CN112125969A - Application of biological factor RIMBP2 in maintaining inner ear hair cell characteristics - Google Patents

Abstract

The invention belongs to the field of biological inner ear research, and particularly discloses application of a biological factor RIMBP2 in maintaining the characteristics of inner ear hair cells. The biological factor is synaptophysin domain protein binding protein RIMBP2, has the function of maintaining the activity of inner ear hair cells, and has an amino acid sequence shown in SEQ NO. 1. The invention is found in the inner ear for the first timeRimbp2Spatio-temporal expression patterns in the inner ear of mice; and the gene can cause capillary after being knocked out in mouse hair cellsApoptosis in turn affects normal auditory function. The invention is as describedRimbp2Lays an important foundation for the subsequent research of the functions of the mouse hair cells.

Description

Application of biological factor RIMBP2 in maintaining inner ear hair cell characteristics

Technical Field

The invention relates to the technical field of inner ear cell protection, in particular to application of a biological factor RIMBP2 in maintaining the characteristics of inner ear hair cells.

Background

Deafness is the most frequently occurring organoleptically deficient disease in the world. However, because of the inexhaustibility of deafness, there is insufficient social interest in the development of deafness treatments, and the research and development efforts for treating deafness have been relatively insufficient. At present, the treatment method for deafness mainly takes the assistance of instruments as main treatment, cannot reach the level of normal people, and has certain damage to spiral neurons. There are a number of effective intervention strategies lacking in deafness treatment. In addition to environmentally induced deafness, more than 50% of sensorineural deafness is thought to be caused by genetic factors in recent studies.

The main causes of sensorineural deafness are: degeneration and death of the helical neuronal neurites following injury and death of inner ear hair cells and damage or death of hair cells. The cochlear hair cells function as sensory cells, and their main function is to convert the mechanical vibration signals of sound into bioelectrical signals. The survival, ciliary movement and synaptic plasticity of inner ear hair cells play important roles in the transmission and amplification of acoustic signals, and the cellular structures need cytoskeletal constitutive proteins as structural bases and are simultaneously regulated and controlled by regulatory proteins of the cytoskeletal constitutive proteins.

RIMBP2 belongs to the synaptotactivity domain protein binding protein family, is a large-scale multi-domain protein, and constitutes a novel protein. The RIMBP family has three very conserved domains, an N-terminal and two C-terminal SH3 domains, and 2 or 3 FN3 domains. The research shows that the SH3 structural domain can be connected with Ca of proline-rich sequence²⁺Channel and Rab3-Interacting molecules (RIMs) interaction, Ca induced by action potential with high fidelity coupling²⁺Influx, and thus is important for inducing extracellular secretion of synaptic vesicles. At present, few studies on the role of Rimbp2 in the inner ear of mice are performed at home and abroad.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the application of a biological factor RIMBP2 in maintaining the characteristics of inner ear hair cells, wherein the biological factor is synaptophysin domain protein binding protein 2(RIMBP2), plays an important role in maintaining hearing and contributes to the restoration of sensorineural deafness caused by heredity of human beings.

The biological factor RIMBP2, the biological factor RIMBP2 is synaptotactic activity domain protein binding protein 2, the amino acid sequence is shown in SEQ NO.1, and the biological factor has the function of maintaining the characteristics of inner ear hair cells.

SEQ NO.1：

A nucleic acid molecule, the nucleotide sequence of the synaptotagmin binding protein 2(RIMBP2) is shown in SEQ NO. 2.

SEQ NO.2：

A recombinant plasmid incorporating the above-described nucleotide sequence encoding synaptic activity domain protein binding protein 2(RIMBP 2).

A recombinant vector, which is transformed with the recombinant plasmid.

The biological factor RIMBP2 can be used for preparing products for treating RIMBP2 deficiency.

A gene targeting drug, the active component comprises any one of the nucleotide sequence for coding synapse activity domain protein binding protein 2(RIMBP2), recombinant plasmid or recombinant vector.

Has the advantages that:

compared with the prior art, the application of the biological factor RIMBP2 in maintaining the characteristics of inner ear hair cells has the following advantages:

1. according to the invention, the Rimbp2 gene is specifically knocked out from a transgenic mouse, and the inner ear hair cells and the regulation mechanism thereof are researched in a knocked-out mouse model, which cannot be regulated by conventional medicines, and the regulation effect is good, and the directivity is very clear;

2. the regulation and control mechanism of Rimbp2 on inner ear hair cells is further and deeply explored on the basis of the existing research, the innovation and the depth on academic theory are provided, the scientific theoretical basis is provided for further treating hereditary sensorineural deafness, and the genetic targeted therapy is adopted to replace an artificial cochlea, protect hair cell injury and treat hereditary sensorineural deafness, so that the strong evidence is provided;

3. the invention has good clinical application value through mechanism research and live animal experiments, and lays an experimental foundation for recovering auditory function by hereditary sensorineural deafness in clinical treatment.

Drawings

FIG. 1 is a schematic diagram of the constructed PB element of Rimbp2 knockout mice;

FIG. 2 is a graph of Rimbp2 expression in the inner ear of P0 (day of birth) mice, where (A) is the Rimbp2 immunofluorescence mapping of the inner ear hair cell nuclear layer and the support cell layer, hair cells were labeled with MYOSIN7A, proteins of interest were labeled with RIMBP2, and support cells were labeled with SOX 2; (B) for frozen sections immunohistochemical results, hair cells were labeled with MYOSIN7A, target proteins were labeled with RIMBP2, and nuclei were labeled with DAPI; scale bar 10 μm; (C) GAPDH is the internal reference of protein, the size is 36kDa, RIMBP2 is the target protein, and the size is 116 kDa; (D) the mRNA expression level of RIMBP2 at 4 time points of mice E16, P3, P14 and P30;

FIG. 3 is an immunofluorescence mapping of inner ear hair cell cytoplasmic layer RIMBP2 in mice at three stages P14, P21 and P30, hair cells labeled with MYOSIN7A, target protein labeled with RIMBP2, nuclei labeled with DAPI, scale bar 10 μm;

