A marker molecule for detecting mouse inner ear progenitor cells and its application
A technology for labeling molecules and progenitor cells, 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 progenitor cells
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Embodiment 1
[0039] Example 1 Isolation and cultivation of mouse inner ear stem cells
[0040] 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, vest...
Embodiment 2
[0044] Example 2 Induced differentiation of inner ear stem cells into inner ear progenitor cells
[0045] 1) The inner ear stem cell spheres formed in Example 1 after being cultured for 5 days were blown into single cells with a pipette gun with a volume range of 200 μL, centrifuged at 1000 rpm for 5 minutes, and the cells were collected, and then 200 μL of cells were resuspended in 2 mL of inner ear stem cell culture medium, Cultured at 37°C for 5 days to form P2 inner ear stem cell spheres. The inner ear stem cells of passage P2 were further subcultured to obtain inner ear stem cell spheres of passage P3.
[0046] 2) Incubate the culture dish with 200 μL of 0.1 mg / ml poly-lysine (Sigma Company) at 37° C. for 5 minutes, and then completely remove the poly-lysine. Rinse the Petri dish 3 times with PBS, 5 min each. Place the dish in a 37°C incubator to dry overnight.
[0047] 3) Suspend P3 inner ear stem cell spheres at a density of 500 spheres / ml in the inner ear progenitor...
Embodiment 3
[0048] Example 3 Detection of inner ear progenitor cell gene expression
[0049] 1) Extraction of total RNA from inner ear progenitor cells: Collect the culture solution containing inner ear progenitor cells in Example 2 in a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes and discard the supernatant; add 1mL TRIzol (TakaRa company), and shake on a vortex shaker until After the inner ear progenitor cells were completely dissolved, transfer them to a 1.5ml EP tube (Axgen); add 200 μL of chloroform (Sinopharm Chemical Reagent Co., Ltd.), vortex vigorously to mix, and place at room temperature for 5 minutes; centrifuge at 12,000 rpm for 15 minutes at 4°C, and absorb the upper layer of water Put about 300 μL into a new 1.5ml EP tube; add an equal volume of isopropanol (Sinopharm Chemical Reagent Co., Ltd.), invert to mix, and let stand at room temperature for 10 minutes; centrifuge at 12,000 rpm for 10 minutes at 4°C, discard the supernatant; Wash the precipitate with 75...
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