (3% pentobarbital sodium) required was calculated according ...
Embodiment 3
[0089] Example 3 Detection of myocardial hypertrophy and fibrosis in myocardial hypertrophy model mice
[0090] 1. Take material
[0091] (1) Preliminary work: Prepare a urine cup containing 20 mL of 10% formaldehyde by volume and label it (mouse number, group, type of surgery, and date of collection). A petri dish filled with 10% KCl solution by mass was placed at the sampling site. Turn on the analytical balance and set it to zero. Mice were sacrificed by weighing again.
[0092] (2) Material sampling: The vascular pedicle below the atrial appendage was clamped by ophthalmic curved forceps, the heart was cut off, and the heart was quickly placed in a 10% KCl solution. After the heart stopped beating in the diastolic phase, put it on sterilized gauze, gently squeeze the fluid in the cardiac cavity, dipped in the surface liquid, weighed and recorded, put the heart into the corresponding urine cup, fix it for 48 hours and then use it. for pathological examination.
[0093]...
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