FIG. 4 is a diagram of auditory brainstem response of a Rimbp2 knockout mouse, wherein (A) the hearing changes of the Rimbp2 knockout mouse are detected in a period P21, and the frequencies are selected from 4KHz,8KHz,12KHz,16KHz,24KHz and 32KHz, (B) the hearing changes of the Rimbp2 knockout mouse are detected in a period P30, and the frequencies are selected from 4KHz,8KHz,12KHz,16KHz,24KHz and 32KHz, and (C) the hearing changes of the Rimbp2 knockout mouse are detected in a period P60, and the frequencies are selected from 4KHz,8KHz,12KHz,16KHz,24KHz and 32 KHz; (D) is an auditory brainstem response oscillogram of a P21 day mouse;

FIG. 5 is a graph showing morphological and quantitative changes of inner ear hair cells of Rimbp2 knockout mice, (A) is a graph showing immunofluorescence staining of inner ear hair cells of mice at stage P21, hair cells were labeled with MYOSIN7A, and nuclei were labeled with DAPI; scale bar 10 μm, (B) immunofluorescent staining of mouse inner ear hair cells at stage P30, MYOSIN7A labeling hair cells, DAPI labeling nuclei; scale bar 10 μm, (C) immunofluorescence staining profile of mouse inner ear hair cells at stage P60, hair cells labeled with MYOSIN7A, nuclei labeled with DAPI; scale bar 10 μm; (D) statistics of the number of mouse inner ear outer hair cells at the period of P21, (F) statistics of the number of mouse inner ear outer hair cells at the period of P30, (H) statistics of the number of mouse inner ear outer hair cells at the period of P60; (E) the statistics of the number of inner ear hair cells of the mice at the P21 stage, (G) the statistics of the number of inner ear hair cells of the mice at the P30 stage, (I) the statistics of the number of inner ear hair cells of the mice at the P60 stage;

fig. 6 is a graph of ciliary morphology and polarity changes of inner ear hair cells of a Rimbp2 knockout mouse, (a) is a graph of mouse inner ear hair cell ciliary immunofluorescence staining at stage P21, scale bar ═ 10 μm, (B is a graph of mouse inner ear hair cell ciliary immunofluorescence staining at stage P30, scale bar ═ 10 μm, and (C) is a graph of mouse inner ear hair cell ciliary immunofluorescence staining at stage P60, scale bar ═ 10 μm;

FIG. 7 shows apoptosis of inner ear hair cells of Rimbp2 knockout mice, (A) Tunel staining, MYOSIN7A labeling of hair cells, DAPI labeling of cell nuclei, and Tunel labeling of apoptotic cells; (B) statistics for cells that were both Tunel and MYOSIN7A positive; (C) the protein expression level of the cleared Caspase-3 in WT mice and Rimbp2 knockout mice is shown in the figure, wherein GAPDH is an internal protein reference with the size of 36kDa, and the cleared Caspase-3 is a target protein with the size of 19 kDa; (D) the grey level statistical result of the clear Caspase-3 protein expression is obtained; (E) is a result of the mRNA expression level of the apoptosis-related factor.

FIG. 8 is a graph showing synapse changes in Rimbp2 knockout mice, (A) shows synapse immunofluorescence staining patterns in Rimbp2 knockout mice and WT mice, with CTBP2 labeling robbon, with GluR2 labeling AMPA receptor, and (B) shows Ctbp2 labeling⁺The statistical result of punca (C) is GluR2⁺The result of statistics of (1), (D) is Ctbp2⁺/GluR2⁺Statistics of punta (g).

Detailed Description

To better study the mechanism of action of Rimbp2, the present invention was validated using the following procedure.

Example 1 spatiotemporal expression of RIMBP2 protein in wild-type mouse cochlea

1.1 the temporal and spatial expression of Rimbp2 in the inner ear of mice of different ages, such as P0, P14, P21, P30, was investigated by taking the temporal bone of wild-type mice of FVB strains of different ages, such as P0, P14, P21, P30, and finely dissecting the basement membrane of the mice, immunofluorescent staining using the hair cell marker MYOSIN7A (DSHB), the support cell marker SOX2(santa, sc-17320), and RIMBP2 antibody (Proteintetech, 15716-1-AP).

And (3) immunofluorescence staining: dissecting wild-type mouse basement membranes of P0, P14, P21 and P30 in precooled HBSS (GIBCO) respectively; coating with cell tab (CORNING) on 10mm cover glass to form a circle with diameter of 5mm, and air drying (coating); putting the coated cover glass into a culture dish with a cochlea, and adhering the cochlea on the glass sheet with the right side facing upwards; placing the adhered cochlea into a 4-hole culture dish filled with PBS (GIBCO), changing the PBS into 4% PFA after all the cochlea are adhered, and fixing for 1h at room temperature; rinsing with 0.1% PBST for 5min 3 times; after Blocking medium is used for sealing for 1h at room temperature, PBT-1 is used for sealing according to the proportion of 1: diluting hair cell marker myostatin 7a, support cell marker sox2 and Rimbp2 antibody with 400, adding into cochlea in a four-hole dish for co-staining, and incubating at 4 deg.C overnight; rinsing with 0.1% PBST for 5min 3 times; the corresponding secondary antibody was then conjugated with PBT-2 at 1:400 dilution (additional DAPI at 1: 1000 dilution) and incubation at room temperature for 1 h; rinsing with 0.1% PBST for 5min 3 times; add 6. mu.L of DAKO (DAKO, S3023) to each slide (8. mu.L for adult mouse cochlea), cover the slides, and seal with nail polish; collecting pictures by using a confocal microscope;

1.2 extraction of protein and RNA from cochlear basement membrane tissue of wild type mice of FVB strains of different ages such as P0, P14, P21, P30, etc., and immunoblotting (Western Blot) and qPCR (quantitative PCR) experiments were carried out to confirm the expression of Rimbp2 in the cochlea of normal mice.

