CN109078169B

CN109078169B - Application of recombinant human Neuritin protein in preparation of medicine for treating sensorineural deafness and related diseases

Info

Publication number: CN109078169B
Application number: CN201810863738.9A
Authority: CN
Inventors: 杨磊; 黄瑾; 汪海燕; 董玉梅; 孙筱品; 桂飞; 宋晓明; 朱井玲; 杨怡; 洪玉; 谌容
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2022-02-11
Anticipated expiration: 2038-08-01
Also published as: CN109078169A

Abstract

The utility model discloses an application of recombinant human Neuritin protein in preparing a medicament for treating sensorineural deafness and related diseases. The unique action of the recombinant human Neuritin protein of the utility model can reach or even exceed the regeneration or nutrition action of damaged hair cells by the administration of a single Notch blocker or the single use of neurotrophic factors, thereby playing the role of treating sensorineural deafness and related diseases. However, it is not necessary for any product to achieve all of the above-described technical effects simultaneously.

Description

Application of recombinant human Neuritin protein in preparation of medicine for treating sensorineural deafness and related diseases

Technical Field

The application belongs to the technical field of biological medicines, and particularly relates to application of recombinant human Neuritin protein in preparation of medicines for treating sensorineural deafness and related diseases.

Background

Sensorineural deafness is a common disease of the ear, and although the pathways and mechanisms leading to sensorineural deafness are diverse, irreversible damage to the inner ear hair cells is the core cause of sensorineural deafness. Although hearing aids and cochlear implants which are commonly used in clinic at present can partially improve the hearing of patients, the structure and function of damaged cells cannot be fundamentally repaired, and the treatment effect also depends on the quantity and quality of residual hair cells and spiral neurons. Therefore, the discussion of methods and strategies for hair cell regeneration after injury has been the focus of recent research in the field of hearing.

Hair cells are sensory epithelial cells that sense acoustic stimuli and function to convert sound into electrical stimuli that are sent to the brain. As the sound waves pass, ciliary beat on the surface of the hair cells converts the mechanical energy of the sound waves into neuro-biological electrical signals, which are transmitted to the brain via nerve fibers. The inner ear hair cells of mammals are constant in number, terminally differentiated cells, irreversible after damage, and have no regenerative repair ability. Studies have shown that cochlear support cells are a major source of hair cell regeneration. Mammalian cochlear support cells (including Hensen cells, Deiters cells, and inner and outer column cells) and hair cells are derived from the same precursor cells during development. The precursor cells are regulated and controlled by various signal pathways in the process of directionally differentiating to hair cells and supporting cells, wherein the Notch signal pathway participates in the differentiation regulation of the hair cells in a lateral inhibition mode and plays an important role in maintaining the quantity and the structural homeostasis of cochlear sensory epithelial cells. Studies have shown that inhibition of the Notch pathway promotes the transdifferentiation of the support cells into new hair cells following mammalian hair cell injury.

Neuritin is a neurotrophic factor closely related to neural plasticity, and can promote the growth of neurites and the development and maturation of branches and synapses of the neurites, regulate the formation of synaptic circuits, inhibit apoptosis and maintain the survival of neurons. Is a common downstream factor for the functioning of neurotrophic factors and neural activity, and is also an essential and key molecule for mediating androgen and electrical stimulation to promote neural regeneration. The compound shows good effects of promoting injured tissue structure and functional recovery in sciatic nerve and spinal cord injury models. In addition, neuron can down-regulate Notch signal pathway and reduce the expression of its receptor NICD and downstream target gene HES 1. The above studies suggest that Neuritin may play an important role in the injury repair of sensorineural deafness.

At present, the therapeutic research on deafness uses small molecule compounds such as Notch pathway blocker DAPT, LY411575 and the like to promote the regeneration of hair cells by blocking Notch, and the hair cells regenerated by the method have ion channel activity but no nerve re-innervation effect, thereby influencing the functions of the regenerated hair cells. And the induction of the re-innervation of hair cells requires the participation of neurotrophic factors such as BDNF, NT-3 and the like. Neuritin is a neurotrophic factor capable of inhibiting Notch pathways, and is also a downstream effector which acts like BDNF, NT-3, NGF and the like. Its unique effect will reach or even exceed the effect of Notch blockers alone or neurotrophic factors alone on damaged hair cells.

