Tagged molecules for detecting mouse inner ear stem cell and application thereof
A technology of inner ear stem cells and marker molecules, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., and can solve the problems of separation, purification and identification of inner ear stem cells
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Embodiment 1
[0036] Example 1 Isolation and culture of mouse inner ear stem cells
[0037]1) ICR mice (Zhejiang Academy of Medical Sciences) were anesthetized in a refrigerator at -20°C for 5 minutes, and then sterilized by immersing them in 75% alcohol. The mice were directly decapitated with surgical scissors, and the fur was removed. Then, the head was divided in half under aseptic condition, the brain and brainstem were removed, and the temporal bone at the base of the skull was fully exposed. Carefully separate the temporal bone with scissors and tweezers, and transfer it to a 3.5 cm Petri dish filled with 4°C pre-cooled PBS (pH 7.4). Under a microscope, a small hole was drilled at the bottom of the cochlea with tweezers, and the shell was cut from bottom to top to expose the complete modiolus and cochlear duct. Clamp the very bottom of the cochlear duct with tweezers and separate the cochlear duct from the modiolus. The resulting cochlear duct includes the spiral ligament, vestibu...
Embodiment 2
[0041] Example 2 Detection of inner ear stem cell gene expression
[0042] 1) Extraction of total RNA from inner ear stem cells: Collect the culture medium containing inner ear stem cell spheres cultured for 5 days in Example 1 in a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes and discard the supernatant; add 1mL TRIzol (TakaRa Company), and vortex Shake on the machine until the inner ear stem cells are completely dissolved, transfer to a 1.5ml EP tube (Axgen Company); add 200 μL of chloroform (Sinopharm Chemical Reagent Co., Ltd.), shake vigorously to mix, and place at room temperature for 5 minutes; centrifuge at 12,000 rpm for 15 minutes at 4°C , pipette about 300 μL of the upper aqueous phase into a new 1.5ml EP tube; add an equal volume of isopropanol (Sinopharm Chemical Reagent Co., Ltd.), mix by inverting, and let stand at room temperature for 10 minutes; centrifuge at 12,000 rpm for 10 minutes at 4°C, discard the upper Clear; wash the precipitate with etha...
Embodiment 3
[0047] Example 3 Detection of 5-bromodeoxyuridine (Brdu) in dry microspheres of the inner ear
[0048] 1) The inner ear stem cell spheres formed by culturing for 5 days in Example 1 were blown into single cells with a pipette gun with a volume range of 200 μL, and the cells were collected after centrifugation at 1000 rpm, and then the cells were washed with 2 mL of 5 μmol / L Brdu (Sigma company) After the inner ear stem cell culture medium was resuspended, cultured at 37°C for 5 days to form progeny inner ear stem cell spheres. The coverslip was incubated with 200 μL of 0.1 mg / ml poly-lysine (Sigma) for 5 minutes, and then the poly-lysine was completely aspirated. Rinse coverslips with PBS 3 times, 5 min each. Place the coverslips in a 37°C incubator to dry overnight. The progeny inner ear stem cell spheres were inoculated on the above-mentioned coverslips pretreated with polylysine, and then incubated in DMEM / F12 medium with 10% (v / v) fetal bovine serum (PBS; GIBCO company) ...
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