Application of sphingosine phosphorylcholine in the preparation of drugs for promoting the differentiation of endogenous cardiac stem cells into cardiac lineage cells
A technology of sphingosine phosphorylcholine and cardiac stem cells, which is applied in the new application field of sphingosine phosphorylcholine, and can solve problems such as unreported
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Embodiment 1
[0044] Example 1 Extraction of Sca-1+ cardiac stem cells and detection of their purity by flow cytometry
[0045] ①The 10-week-old C57 / BL mice were killed by neck dislocation, and the heart was quickly disinfected with alcohol, cleaned, and cut into pieces to about 1mm 3 size.
[0046] ② Digest with trypsin solution at 37°C for 30 minutes.
[0047] ③ After digestion, add a little fetal bovine serum to stop the digestion, filter with a 200-mesh cell sieve, add a small amount of cellstaining buffer to suspend the filtered cells, and count the cells.
[0048] ④ Centrifuge the filtrate at 350g for 8min, and resuspend the cells with cell staining buffer to make the density reach 2×10 7 / ml, add biotin-labeled sca-1 antibody to the solution., per 1×10 7 Add 5 μl antibody to each cell and incubate on ice for 20 min.
[0049]⑤Use an excess volume of 1xBD IMag TX The labeled cells were washed with buffer, centrifuged at 350g for 8min, and the supernatant was carefully sucked off. ...
Embodiment 2
[0055] The preparation of embodiment 2 SPC storage solution
[0056] A stock solution with a concentration of 5 mg / ml was prepared from the SPC powder provided by sigma, dispensed into 1.5 ml eppendorf tubes, dried with liquid nitrogen, and stored at -20°C.
Embodiment 3
[0057] Example 3 SPC to sca-1 + Examination of cardiac stem cell morphology effects
[0058] The extracted sca-1 + Cardiac stem cells were seeded in 24-well plates, and after 7 days they were congested, evenly introduced into small dishes, cultured with IMDM containing growth factors and 10% fetal bovine serum, passaged after confluence, and stem cells of passage 1-5 were used for experiments. The cells attached to the wall for 24 hours were added with different concentrations (1, 2, 5 μM) of SPC as the experimental group. Add ethanol as the solvent control group, without any treatment as the normal group. Culture conditions: 37°C, CO 2 Cultured in an incubator.
[0059] Change the culture medium every three days and continue to add the SPC of the concentration, and observe regularly under an inverted phase contrast microscope after cultivating for 2-3 weeks (results see figure 2 ).
[0060] The results showed that compared with the normal group and the solvent control ...
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