1.2.1 Western Blot experiment:

a) protein extraction: temporal bones of 10P 0 wild-type mice were harvested, dissected in precooled HBSS to remove basement membrane, and placed in 1.5mL centrifuge tubes containing PBS. PSB was discarded immediately after the dissociation, 150. mu.L of RIPA lysate (Fried biosystems, FD008) (containing protease inhibitor Cocktail) was added, followed by grinding until a homogenized state (20S-30S for each 30S grinding), centrifugation at 4 ℃ (12000g, 10min) was performed after leaving on ice for 10 min; the supernatant (ca. 100. mu.L) was transferred to a new 1.5mL centrifuge tube and 25. mu.L of 5 × loading buffer (Solebao, P0015L) was added; the mixture is frozen and stored in a boiling water bath for 15min at the temperature of-20 ℃ or Western Blot.

b) Western Blot i) preparing PAGE gel with corresponding concentration according to the size of protein, firstly adding the separation gel of the lower layer into a glass plate, pressing the gel with isopropanol, removing the isopropanol after the gel is solidified for about 20min at room temperature, preparing the concentrated gel of the upper layer, inserting a 10-hole comb with the diameter of 1.5mm, and standing for 15min at room temperature for solidification; ii) clamping the glass plate by a clamping plate, placing the glass plate into an electrophoresis tank, pouring 1 × Running Buffer to an indicated position, pulling out a comb, adding a fully denatured protein sample and a protein ladder (thermols, 26616) into a hole tank, performing electrophoresis at a constant voltage of 80V for about 30min until the sample completely enters the lower layer gel, then changing the electrophoresis to constant voltage of 120V, and observing the protein ladder according to the size of the protein to be detected to determine when to finish SDS-PAGE; iii) shearing PVDF membrane with proper size, and activating in methanol for 1-5 min; cleaning a glass dish, pouring a proper amount of ice-cold 1 × Transfer Buffer, taking out separated PAGE gel, removing redundant parts, putting a Transfer clamp, enabling a black side (a negative electrode) to be at the bottom, sequentially placing a sponge, three layers of filter paper, PAGE gel, a PVDF membrane, three layers of filter paper and a sponge, finally closing and fastening a white side (a positive electrode), and transferring in a Transfer tank filled with the pre-cooled 1 × Transfer Buffer in ice (300mA constant current Transfer for 2 hr); iiii) preparing 5% skimmed milk powder (illite) sealing solution by using TBST, placing the transferred PVDF membrane with the right side facing upwards in the sealing solution, and sealing for 1hr at room temperature; removing the confining liquid, adding primary antibody diluted by TBST in proportion, and incubating overnight in a shaking table at 4 ℃; washing the residual primary antibody on the membrane with TBST 4 times for 10-15min the next day; diluting the secondary antibody with TBST at a ratio of 1:4000, and incubating for 1hr at room temperature in a shaking table; the secondary antibody remained on the membrane was washed with TBST 4 times for 10-15min each time. V) add the appropriate amount of chemiluminescent solution to the PVDF membrane, observe the results in a gel imaging system and record the photographs.

1.2.2 qPCR experiments:

a) RNA extraction: selecting a wild mouse of an FVB strain, dissecting and taking a cochlea basement membrane tissue in HBSS, cleaning the cochlea basement membrane tissue by PBS, adding 1ml of Trizol (life, 15596-018) for fully grinding, placing the obtained product on ice for 10min, then centrifuging the obtained product at 12000rpm for 15min at 4 ℃, adding 0.2ml of chloroform into the obtained supernatant, shaking the obtained product for uniform mixing, placing the obtained product at room temperature for 5min, and centrifuging the obtained product at 12000rpm for 15min at 4 ℃. The solution is evenly layered after centrifugation, then the upper water phase containing RNA is carefully taken, added with isopropanol and fully mixed, then kept stand on ice for 10min, then centrifuged for 5min at 12000rpm under 4 ℃, the supernatant is discarded, 1ml of 75% ethanol (prepared by DEPC water) is added for washing, centrifuged for 10min at 7500rpm under 4 ℃, and 50 mul of DEPC (Bio-Industrial, B501005) water is added for dissolving after the supernatant is discarded.

b) qPCR: adding the extracted total RNA sample and sterile water without RNase into a reverse transcription primer mix (Oligoldt)18) (thermo, K1622) to construct a mixed system, uniformly mixing, placing at 70 ℃ for 5min for RNA denaturation, and immediately placing on ice for 10min to terminate the reaction after the reaction is finished. Adding 6 μ l reverse transcription buffer solution (thermo, K1622) and 2 μ l reverse transcriptase (thermo, K1622) to the reaction mixture to construct a reverse transcription system, mixing well, performing reverse transcription at 42 deg.C for 1hr, and performing reverse transcriptase denaturation at 95 deg.C for 5 min. The Reverse Transcription PCR reaction (RT-PCR) was performed using a Reverse Transcription PCR enzyme (thermo, K1622) mixed reagent to obtain cDNA, and the concentration of the cDNA was measured. Using Real-Time quantitative PCR buffer reagent SYBR Green (ROCHE, 17747200) to carry out Real-Time quantitative PCR reaction (RT-PCR), diluting cDNA obtained by reverse transcription according to the measured concentration to make the concentration of each group the same, constructing a reaction system, adding 2 mul of diluted cDNA, 10 mul of SYBR, 0.6 mul and 6.8 mul of sterilized water respectively for PCR upstream and downstream primers, fully mixing uniformly, using a Bio-Rad IQ5Real-Time PCR instrument to carry out reaction and obtaining corresponding Ct value data.

1.3 taking the wild-type mouse temporal bones of FVB strains of different age groups such as P0, P7, P14, P21, P30, etc., and further studying the temporal and spatial expression of Rimbp2 in the inner ear of mice of different age groups such as P0, P7, P14, P21, P30 by a frozen section method.

