Application of miR-135a in preparation of drugs for treating myocardial fibrosis
A technology of 1.mir-135a and 2.mir-135a, applied in the field of biomedicine, can solve the problem of no research on the influence of fibrosis-related pathways
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Embodiment 1
[0044] Example 1: Detection of microRNA in plasma of patients with atrial fibrillation
[0045] 10 cases of plasma from healthy and simple atrial fibrillation patients were obtained from Jiangsu Provincial People's Hospital. The sample age was 50±5 years old. The experiment was approved by the Ethics Committee of Jiangsu Provincial People's Hospital and China Pharmaceutical University.
[0046] Medicines and reagents: DEPC water (Biyuntian Bio), miRNeasy Serum / Plasma kit (QIAGEN, Germany), miRNA first strand cDNA synthesis (tailing method) kit (Biotech), Premix Ex Taq TM II (Dalian Biotech)
[0047] 1. Extraction of plasma microRNA
[0048] (1) Plasma sample processing
[0049] Medical anticoagulant tube (containing EDTA-K2 anticoagulant) to collect fresh blood samples, centrifuge at 3000rpm for 10min, take the upper plasma into a new RNase-free centrifuge tube, centrifuge at 12000rpm for 10min at high speed to remove cell debris, and take the upper plasma (don’t The cell debris suck...
Embodiment 2
[0075] Example 2: MicroRNA-135a targets TRPM7 channel to inhibit hypoxia-induced myocardial fibrosis
[0076] Experimental animals: 1 to 3 days old suckling rats, male or female, provided by the Center of Comparative Medicine of Yangzhou University, animal certificate number: SCXK (苏)2016-0001.
[0077] Medicines and reagents: FBS (Gibico, USA), low-sugar DMEM (HyClone, USA), penicillin, streptomycin, Trypsin, EGTA (Biosharp), HEPEs, DEPC water, Trizol, BeyoECL Plus developer, RIPA lysis solution (strong) , BCA protein quantification kit (enhanced) (Biyuntian Bio), Cs-methanesulfonate (cesium methanesulfonate), 2-APB, Carvacrol (carvacrol), DMSO (Sigma Aldrich, United States), type II collagenase ( Worthington, USA), miRNA first strand cDNA synthesis (tailing method) kit, 5×SDS-PAGE loading buffer (Biotech), PrimeScript TM RTMaster Mix, Premix Ex Taq TM II (Dalianbao Bio), rabbit anti-TRPM7 protein primary antibody (Abcam, UK), rabbit anti-GAPDH internal control primary antibody...
Embodiment 3
[0147] Example 3: MicroRNA-135a targets TRPM7 channel to inhibit isoprenaline-induced myocardial fibrosis
[0148] Experimental animals: SPF grade SD rats, male, weighing 200-220g, purchased from the Comparative Medicine Center of Yangzhou University, certificate number: SCXK (Su) 2015-0002. Breed in an environment of 20±2℃, keep 12h light / 12h dark conditions, and freely feed in water. 1-3 days old suckling rats, male or female, purchased from the Center of Comparative Medicine, Yangzhou University, animal certificate number: SCXK (苏)2016-0002.
[0149] Medicines and reagents: I Reduced Serum Medium (Gibco, USA), lipo 6000 transfection reagent (Biyuntian Biotechnology), Isoprenaline hydrochloride (Sigma, USA), bovine serum albumin (Biosharp), the sources of other reagents and medicines are the same as in Example 2.
[0150] 1. Solution and culture medium
[0151] TRPM7 current extracellular fluid (mM): 140NaCl, 5KCl, 2CaCl 2, 20HEPEs, 10Glucose, NaOH adjusted to pH 7.4, stored at 4°...
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