Disclosure of Invention

In view of the above, the present application provides an application of a recombinant human Neuritin protein in the preparation of a medicament for treating sensorineural deafness and related diseases.

In order to solve the technical problems, the utility model adopts the following technical scheme:

compared with the prior art, the application can obtain the following technical effects:

application of the recombinant human Neuritin protein in preparing medicines for treating sensorineural deafness and related diseases.

Sensorineural deafness is a disease in which cochlear hair cells are damaged due to noise damage, drug damage, aging and the like, while supporting cells are not obviously damaged.

After the recombinant human Neuritin protein is added into a culture medium for in vitro culture of cochlear tissues,

the recombinant human Neuritin protein dose-dependently promotes a large amount of supporting cells in the cochlear cotti tissue of the newborn mouse to be proliferated and transdifferentiated into hair cells, so that the number of the hair cells is increased; the administration concentration range of the recombinant human Neuritin protein is 2, 4, 8, 16, 32 and 64 mu g/mL; the induction time was 3 days.

The recombinant human Neuritin protein induces the regeneration and arrangement of damaged hair cells in the cochlear corti tissue in a dose and time-dependent manner, and the shape and arrangement of the regenerated hair cells are gradually mature along with the prolonging of the action time. The administration concentration range of the recombinant human Neuritin protein is 4, 16 mug/mL; the induction time is 3, 5 and 7 days after hair cell damage.

Half of the liquid change is carried out every day in the induction culture process, and the specific method is to prepare 2 times of the recombinant human Neuritin protein (2 x) with the required final concentration in a culture medium, and to change half of the culture medium in the original culture medium into a newly configured culture medium until the concentration of the recombinant human Neuritin protein is 1 time (1 x).

The unique action of the recombinant human Neuritin protein of the utility model can reach or even exceed the action of a single Notch blocker or a single neurotrophic factor on damaged hair cells, thereby playing the role of treating sensorineural deafness and related diseases, and certainly, any product implementing the application does not necessarily need to simultaneously achieve all the technical effects.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

fig. 1 is a schematic diagram of Neuirin expression and localization in the cochlea under normal conditions.

Wherein A is the protein expression condition of Neuirtin detected by a Western blot method. B, the location of Neuritin in cochlear cells is detected by immunofluorescence, and the result shows that Neuritin is expressed in both cochlear support cells (sox2) and hair cells (phallodin).

FIG. 2 is a schematic representation of the identification of a drug-induced deafness model in CBA mice.

Wherein A is the statistics of hearing threshold before and after modeling of drug-induced deafness of CBA mice, and after modeling, before modeling vs: p is less than 0.01.

B is the cochlear hair cell (myo7a) and the supporting cell (sox2) status before and after modeling.

FIG. 3 is a schematic diagram of correlation test of Neuritin expression and deafness.

Wherein A is the protein expression change condition of Neuirtin before and after the injury detected by a Western blot method at different time periods. And B is the quantification result of the A picture.

FIG. 4 is a schematic diagram of the establishment of a new born mouse cochlear cotti tissue in vitro culture method.

Wherein A is a whole cochlea pattern (whole mount); and B is the number, shape and position of hair cells and supporting cells under normal culture conditions.

FIG. 5 is a graphical representation of the effect of different concentrations of Neuritin on hair cells and supporting cells of corti cultured in vitro to determine the effective concentration of Neuritin protein. .

From left to right are the control group without the Neuritin protein, the Neuritin protein 2, 4, 8, 16, 32, 64 μ g/mL treatment group, and the Neuritin group is occupied by hair cells where it supports cytopenia.

FIG. 6 is a schematic diagram of the establishment of a hair cell damage model in corti cultured in vitro and the study of the regenerative repair effect of Neuritin.

A is a cochlea pattern of 3-day PBS culture after 24-hour injury of 3mM gentamicin; b is a cochlea pattern of 4 mug/mL Neuritin which is cultured for 3 days continuously after 3mM gentamicin and 4 mug/mL Neuritin are co-cultured for 24 hours; c is a cochlea pattern of 16 mu g/mL Neuritin which is cultured for 3 days after 3mM gentamicin and Neuritin are cultured together for 16 mu g/mL for 24 hours.