Freezing and slicing: taking temporal bones of FVB strains of P0, P7, P14, P21, P30 and the like of different age groups, sequentially putting the temporal bones into sucrose solutions with concentrations of 15%, 20% and 30% for vacuum dehydration treatment, and then putting the temporal bones in a refrigerator at 4 ℃ for overnight; placing into 30% sucrose solution and O.C.T (oriental cherry) mixed solution (containing three gradients, 50:50, 30:70, and 15:85) for vacuum dehydration treatment, embedding with 100% O.C.T, placing in a right position, vacuumizing, removing bubbles, and placing in a refrigerator at 4 deg.C overnight; and (4) after the next day, performing vacuum pumping after the position is adjusted again, then putting the mixture into a refrigerator with the temperature of 20 ℃ below zero, freezing the mixture, and then putting the frozen mixture into an ultra-low temperature refrigerator for freezing. The embedded tissue was sectioned at 10 μm, then immunofluorescent stained, and observed with a confocal laser microscope.

The results show that: rimbp2 is expressed in the cytoplasm of inner ear hair cells, suggesting that Rimbp2 plays an important role in inner ear hair cells.

1.4 changes in expression of apoptosis-related factors

Cochlear protein and RNA of Rimbp2 knockout mice and wild-type mice of FVB strains of different age groups such as P0, P7, P14, P21 and P30 are respectively extracted, and the expression change of main signal molecules such as Caspase, Bcl2, Bak, Bax and the like in the apoptosis signal pathway in the cochlea of the Rimbp2 knockout mouse is detected by Western Blot (refer to 1.2.1) and q-PCR (refer to 1.2.2) technologies.

Tunel staining: wild-type mice and Rimbp2 knockout mice of P18 were dissected in precooled HBSS, respectively, for basement membrane; coating a 10mm cover glass with cell tak to form a circle with the diameter of 5mm, and airing (coating at present); putting the coated cover glass into a culture dish with a cochlea, and adhering the cochlea on the glass sheet with the right side facing upwards; placing the adhered cochlea into a 4-hole culture dish filled with PBS (phosphate buffer solution), changing the PBS into 4% PFA (Perfluoro fluoro ethylene) after all the cochlea are adhered, and fixing for 1h at room temperature; rinsing with 0.1% PBST for 5min 3 times; preparing a system: 34 μ lddH₂O, 5. mu.l FITC mix, 10. mu.l 5X Equisition buffer, 1. mu.l TdT Enzyme (Novozam, A111-03), adding 80-100. mu.l of the mixed system to each well, and incubating at 37 ℃ for 1 hour; wash 2 times with PBS for 5min each. After Blocking medium is used for sealing for 1h at room temperature, PBT-1 is used for sealing according to the proportion of 1: diluting hair cell marker myostatin 7a at 400 deg.C, adding into cochlea in four-hole dish, co-staining, and incubating at 4 deg.C overnight; rinsing with 0.1% PBST for 5min 3 times; the corresponding secondary antibody was then conjugated with PBT-2 at 1:400 dilution (additional DAPI at 1: 1000 dilution) and incubation at room temperature for 1 h; rinsing with 0.1% PBST for 5min 3 times; add 8. mu.L of DAKO (DAKO, S3023) to each slide, cover the slide, and seal with nail polish; collecting pictures by using a confocal microscope;

the sequences of the primers used are shown in the following table:

GAPDH Forward sequence	GCAAGAGAGAGGCCCTCAG
		GAPDH Reverse sequence	TGTGAGGGAGATGCTCAGTG
Bcl-2 Forward sequence	GGTGAACTGGGGGAGGATTG
		Bcl-2 Reverse sequence	AGAGCGATGTTGTCCACCAG
Caspase-8 Forward sequence	AGCCTATGCCACCTAGTGAT
		Caspase-8 Reverse sequence	GGAGAGCTGTAACCTGTCGC
Fadd Forward sequence	ACAATGTGGGGAGAGACTGG
		Fadd Reverse sequence	CCCTTACCCGATCACTCAGG
Apaf1 Forward sequence	AGGGTGTGAGAGGAGTGTGT
		Apaf1 Reverse sequence	ATCACCTCGATGGACTTGCC
Caspase-2 Forward sequence	CTGACAGGAGGAGCAGGATTTT
		Caspase-2 Reverse sequence	CACCGAGAAGGGGAGACTTG
Caspase-3 Forward sequence	AATCATGCCATTTGCCCAGC
		Caspase-3 Reverse sequence	CTCAAGTGTGTAGGGGGAGG
Rimbp2 Forward sequence	TGAGCAGTTTAACTTGTTGTCCC
		Rimbp2 Reverse sequence	GGAGGTGGCTAGTCCATTCAT

the results show that: apoptosis of mouse hair cells occurred following Rimbp2 knock-out.

1.5 changes in the molecules involved in synaptic Transmission

Cochlear protein and RNA of Rimbp2 knockout mice and wild mice of FVB strains of different age groups such as P0, P7, P14, P21 and P30 are respectively extracted, and expression change conditions of main signal molecules such as RIM, Rab3, Munc13, Bassoon and the like in cochlea of the Rimbp2 knockout mice in synaptic transmission are detected by Western Blot and q-PCR technology.

1.6 in the inner ear proteins that have been reported to interact with RIMBP2 were explored

Cochlear proteins and RNAs of Rimbp2 knockout mice and wild mice of FVB strains of different age groups such as P0, P7, P14, P21 and P30 are respectively extracted, and proteins (such as MYOSIN7A, BASSOON, Ca) interacting with RIMBP2 in cochlea of the Rimbp2 knockout mice are detected by Western Blot, q-PCR, Co-IP and other technologies²⁺Channels, etc.).

