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Biological factor and application thereof in promoting regeneration of inner ear hair cells

A technology of biological factors and inner ear stem cells, applied in the field of biological inner ear research, can solve the problems that the effect has not been reported, and achieve the effect of regenerative recovery and great clinical application value

Active Publication Date: 2021-01-05
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of this gene in the inner ear has not yet been reported

Method used

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  • Biological factor and application thereof in promoting regeneration of inner ear hair cells
  • Biological factor and application thereof in promoting regeneration of inner ear hair cells
  • Biological factor and application thereof in promoting regeneration of inner ear hair cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Spatiotemporal expression pattern of Usp16 gene in mouse inner ear

[0042] See Table 1 and Table 2 for the reagents and antibodies used below.

[0043] Table 1 Main Reagents and Drugs

[0044]

[0045]

[0046] Table 2 Antibody and dye information and dilution ratio

[0047]

[0048] 1. The expression of Usp16 gene in the inner ear:

[0049] a) Protein extraction:

[0050]Three P30 and P60 wild-type mice were selected, and the temporal bones were taken out quickly after the neck was broken, and the tissues were dissected in pre-cooled HBSS. Protease inhibitor cocktail of corresponding proportion (place the centrifuge tube on ice), then add 2 steel balls soaked in medical alcohol to each tube, grind in an ice-cold grinder until homogenized state, place on ice for 10min, and then carry out at 4°C Centrifuge (12000g, 10min); take the supernatant (about 280μL) into a new 1.5mL centrifuge tube, add 70μL 5×loading buffer; clip the explosion-proof clamp...

Embodiment 2

[0065] Embodiment 2 Knockout mouse preparation and identification method

[0066] GFI-cre and Lgr5-CreER mice were bred in this laboratory, and USP16-Loxp\Loxp mice were obtained from Jin Jin Laboratory, School of Life Sciences, Zhejiang University.

[0067] 1. Genotype identification of USP16-Loxp\Loxp mice

[0068] (1) Cut off the corresponding numbered toe of the mouse and put it into a 200 μL centrifuge tube, add 180 μL of 50 mM NaOH solution, and digest in a PCR instrument at 98 ° C for 1 hour. The tissue is thoroughly mixed. The primer sequences are as follows:

[0069] Table 3 USP1 6-Loxp mouse identification primers

[0070]

[0071] (2) PCR amplification system

[0072]

[0073] (3) PCR amplification program

[0074]

[0075] (4) DNA agarose gel electrophoresis and imaging: 6 μL of DNA Ladder (Vazyme, PM107-02), 10 μL of amplified product, 120V electrophoresis for 35 min, and photographed by the gel imaging system after completion.

[0076] (5) Genotype...

Embodiment 3

[0078] Example 3 Effect of Usp16 gene deletion on mouse hearing and inner ear structure

[0079] 1. Gfi-Cre, Usp16-loxp / loxp and Gfi-Cre, and Usp16-loxp / + knockout mice were obtained by crossing Gfi-Cre mice with Usp16-loxp / loxp mice. Cre The enzyme can specifically knock out the Usp16 gene in the middle of the two loxp sequences in the hair cells, so as to achieve the knockout effect. Unfortunately, the Usp16-loxp / loxp mice will die shortly after birth, so our Gfi-Cre , Usp16-loxp / + mice were subjected to ABR hearing test and immunofluorescence experiment of inner ear structure.

[0080] Parameters such as auditory response threshold, latency, and wave interval were detected by ABR, and short sounds (Click) and short sounds of different audio frequencies (especially high frequency and ultra-high frequency) were used as stimuli to analyze the hearing sensitivity of mice.

[0081] in,

[0082] Table 4 Main Instruments of ABR Test

[0083]

[0084]

[0085] The specific...

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Abstract

The invention belongs to the field of biological inner ear research, and particularly discloses a biological factor and application thereof in promoting regeneration of inner ear hair cells. The biological factor is a histone deubiquitination enzyme USP16 in a single dose state, has the activity of promoting proliferation and differentiation of inner ear stem cells into hair cells, and has an amino acid sequence as shown in SEQ NO.1. The inventor finds the space-time expression mode of USP16 in the inner ear of a mouse for the first time, subsequently finds that after the gene is knocked downin mouse hair cells, hair cell apoptosis is caused such that the normal auditory function is influenced, and finally finds that after the USP16 gene is knocked down from the inner ear Lgr5 positive inner ear stem cells, proliferation of the inner ear stem cells is promoted such that hair cells proliferate. The USP16 gene disclosed by the invention lays an important foundation for subsequent research on mouse hair cell regeneration.

Description

technical field [0001] The invention belongs to the field of biological inner ear research, and relates to a biological factor and its application in promoting inner ear hair cell regeneration. Background technique [0002] Loss of mammalian hair cells will directly lead to hearing loss. At present, the main technology is to assist patients to restore certain hearing functions through cochlear implants, but this is only a temporary solution, not the root cause. In addition, the effect of cochlear implants varies from person to person, and only some people can reach or approach the hearing level of normal people. If it can maintain the homeostasis of hair cells and promote the regeneration of hair cells, it has great research prospects. At present, many scholars have studied the regeneration of hair cells, but the regeneration effect is not ideal. In addition, it is even more difficult to know whether the hair cells have normal hearing function after regeneration. The regen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12N15/57A61K38/48A61P27/16
CPCC12N9/485A61P27/16C12Y304/19012A61K38/00
Inventor 柴人杰洪国栋高下唐明亮付小龙
Owner SOUTHEAST UNIV
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