FIG. 7 is a schematic representation of the morphology of the cochlear apical hair returning cells and the supporting cells of the respective groups of FIG. 6. .

Fig. 8 is a schematic diagram of the morphology of hair-returning cells and supporting cells in each group of cochlea in fig. 6. .

FIG. 9 is a schematic representation of the morphology of the cochlear bottom hair returning cells and the supporting cells of the respective groups of FIG. 6. .

Fig. 10 is a schematic diagram of the phenomena observed in the neurotin group of hair cells in the mitotic phase and proliferating support cells. .

FIG. 11 is a schematic representation of the morphology of hair cells and supporting cells from each group of FIG. 6 cultured for 5 days. .

FIG. 12 is a schematic representation of the morphology of hair cells and supporting cells from the groups of FIG. 6 cultured for 7 days. .

Detailed Description

Embodiments of the present application will be described in detail with reference to the drawings and examples, so that how to implement technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.

Example 1: neuritin expression and localization in cochlea

(1) Neuritin expression in CBA mouse cochlea

1) Extraction of total cochlear protein

After 2-3 CBA mice are anesthetized, skin, muscles and cervical vertebra of the neck are cut off by common scissors at the position of the neck close to the occiput, the scissors are inserted into the macropores of the occiput after head breaking, skull fossae on the left side and the right side are respectively cut off and exposed along the upper parts of the temporal bones on the two sides, the rock tips on the two sides and the temporal scaly parts on the two sides are folded by middle fingers and thumbs of the two hands, the auditory meatus is completely extracted, the hemostatic forceps open the auditory meatus, the tympanic cavity is exposed, the outer wall of the tympanic cavity is cut off, the inner wall of the tympanic cavity is fully exposed, and the cochlea is separated and exposed. The tissue preserved by the method can be used for Western blot experiments.

2) Processing of protein samples

The magnetic beads and the homogenizing tube are cleaned at the night and soaked in 75% ethanol. Taking out the magnetic beads and the homogenizing tube in the morning, and naturally drying; the preparation of the cracking liquid, RIPA and PMSF are 100: 1, and the cracking liquid is prepared in situ. (1000. mu. LRIPA + 10. mu. LPMSF). Put on ice at 4 ℃. Protein extraction was performed on ice.

a. And taking the cochlear tissue out of the liquid nitrogen, putting the cochlear into a mortar, adding a certain amount of liquid nitrogen for grinding, collecting the ground tissue into an EP tube, and adding a lysis solution.

b. The tissue was homogenized in a 1.5mL EP tube and allowed to stand for 10min, and this was repeated three times. The homogenized tissue was centrifuged at 12000r/min4 ℃ for 15 min. After centrifugation, the liquid in the EP tube is divided into three layers, and the intermediate colorless liquid phase is extracted and transferred into a new EP tube. Can be stored in a refrigerator at-80 deg.C.

c. The concentration of the extracted protein was determined spectrophotometrically.

d. Subpackaging the protein solution, and storing at-80 deg.C for use.

3)Westernblot

a. Preparing glue: 15%, 1.5mm, mini glue

SDS-PAGE electrophoresis gel was prepared from the EpiZyme PAGE gel Rapid preparation kit PG 114.

Composition and preparation of kit

b. Adding the prepared separation gel into a gel tank according to the proportion, then adding a little distilled water, and standing for 30 minutes at room temperature.

c. After the separation gel is solidified, sucking dry distilled water, adding the prepared concentrated gel in proportion and inserting a comb.

d. After the concentrated gel is solidified, the comb teeth are pulled out to carry out protein electrophoresis.

e. Loading: adding a 5 xSDS-PAGE protein loading buffer into a protein sample; cooking at 100 ℃ for 8 minutes to fully denature the protein.

f. Electrophoresis: 1 × Tris-Gly solution with pH of 8.3, 110V, 30 min, after the sample is pressed into a straight line in the concentrated gel, 80V running is continued until the bottom of the gel.

g. Film transfer: transferring the protein from the gel to the membrane by a wet-to-membrane converter, sequentially placing thin-layer sponge, thin filter paper, protein gel, PVDF (polyvinylidene fluoride) membrane, thin filter paper and thin-layer sponge from black to white, removing bubbles from each layer by using a test tube, and setting the voltage and time to be 110V and 90min respectively.