Example 2 morphological analysis of cochlea of Rimbp2 knockout mouse

2.1 taking Rimbp2 knockout mice of different age groups such as P21, P30, P60 and the like and wild type temporal bones of FVB strains, finely dissecting basement membranes of the mice, and detecting the changes of hair cells and the number of supporting cells and cilium morphology and polarity in the inner ears of the Rimbp2 knockout mice of different age groups by immunofluorescence staining with hair cell markers MYOSIN7A, phalloidine and supporting cell marker SOX2 (the specific operation can be referred to the method in 1.1 of example 1);

the results show that: there was a partial deletion of hair cells following the Rimbp2 knock-out, and there was no change in ciliary morphology and polarity.

2.2 taking Rimbp2 knockout mice of different age groups such as P14, P21 and P30 and wild type temporal bones of FVB strains, the change of synapse number of inner ear hair cells of the Rimbp2 knockout mice of different age groups is detected by immunofluorescence staining with a presynaptic marker CTBP2 and a postsynaptic marker GluR2 (the specific operation can be referred to the method of 1.1 in example 1).

The results show that: there was no change in the number of CTBP2 and GluR2 after Rimbp2 knockout.

Example 3 analysis of cochlear function in Rimbp2 knockout mice

Changes in mouse audiometric Response (ABR) and diagnosis Product ocular emulsions (DPOAE) were analyzed using littermate wild-type mice as controls to evaluate the overall hearing status and cochlear hair cell function of Rimbp2 knockout mice as fully as possible.

3.1 Auditory Brainstem Response (ABR) threshold analysis

Parameters such as auditory response threshold, latency and interphase are detected through ABR, short sound (Click), particularly short sounds (4KHz, 8KHz,12KHz,16KHz,24KHz and 32KHz) of different audios are respectively used as stimulating sound, the hearing thresholds of the Rimbp2 gene-deficient mice on the different audios are detected, and the hearing sensitivity of the mice is analyzed, so that whether the Rimbp2 gene-deficient mice have normal auditory function from hair cells to cerebral cortex is integrally judged.

Mice were anesthetized with an i.p. injection of 10mg/ml sodium pentobarbital (bioshapr) at a mouse body weight ratio (1mg/g) and then the corresponding test and recording was performed on anesthetized mice placed in a double-walled glottic system using the Smart EP evoked potential system. The electrode used in the experiment is a hypodermic needle, the used detection electrode is implanted into the top position of the mouse brain, and the reference electrode and the grounding electrode are respectively implanted on the outer sides of the left ear and the right ear. The body temperature of the experimental animals needs to be kept constant during the experiment. Signals of 100Hz or less and 3000Hz or more processed by band-pass filtering are used as the biological signals, and the signals are amplified 200000 times. The anti-interference level is set to 31.00 mu V when data are collected, and the collected results are averaged by a computer and then directly displayed. The operation time of the computer is basically stabilized at about 10 ms. The determination of the threshold is made by increasing the Sound Pressure Level (SPL) by 5dB increments each time. Detecting parameters such as auditory response threshold, latency and wave interval, and analyzing auditory sensitivity of the mouse.

The results show that: ABR thresholds were significantly increased after Rimbp2 knockdown at each frequency, indicating severe loss of auditory function in Rimbp2 gene-deficient mice.

3.2 outer hair cell function analysis (diagnosis Product oxygen extrinsic emulsions, DPOAE)

The amplitude and the threshold value of mouse DPOAEs are measured through distortion product otoacoustic emission, two long-term pure tones (2f1-f2) with different frequencies and a certain frequency ratio relation are used for induction, and whether the cochlear outer hair cells of the mice with the Rimbp2 gene defect have normal functions is further analyzed. Mice were anesthetized with sodium pentobarbital at a concentration of 10mg/ml, and then the anesthetized mice placed in a double-walled sound chamber system were tested and recorded using an IL092 otoacoustic transmitter. The body temperature of the experimental animals needs to be kept constant during the experiment. In the experimental process, the good tightness of the external auditory canal and the probe (equipped with instruments) of the mouse needs to be ensured. The probe was wired into IL092 with relevant parameters set to f2/f 1-1.207 and L1-L2, with the threshold set to just 3dB above the local noise with DPOAE strength. In testing the DPOAE input and output curves, the raw tone intensity needs to be attenuated from 80dB SPL to below the threshold by 1.5dB each. Record f2 as DPOAE thresholds at 5, 6, 7, and 8 kHz; the intensity of 2f1-f2DPOAE extracted when L1 ═ L2 was recorded as 70dB SPL and 60dB SPL. When DPOAE latencies were analyzed, the original note L1 ═ L2 was 70dB, and f2 was recorded as DPOAE latencies at 5, 6, and 7 kHz. And meanwhile, the obtained data is directly processed by using corresponding software, and the function of the cochlear outer hair cells of the mouse is preliminarily analyzed.

The research shows that the biological factor Rimbp2 has good application in maintaining the characteristics of inner ear hair cells, replaces artificial cochlea, protects hair cell injury and treats hereditary sensorineural deafness through gene targeting treatment.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

<110> university of southeast

<120> use of the biological factor RIMBP2 for maintaining the properties of inner ear hair cells

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Pro Ala Gly Pro Pro Ala Pro Pro Gln Asp Val Thr Val His Ala Gly

