Application of metformin (MET) combined with adult stem cells in preparing medicine for treating myocardial infarction (MI)
An adult stem cell and combined adult technology, which is applied in drug combinations, medical preparations containing active ingredients, pharmaceutical formulations, etc., can solve the problems of limiting the efficacy of myocardial infarction and low survival rate, and achieve the effect of increasing survival and improving ability.
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Embodiment 1
[0018] Extraction, Culture and Identification of Cardiosphere-derived Cells (CDC)
[0019] 16-week-old C57 mice (C57BL / 6-Tg(CAG-EGFP)C14-Y01-FM131Osb) were labeled with GFP, the isolated heart of the mouse was taken, and the mouse heart was cut into pieces smaller than 1mm 3 The tissue block was digested with 0.05% trypsin, and then the tissue was seeded in a 60mm small dish pre-incubated with fibronectin, and then 1ml of CEM (80% IMDM, 20% serum, 1% double antibody) was added, and the trypsin was used 7 days later. The incubated CDCs were digested enzymatically.
[0020] Centrifuge the above CDC cells at 1000r / min for 3min, discard the supernatant medium, wash twice with PBS, add CD29, CD105, CD90, CD45, CD34, CD31 antibodies, and FITC and PE isotype antibodies and incubate at 4°C for 30min, then PBS Wash twice, use C6 flow cytometer to identify, and the sample volume is 10000. The identification results were positive for CD29, CD105, and CD90, and negative for CD45, CD34, ...
Embodiment 2
[0022] Metformin (MET) combined with CDC can improve cardiac function in mice after myocardial infarction
[0023] Mice were randomly divided into four groups: MI+PBS, MI+MET, MI+CDC and MI+MET+CDC. Metformin 125 mg / kg was injected intraperitoneally 6 hours before myocardial infarction. After induction of anesthesia by isoflurane, the mice were fixed on a heating blanket, the skin and muscles of the neck were cut longitudinally, the trachea was exposed, and the trachea was intubated. The skin and muscles were cut transversely in the fourth intercostal space of the mouse, the ribs were exposed, the intercostal muscles were cut open, the heart was exposed with a spreader, the pericardium was torn, and the coronary artery was ligated 2-3mm below the left atrial appendage with 7-0 silk thread. Seeing that the color of the heart turned white, it indicated that the ligation was successful. In the experimental group, CDC was injected at 8 points at the edge of the myocardial infarct...
Embodiment 3
[0025] Metformin promotes CDC survival and reduces infarct size
[0026] Extract the cardiomyocytes of each group of mice in Example 2 and count the CDCs with green fluorescence under a microscope, specifically as follows: take the isolated hearts of mice in each group, place them in PBS at 4°C, and move them under the microscope; remove them with ophthalmic forceps For fat and other tissues around the aorta, quickly hang the aorta on the needle and tie it with a thread to fix it. The position of the needle should not exceed the root of the aorta; inhale No. 4mM KCl, 1mM MgCl 2 .6H 2 0, 10mM Hepes, 10mM taurine, 10mM BDM) to flush out the blood in the heart; remove the needle, hang it on the perfusion device, remove the air bubbles in the perfusion device, perfuse with No. 1 solution for 2min, and then change No. 2 solution ( 50ml No. 1 solution, 50mg collagenase, 6mg trypsin, 0.5M CaCl 2 2uL) to wash and digest for 10 minutes until the heart tissue becomes soft and collap...
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