h. And (3) sealing: the membrane was placed in a blocking solution containing 5% skimmed milk powder prepared with 1 XTSST and blocked on a shaker at room temperature and low speed (40-60rpm) for 1-2 h.

i. Incubating the primary antibody: neuritin monoclonal antibody is diluted to a newly prepared confining liquid containing 5% skimmed milk powder according to the recommended proportion of the instruction at a ratio of 1: 500, and is shaken overnight at 4 ℃.

j. Washing: the membrane was transferred from the primary antibody to a membrane washing chamber, face up, with 1 × TBST added, and washed 5 times (70-90rpm) with rapid shaking, the first 5min, followed by 4 times 15min each.

k. Incubation of secondary antibody: adding goat anti-rabbit IgG secondary antibody into newly-prepared confining liquid containing 5% skimmed milk powder at a ratio of 1: 3000 according to the recommended proportion of the instruction, and placing on a shaking table to incubate at room temperature and low speed (40-60rpm) for 1 h.

1. The front side of the membrane is placed upwards, put into TBST solution, and washed on a shaking table for about 15min each time for 3 times.

m. mixing the developers A and B in equal volume, dropping the developer on the membrane in a dark room, developing through a film, and analyzing the results.

(2) Neuritin localization in CBA mouse cochlear cells

1) Obtaining a basilar membrane of a cochlea of an adult mouse: after a mouse is killed, taking out bilateral auditory follicles, peeling temporal bone tissues under a dissecting microscope, exposing cartilage surfaces, taking out the whole inner ear labyrinth, carefully separating a volute from a spiral ligament by using forceps and removing the volute, then completely taking down cochlear epithelium together with the spiral ligament and the blood vessel veins on the outer side from a volute shaft, separating a structure connected with cochlear epithelial tissues to obtain a complete cochlear basilar membrane, placing a specimen in 4% paraformaldehyde fixing liquid, and fixing in a refrigerator at 4 ℃ for more than 0.5h to be subjected to immunofluorescence staining;

2) and (3) immunofluorescence staining:

a. the fixed specimen was removed, the 4% paraformaldehyde solution was discarded, and the sample was washed with 1 × PBS 3 times for 15 minutes each at room temperature.

b.10% horse serum + 0.03% saponin (formulated with 1 × PBS of 0.1% TritonX 100) blocked for 1 hour at room temperature.

c. Primary antibodies were MyoVIIa (rabbit-derived polyclonal antibody, 1: 1000) or SOX2 (mouse-derived monoclonal antibody, 1: 500) formulated using 3% horse serum + 0.03% saponin + 3% BSA (formulated with 1 XPBS of 0.1% TritonX 100). The mixture was left overnight in a refrigerator at 4 ℃.

d. The next day, primary antibody was discarded and washed 4 times with 0.1% PBST (1 XPBS with 0.1% Triton X100) for 2 hours each.

e. Secondary antibodies were formulated with goat anti-rabbit Alex Fluro 488 or goat anti-mouse Alex Fluro 555, 3% horse serum + 0.03% saponin + 3% BSA (formulated with 0.1% triton x100 in 1 × PBS). And the temperature is normal for 2 hours.

f. The secondary antibody was discarded and washed 3 times with 1 × PBS for 15 minutes each at room temperature.

Phallodin was incubated for 30 min, washed 3 times with 1 × PBS, 5min each, at room temperature.

h. Tissues were transferred to slides, 20 μ L DAPI (containing mounting medium) was added, mounted, and confocal images were taken as shown in fig. 1.

Example 2: establishment and identification of drug-induced deafness model of CBA mouse

(1) Establishment of drug-induced deafness model of CBA (CBA) mice

After 60 healthy CBA mice (male, 4-6 weeks old) are purchased, conventional feed is fed into SPF animal rooms of the center of Hangzhou university laboratory animals to adapt to the environment for 1 week, and the animals are randomly divided into 8 groups and marked by numbers: groups of 10 animals per group were normal, 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 28 days after molding. The normal group did not inject any drug; a drug-induced deafness injury model of the CBA mice is established by a method of injecting kanamycin sulfate (1mg/g) subcutaneously for half an hour and then injecting furosemide (0.5mg/g) into the abdominal cavity.