500 505 510

Ala Thr Ala Ala Ser Val Gln Val Ser Trp Lys Pro Pro Ala Leu Thr

515 520 525

Pro Thr Gly Leu Ser Asn Gly Ala Asn Val Thr Gly Tyr Gly Val Tyr

530 535 540

Ala Lys Gly Gln Arg Val Ala Glu Val Ile Ala Pro Thr Ala Asp Gly

545 550 555 560

Thr Ala Val Glu Leu Ile Arg Leu Arg Ser Leu Glu Ala Lys Ala Val

565 570 575

Ser Val Arg Thr Leu Ser Val Gln Gly Glu Ser Met Asp Ser Ala Leu

580 585 590

Ala Ala Ile Pro Pro Asp Leu Leu Val Pro Pro Ala Pro His Pro Arg

595 600 605

Thr Ala Pro Pro Pro Lys Pro Leu Ala Ser Asp Met Asp Thr Lys Asp

610 615 620

Gln His Leu Gly Pro His Val Lys Val Asp Glu Ser Trp Glu Gln Ser

625 630 635 640

Arg Ser Pro Gly Pro Ala His Gly His Met Leu Glu Pro Pro Asp Met

645 650 655

His Ser Ala Gly Pro Gly Arg Arg Ser Pro Ser Pro Ser Arg Ile Leu

660 665 670

Pro Gln Pro Gln Gly Ala Pro Val Ser Thr Thr Val Ala Lys Ala Met

675 680 685

Ala Arg Glu Ala Ala Gln Arg Val Ala Glu Ser Asn Arg Leu Glu Lys

690 695 700

Arg Ser Leu Phe Leu Glu Gln Ser Ser Ala Gly Gln Tyr Thr Asn Ser

705 710 715 720

Asp Glu Glu Asp Gly Tyr Ala Ser Pro Glu Val Lys Arg Arg Gly Thr

725 730 735

Ser Val Asp Asp Phe Leu Lys Gly Ser Glu Leu Gly Lys Gln Pro His

740 745 750

Cys Cys His Gly Asp Glu Tyr His Thr Glu Ser Ser Arg Gly Ser Asp

755 760 765

Leu Ser Asp Ile Met Glu Glu Asp Glu Glu Glu Leu Tyr Ser Glu Met

770 775 780

Gln Leu Glu Asp Gly Gly Arg Arg Arg Pro Ser Gly Thr Ser His Asn

785 790 795 800

Ala Leu Lys Ile Leu Gly Asn Ser Thr Leu Met Gly Arg Ala Asp Arg

805 810 815

Met Glu His Val Ser Arg Arg Tyr Ser His Ser Gly Gly Gly Ser His

820 825 830

Arg His Arg Pro Ala Met Ala Pro Ser Ile Asp Glu Tyr Thr Gly Arg

835 840 845

Asp His Leu Ser Pro Asp Phe Tyr Asp Glu Ser Glu Thr Asp Pro Gly

850 855 860

Ala Glu Glu Leu Pro Ala Arg Ile Phe Val Ala Leu Phe Asp Tyr Asp

865 870 875 880

Pro Leu Thr Met Ser Pro Asn Pro Asp Ala Ala Glu Glu Glu Leu Pro

885 890 895

Phe Lys Glu Gly Gln Ile Ile Lys Val Tyr Gly Asp Lys Asp Ala Asp

900 905 910

Gly Phe Tyr Arg Gly Glu Thr Cys Ala Arg Leu Gly Leu Ile Pro Cys

915 920 925

Asn Met Val Ser Glu Ile His Ala Asp Asp Glu Glu Met Met Asp Gln

930 935 940

Leu Leu Arg Gln Gly Phe Leu Pro Leu Asn Thr Pro Val Glu Lys Ile

945 950 955 960

Glu Arg Ser Arg Arg Ser Gly Arg Gly His Ser Val Pro Thr Arg Arg

965 970 975

Met Val Ala Leu Tyr Asp Tyr Asp Pro Arg Glu Ser Ser Pro Asn Val

980 985 990

Asp Val Glu Ala Glu Leu Pro Phe Cys Thr Gly Asp Ile Ile Thr Val

995 1000 1005

Phe Gly Glu Ile Asp Glu Asp Gly Phe Tyr Tyr Gly Glu Leu Asn Gly

1010 1015 1020

Gln Lys Gly Leu Val Pro Ser Asn Phe Leu Glu Glu Val Pro Asp Asp

1025 1030 1035 1040

Val Glu Val His Leu Ser Asp Ala Pro Pro His Tyr Ser His Asp Pro

1045 1050 1055

Pro Met Arg Ser Lys Ala Lys Arg Val Ser Gln Pro Pro

1060 1065

<210> 2

<211> 7020

<212> DNA

<213> Gene Sequence (Gene Sequence)

