Exosome for highly expressing miR-214 as well as preparation method and application of exosome
A 1. mir-214, exosome technology, applied in the field of cell biology, can solve problems such as research that has not yet been reported
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Embodiment 1
[0037] Example 1 The cultivation of mesenchymal stem cells and the isolation of exosomes
[0038] (1) After resuscitation, umbilical cord mesenchymal stem cells were passaged to passages P3-P6 using DMEM:F12+10% FBS, and the expression of their surface marker molecules was identified by flow cytometry.
[0039] (2) Cells were expanded to about 90% confluence, digested with 0.25% trypsin (containing 0.02% EDTA), and passaged at a ratio of 1:2.
[0040] (3) Normoxic conditions (95% air, 5% CO 2 ) after cultivating overnight, remove the original medium, replace the medium with exo-free medium, and place it in a hypoxic environment (1% O 2 , 5% CO 2 , 94%N 2 ) for 48 h.
[0041] (4) Collect the cell culture supernatant for the isolation of exosomes.
[0042] (5) Centrifuge at 2000 g for 30 minutes to remove dead cells and cell debris, take the supernatant and filter it into a new centrifuge tube using a 0.22 micron filter.
[0043] (6) Add 0.5 times volume of exosome isolati...
Embodiment 2
[0044] Example 2 Identification of exosomes
[0045] (1) TEM observation
[0046] The isolated exosomes were resuspended with 1×PBS, and then 1 / 9 volume of glutaraldehyde at a concentration of 25% was added to the suspension for fixation (final concentration of glutaraldehyde was 2.5%). Take 30 μL of the fixed exosome suspension and drop it on the carbon film-coated copper grid, and let it stand at room temperature for 1 min. Dry under infrared light for 5 minutes. Add a drop of 1% phosphotungstic acid solution (pH 6.8) onto the copper grid, and stain for 5 minutes at room temperature. The filter paper was used to absorb the phosphotungstic acid solution, and the copper grid was placed under an infrared lamp to dry for about 10 minutes. Observe and collect photos under a transmission electron microscope.
[0047] (2) Western blot identification
[0048] Take out the frozen exosomes from the -80°C refrigerator, add 1 / 4 volume of 5×SDS non-reducing loading buffer to the sus...
Embodiment 3
[0051] Example 3 Study on the effect of MSCs-derived exosomes on the treatment of myocardial infarction after hypoxia induction
[0052] (1) The rat myocardial infarction model was used to evaluate the myocardial repair effect of exosomes with different miR-214 expression. Male SD rats with a weight of about 250 g were selected. After anesthesia, tracheal intubation was performed, and small animal ventilator support (breathing ratio 1:1, tidal volume 6 mL, frequency 70 times / min).
[0053] (2) Cut the skin along the left side of the sternum, select the 3rd-4th intercostal space for thoracotomy, and use 6-0 silk thread to ligate the left anterior descending coronary artery at 1-2mm below the left atrial appendage.
[0054] (3) According to different groups, exosomes derived from MSCs cultured in hypoxia and normoxia were transplanted into the peripheral area of myocardial infarction according to the volume of 20 μL (20 μg) at each point.
[0055] (4) VisualSonics Vevo 2100 s...
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