(2) Identification of drug-induced deafness model of CBA mice (ABR hearing function test)

In the auditory brainstem response test (ABR), 5 percent chloral hydrate is used for anesthesia according to intraperitoneal injection of 0.01mL/g, and after 5-10min, the rat is observed to have no stabbing pain reflex and weak respiration, so that the experiment can be started when the anesthesia is proper. The mouse with moderate anesthesia is fixed on a laboratory table in a sound insulation chamber in a prone position; ABR detection is carried out by using an ABR instrument of the American TDT company, a measuring electrode is inserted under the mastoid of a detected square ear, a recording electrode is inserted under the skull crest of the middle of two ears, and a grounding electrode is inserted under the mastoid of the opposite side ear of the detected ear; the impedance is required to be less than < 3k omega, and the tip of each electrode needle is left to penetrate into the subcutaneous tissue by 5 mm. The earphone is placed at a position 10cm away from the external auditory canal of a mouse detection ear, the stimulation sound is alternate short sound, the repetition rate is 21.1counts/s, the recording and analyzing time is 15ms, the superposition times are 1024 times, and the band-pass filtering is 100-3000 Hz. The initial stimulation level was 90dB, decreasing every 5dB, and the II wave with the minimum stimulation level was labeled as the hearing threshold of the animal. ABR hearing thresholds were recorded for the left and right ears of each group of mice.

(3) Identification of drug-induced deafness model in CBA mice (histomorphism detection)

After the ABR detection, the mice were sacrificed by removing their necks and taking the two lateral auditory bulbs. Opening the auditory bulb to expose the cochlea of the mouse, drilling a hole at the cochlea tip by using a 1mL syringe sharp needle, injecting 4% paraformaldehyde solution from the top of the cochlea spiral for fixing, after seeing that the 4% paraformaldehyde solution flows out of the round window hole, placing the specimen in 4% paraformaldehyde fixing solution, placing the specimen in a refrigerator at 4 ℃ for fixing for more than 0.5h, and taking a basement membrane for immunofluorescence staining and plating. The staining procedure was as before, as shown in FIG. 2.

Example 3: correlation detection of Neuritin expression and deafness by Western blot method

The protein level expression of Neuirtin changes at different time periods after the injury, and the Western blot method is the same as that of the above method, and is shown in FIG. 3.

Example 4: determination of the effective concentration of Neuritin protein to promote hair cell regeneration

(1) Establishment of new-born mouse cochlea cotti in-vitro culture method

1) Preparation of a culture dish: according to the rat tail collagen coating method of the professor tinctoria, 5mg/mL rat tail collagen, 10 multiplied by BME culture medium and 2% Na2CO3 are sequentially added and mixed uniformly according to the proportion of 9: 1 (air bubbles are avoided), 10 mu L of the rat tail collagen, the BME culture medium and the 2% Na2CO3 are placed in the center of a 35mm dish, a cover is opened, ultraviolet irradiation is carried out for 40-50min, after a glue drop is coagulated, 1mL of 1 multiplied by serum-free culture medium is added, and the rat tail collagen is placed in a 37 ℃ culture box with 5% CO2 for standby application.

2) The material of a cochlea basal membrane of a newborn mouse is as follows: disinfecting a 0-3D postnatal mouse with alcohol, placing a head of the mouse into D-HANKS anatomical solution in an ice bath in a super clean bench, cutting skin along the center of the vertex of a skull with a dissecting scissors, cutting the skull from a large hole of an occipital bone, removing brain tissues, taking down temporal bones at two sides, and placing the temporal bones into the D-HANKS anatomical solution in the ice bath; peeling temporal bone tissue under a dissecting microscope, exposing the cartilage surface, taking out the whole inner ear labyrinth, carefully separating the volute from the spiral ligament by using forceps and removing the volute, then completely taking down the cochlear epithelium together with the spiral ligament and the blood vessel veins on the outer side from the spiral shaft, separating a structure connected with the cochlear epithelium tissue to obtain a complete cochlear basilar membrane, placing the cochlear basilar membrane in 4% paraformaldehyde fixing solution, placing the cochlear basilar membrane in a refrigerator at 4 ℃ for fixing for more than 0.5h, and waiting for immunofluorescence staining;