<400> 2

attcctgccc tgcttttgac caccgcttcg tgagagacac cggcagggaa gggacagagc 60

catcttggga tgcgtgtgtg catgcgcgtt tgtgtgcgca cgtgtgtgtg tgtgtgtgtg 120

tgtgggtgtg tgtgtgtgtg tgtgttaaaa cactgccgtt tccaagcttt ggctactgct 180

ctctggctgg tgactcttgc tcatgcctct ctctctctct ctctctctct ctctctctct 240

ctctctctct ctctttctct ccctccctgc aaaaattaaa tggataattt ctagctgata 300

ccaagcggtg cccacacacc cgtgtccttc agtgttgatt ggatctctct ctctctctct 360

ctctctctct ctctctctct ctctctctct ctctcctctc cactctcctc ccctccctct 420

ggccccagtt tgtaatctga cacagtgcac tgcagtggct gtctctgtct ctctcttgga 480

gccttggagc ccgggaggct gggcaccgag aacatccaag gatgatgaac taaaggccgc 540

tgtgcagaga gagcatcacc tgaagcccct ggaaaggcct tcccttgagg gcccggcagc 600

atgaccaggt gaaggctgaa catgcctggc tctccgtggt gacctgagag gagagttcga 660

ttcactgtgc gggagaaacg ccagctccaa gccaaactgg agaacttgga acaggtcctg 720

aagcatatgc gagaggctgc tgagcggcgg caacagctgg agttggagca tgagcaagcc 780

ctggccttcc tcaatgccaa gcagcaggag atccagctac tgcagcaggc tcaggttgaa 840

gctaagaaag agcatgaagg tgctgtgcag ctgttagaga acaccctgga ctgcatgcag 900

tccaaggtcc gagagctgga agagaaatgc cgtgtgcaga gtgagcagtt taacttgttg 960

tcccgggacc tggaaaagtt ccggcaacac actgggagta ttgacctgct gggcagcagc 1020

tcagtggccc tgctggatgt tcccttggcc cctggcaagc ctttccctca gtacatgaat 1080

ggactagcca cctccatcca caaaggtcac gagggcccca ctggacacta ctctgtgatt 1140

ggtgactata ttccgctgtc tggggacaag ctggagtctc cgtgtgtgaa gccctccttc 1200

ctcttgcgat ccagcagccc aagatgcaga tttgagtccg agatggacaa tgaccggaac 1260

tctaacaact ccaagcaaag cagctcgggg aaggtgcacc tgtgtgtggc ccgctacagt 1320

tacaacccct tcgatgggcc caatgagaac ccagaagctg agctgcctct cacggcggga 1380

aagtacctct atgtctatgg ggacatggat gaagatgggt tctatgaagg agagcttctg 1440

gatgggcaaa ggggcttggt gccctccaac tttgtggatt ttatccagga caatgagtcg 1500

cggctggctg gtactctggg gagtgagcag gaccagaact ttctcaacca ctctggcatc 1560

agtctggagc gcgacagcat ccttcacctt cactccccaa ctcaagtgga ctcggggatc 1620

accgacaatg gtggggggac cctggacgtg aacatcgacg acatcggaga agacaccgtg 1680

ccttacccta ggaaaatcac ccttatcaaa cagcttgcca aaagtgtcat cgtgggctgg 1740

gagccccctg ctgtgcctcc cggctggggc accgtgagca gttataatgt gctggtggac 1800

aaagagacac gcatgagcct tgccctgggt aggagaacta aggcgctgat cgagaaactt 1860

aacacagctg cctgcaccta ccgcatctcc gtgcagtgcg tcacgagccg gggaaactca 1920

gacgagctgc agtgcactct gctggtgggc aaggatgtgg tggtggcacc gtcccagctg 1980

cgtgtggaca acatcacaca gatctctgcc cagctctcgt ggctgccgac caacagtaac 2040

tacagccata tcatcttcct caacgaagaa gaactggaca tcgtgaaggc agccaggtac 2100

aagtaccaat tcttcaacct caggcctaat atggcctaca aggtgaaggt cttggcccag 2160

ccacaccaga tgccctggca gctgccgctg gagcagagag aaaagaagga ggcctgtgtg 2220

gagttctcca cgctgcctgc aggacctcca gccccaccac aagatgtcac tgtccacgct 2280

ggggccacag ccgcctctgt tcaggtctcc tggaagcccc ctgcactgac tcccactggg 2340

ttgtccaatg gagcaaatgt cacaggatac ggcgtgtacg ccaaagggca gagggtggct 2400

gaggtcatcg cccccacggc agatggcacg gcagtggagc tgatccggct gcgaagccta 2460

gaggccaagg ccgtgagtgt gcgtaccctg tctgtacagg gagagtccat ggattctgcc 2520

ctcgctgcca tcccccctga cctcctggtg cctccagccc cccacccgag gactgctccc 2580

ccaccaaagc cattagcaag tgacatggat accaaagacc aacacctggg gccccacgtc 2640

aaagtggatg agtcctggga gcagagccgg tcaccgggcc ctgcacatgg ccacatgttg 2700

gaaccacctg acatgcacag cgctggccca ggcagaaggt caccctcgcc cagccggatc 2760

cttcctcagc cacaaggagc ccccgtgtct accactgtcg ccaaggccat ggcccgtgaa 2820

gctgcacaga gggtggctga gagcaacagg ttagagaaaa ggagcctctt tctagagcaa 2880

agcagtgcgg ggcaatatac caactcggac gaggaggacg gctacgcctc ccccgaggtc 2940

aagaggagag gcacctcagt ggatgacttc ctcaaagggt cagagctggg caagcagccc 3000

cactgttgcc atggagatga gtaccacaca gagagcagcc gggggtcaga cctgtcggac 3060

atcatggagg aggatgagga ggagctatac tcagagatgc agctggagga tgggggccgc 3120

cgtcggccca gcggtacctc tcacaacgcc ctcaagattt taggaaactc cacgttgatg 3180

ggacgagcag accggatgga acacgtgagc cgaaggtatt cacacagtgg cggagggtct 3240

cataggcacc gcccagcgat ggctccatcc attgatgaat acaccgggcg agaccatctt 3300

tctccagact tctatgatga gtccgaaact gaccctggtg ctgaggagct cccagcccgt 3360

atctttgtgg ctctgtttga ctatgaccca ctgaccatgt ccccaaaccc agacgctgct 3420

gaagaggagc ttcccttcaa agaaggacag atcatcaagg tatatggaga caaagacgca 3480

gatggcttct accgtgggga gacctgtgcc aggctcggcc tcattccctg taacatggtc 3540

tctgagatcc atgcggatga tgaggagatg atggatcagc tgctgaggca aggcttcctc 3600

cctctgaaca cgcctgtgga gaaaatagag agaagtagaa gaagcggccg gggtcactct 3660

gtacccacac gaagaatggt ggctctctac gactatgatc ctagggaaag ctctcctaac 3720

gtggatgttg aggctgaact tccattttgc acaggagaca ttattactgt ttttggtgaa 3780

atcgatgaag atggatttta ttatggagag ctgaatgggc aaaaaggcct cgtgccttcc 3840

aatttcctgg aagaagtgcc tgatgatgtg gaggtccacc tttctgatgc tccgccccac 3900

tactcccacg acccgcccat gcgctccaag gccaaaagga agaagagtgt tcatttcaca 3960

ccctaatcag gcatgtagcc gtcacgtaag tgagcaaccg aagacacctt agagagatac 4020

cgacttaagc taccctaagc ggacagcacg gtagtcttaa gacgtcaatg tgggcttaaa 4080

gaaagagaga gaaaagaaat atgtctcaaa acaaacaaac aaaaaaatca ttggacctaa 4140

agtattaggc tatctcacct ggtttcagtt gcctggcaac tagattcata gtgaaatgaa 4200

