Diabetic cardiovascular pathological change related gene and its use
An amino acid, cardiomyocyte technology, applied in genetic engineering, plant genetic improvement, application, etc.
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Embodiment 1
[0117] Embodiment 1. Preparation of diabetic rat model
[0118] 1.1 Animals and materials
[0119] 100 clean-grade male SD (Sprague-Dawley) rats, 2 months old, weighing (250±20) g, were purchased from BK Company, animal certificate number: Shanghai Dong 152; purchased from STZ (streptozotocin) Provided by Sigma Company; blood glucose, triglyceride, cholesterol and other biochemical detection kits were purchased from Mingdian Bioengineering Co., Ltd.; neutral unimodal pure insulin was purchased from Wanbang Biochemical Pharmaceutical Co., Ltd.; insulin primary antibody and related immunohistochemical reagents were purchased from From Maixin Company; Insulin Radioimmunization Kit for Rats was purchased from Linco Company.
[0120] Dietary ingredients: the average calorie of common feed is 3.9cal / g, of which carbohydrates account for 60%, protein accounts for 22%, fat accounts for 10% (mainly soybean oil), other ingredients including cellulose account for 8%; high-fat feed avera...
Embodiment 2
[0140] Example 2 Screening of genes related to early cardiomyopathy in rats with type 2 diabetes
[0141] 2.1 Materials
[0142] 2.1.1 Main instruments and equipment
[0143] Genomyx LRS fluorescence differential display analysis system (Beckman), the system includes (1) Genomyx LR electrophoresis instrument, including automatic gel drying device (2) Genomyx SC scanner (3) analysis software system includes Acquire SC, Clarity SC, ExtendLRS, Virtual (4) Glue cutting workstations such as Grid System. Spectrophotometer (Beckman DU650), 9700 PCR instrument (PE), Fluor-s TM multilmager gel image analyzer (Bia-RAD), low-temperature high-speed centrifuge (Beckman), hybridization oven (Robbins Scientific 2000).
[0144] 2.1.2 Main reagents
[0145] The HIEROGLPH mRNA Profile Kit (Beckman) includes: (1) 12 T7 (dT12) anchor primers (Anchored Primer, AP) with 3' end unlabeled fluorescence (2) 20 5'M13r random primers (Arbitrary Primer, ARP ). Fluoro-DDRT-PCR kit (Beckman) includes:...
Embodiment 3
[0212] Embodiment 3, neonatal rat primary cardiomyocyte isolation and culture
[0213] Take 12 newborn S-D rats 1-2 days old, soak them in 75% alcohol for 1 minute, and then take them out. They were wiped dry with alcohol cotton balls and executed by breaking the cervical vertebrae. A two-centimeter incision was made obliquely upward on the chest wall at the junction of the xiphoid process and the left 5th-6th rib to expose the apex of the heart, and the ventricle was quickly cut off and soaked in D-Hanks solution. After obtaining all twelve hearts, they were washed 6 times in ice D-Hanks solution to wash away the congestion in the ventricular cavity and destroy the endothelial cells of the ventricle. Use ophthalmic scissors to cut the above myocardial tissue into 1mm×1mm pieces, transfer it into a 50ml glass Erlenmeyer flask, continue to wash with D-Hanks solution, wait for the tissue to settle, and suck off the supernatant. Repeat twice. Add 0.125% trypsin + 0.01% EDTA (d...
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