3) taking out the preheated 35mm dish containing the glue drops and the culture medium from the incubator, moving the basement membrane to the center of the glue drops, taking the net surface as the upper part, putting the basement membrane in a spiral shape, discarding about 400-600 mu L of the culture medium, shaking the culture medium up and down, wherein the basement membrane does not move, placing the basement membrane in the incubator at 37 ℃ and culturing in a 5% CO2 incubator, and supplementing the culture medium after about 2-4 h;

4) and after the tissue is cultured for 24 hours by adherence, the culture solution containing different medicines is replaced.

(2) Neuritin protein treatment of in vitro cultured corti

Preparing 2 times Neuritin protein culture solution with the set concentration, reserving 500 mu L of original culture solution when replacing the culture medium, adding 500 mu L of 2 times Neuritin protein culture solution, replacing the culture solution once a day, and adopting a half-amount solution replacement method to maintain the effective concentration of the protein.

(3) Identification of changes in hair cell number

Adding Neuritin protein, collecting sample, placing in 4% paraformaldehyde fixing solution, fixing in refrigerator at 4 deg.C for 0.5 hr or more, and performing immunofluorescence staining as shown in FIG. 4.

Example 5: research on regeneration effect of Neuritin protein on damaged hair cells in cochlear cotti tissue of newborn mouse caused by aminoglycoside drugs

(1) Establishment and identification of cochlear hair cell in-vitro injury model

1) The in vitro culture method of the cochlear tissue of the newborn mouse is the same as that of the cochlear tissue of the newborn mouse

2) Grouping experiments: a normal control group, a 3mM gentamicin lesion group, a 3mM gentamicin + Neuritin 4. mu.g/mL protection group, and a 3mM gentamicin + Neuritin 16. mu.g/mL protection group. Culturing each group for 24 hours under the condition of the medicine, discarding the original culture medium, continuously culturing the normal control group for 3 days, replacing a fresh culture medium for 3 days for a 3mM gentamicin damaged group, replacing a culture solution containing 4 mu g/mL Neuritin for a 3mM gentamicin + Neuritin 4 mu g/mL protective group, and culturing for 3-7 days, replacing a culture solution containing 16 mu g/mL Neuritin for a 3mM gentamicin + Neuritin 16 mu g/mL protective group, and culturing for 3-7 days, wherein the solution replacement mode is the same as the previous mode. As shown in fig. 5 and 6.

(2) Research on regeneration and repair effects of Neuritin protein on damaged hair cells

After staining, the number and morphology of hair cells and supporting cells were observed. As shown in fig. 7-12.

As used in the specification and in the claims, certain terms are used to refer to particular components. As one skilled in the art will appreciate, manufacturers may refer to a component by different names. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. Thus, if a first device couples to a second device, that connection may be through a direct electrical coupling or through an indirect electrical coupling via other devices and couplings. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. The term "comprising", without further limitation, means that the element so defined is not excluded from the article or system in which the element is included.

While the foregoing description shows and describes several preferred embodiments of the utility model, it is to be understood, as noted above, that the utility model is not limited to the forms disclosed herein, but is not intended to be exhaustive or to exclude other embodiments and may be used in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the utility model as defined by the appended claims.

Claims

1. The recombinant human Neuritin protein is applied to the preparation of the medicine for treating the sensorineural deafness, the sensorineural deafness is cochlear hair cell damage caused by noise damage, medicine damage, aging and the like, and the support cells have the capacity of differentiating into hair cells.

2. The use according to claim 1, wherein the recombinant human Neuritin protein is dose and time dependent in inducing in vitro differentiation of the supporting cells into hair cells and hair cell maturation in mouse cochlear cotti tissues.

3. The use according to claim 2, wherein the recombinant human Neuritin protein is added to the tissue culture medium of mouse cochlea corti containing the drug at different concentrations, and the effective action concentration of the recombinant human Neuritin protein for promoting hair cell regeneration is determined by counting the number of hair cells at each concentration.

4. The use according to claim 2, wherein the recombinant human Neuritin protein is administered at a concentration of 4, 16 μ g/mL induced in vitro in mouse cochlear cotti tissue.