tcgtctcgaa caattttttt tttaaatgtg tccgttgaga atacgggaaa ggccaacctt 4260

gggaacatat ttcttaaagc tgtttgttta cacaagagtg cgccagcgtg tactcacaag 4320

gagagttgcc tattgcattg ttagctaccc tcagtcaaaa agcacaaccg gatccatgtg 4380

tttaccgtag cagggttgac tcatggtgag agccaagaca aaactttaat cccagatcaa 4440

gccccaaggg gtggccttgg ctctgggcat gccaaagctg gtggaatacc cagctgcagc 4500

cccttggcct taggagcagg tgttccctct cctcaaagca caattgtccc gagctcatgg 4560

aggtacgcat gtacctacga ggtgctggca tactaatgca atagtatctc cggatgcggc 4620

agctcccatg actgcgctag ctggccccat ggtatcttca ggtgtgcttt gggtgcgccc 4680

taggtgttct ccattagagt tagaccttga ttttcaatcc aagcaagctt gggggcccct 4740

tgccttgtat tgtgtgcccg ctgagtgccc tggtctcact cgccaagttt tgctctgggg 4800

agattactga accagaagcc tggcgatccc tggccacgcc caggcgtgtg cctagctgca 4860

gaatgaagtg aaattggcac acagtgttcc tgaatcaaat tccaccccct tggaagcacc 4920

gttggtcggc cacctaaagt ctctcaaagc ccaccgtctc tctgtgctgg ggaaagtgca 4980

cactcctcca ttgggaaatg gtgagagtct tcaagaaggg cctttgagct tccttagatt 5040

acaattaagg ttcatttccg tttttttttc tttcaaacag tgtgccatcc ccaaagtttc 5100

tgtaggactc taactgaccc aagggcttac tccaccatgg cagggcctta tcgaccccac 5160

ctagttcatg atccatccaa attcccactg agagcttttt tgatttacaa tgattgatgg 5220

gtatctcggc caccttgcct atccatctga ttgacaataa cccctcccct gcttttagtc 5280

tgaatttcca gagtaaatta tgacctggct ttctttgcca aagcgatggc caccccgtct 5340

cccaagacaa agacggcgaa agcccatgga gggccagccc ctctctctca gaggggcctt 5400

gggagagggt gtgtgcttct catggcatac tttaaaaatt gaaagaagaa acgaaccacc 5460

acccaaccta ccctttcccc tggctggcgg gagtccgggg cagtgttacc cagtgaattg 5520

ttgttggcgc tgacgcactc aataaaactg tcgcaatgta cctactaggt tcctcccgag 5580

ggttcaggga cagcaaggag agctccatcc cccacagccc atctccattc ggggtcacct 5640

acgtcatcta tgggttctgg tagtcctggg agaggcaggg aaatgtcctc gaaaaagaaa 5700

aaagggctgc tctccagagg caagaaattg ctgcaaaggc tggccgcagt gaagtgaccc 5760

ctgagcctcc ggacacttcc aaatccatgt cactggcttt aattccaaac gaggactccc 5820

aaaacccaac cttgaccccc accccccacc ccagcagtag agagctacac tcttacacta 5880

ccattcacat gataccactg gagagatcag ggaaggggct gcaccgtggt cccctccccc 5940

tccgctccca aggttctgtc caaggtctgg ggtggggagg tgggtggggg gcagctggct 6000

gagcagcaga aggggcaggg ggagggctct ctgcagccac ctcccctcca cctgcagttc 6060

cagcctgcac gcattagagt gtagccctga caggctttct gacctctccc cctctccccc 6120

cccccccacc tctcaagacg tttgcctcat tacacattat tgtcattttc acgcggctct 6180

tgagattcct agtttaactc gccttgatgt tctggcctta taactgcaca gtggtttgta 6240

ctgtcgaata aaaaacaccg tgtatacttc gcatgtgttg tgcattcctg gtcttcatcc 6300

tcacttgaca cagtggcttc acctcacagt ggcccaggcc ctacactttc agatgcctgt 6360

cttgggtctt tcaacgcaag ccagcaacac acaggacatg tttacttagc ccaccacacg 6420

agagacaggg gaaaggagcg ggagcaactg ggcagcatgc atgtcctcta gccagccagg 6480

gagctgacca atctttgcct ttagtgtcct atcctgtggc ttttagcttt ctgttccttt 6540

cattatttgt tgaaatgcct tgccccgaga caccatccac agcacacata gcaaagccac 6600

cacacgcttc attagccaga ccagaaggag cctaaattga aatctctcag acaccccaca 6660

cccatcttta tcattagcct cgaacagcaa acctccccag acgctggctg atgggatggc 6720

cacaactttg taattcctgt ccactgtaaa ctgtttttac ttctttatac atacttttca 6780

gactgccttt cttttgtaat ttctggaagg tttgtaaatg aatggcaaag ctttccctgt 6840

gcgtcttcag aaactatatt gtacattgta atataattgt atgtggtaga ttgattaaaa 6900

ggtcgttcct cggtgtgtga agtactgtgc gggtgtgtgc atgtgtacag ctagggtgac 6960

atattttcta gaaataaact ttgttatttt gtacaacatt aaaaaaaaaa aaaaaaaaaa 7020

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcaagagaga ggccctcag 19

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgtgagggag atgctcagtg 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggtgaactgg gggaggattg 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agagcgatgt tgtccaccag 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agcctatgcc acctagtgat 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggagagctgt aacctgtcgc 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acaatgtggg gagagactgg 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cccttacccg atcactcagg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agggtgtgag aggagtgtgt 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atcacctcga tggacttgcc 20

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ctgacaggag gagcaggatt tt 22

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caccgagaag gggagacttg 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aatcatgcca tttgcccagc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ctcaagtgtg tagggggagg 20

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tgagcagttt aacttgttgt ccc 23

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggaggtggct agtccattca t 21

Claims (6)

1. The biological factor RIMBP2 is characterized in that the biological factor RIMBP2 is synaptophysin domain protein binding protein 2, the amino acid sequence is shown as SEQ NO.1, and the biological factor has the function of maintaining the characteristics of inner ear hair cells.

2. A nucleic acid molecule, wherein the nucleotide sequence encoding the synaptophysin domain protein binding protein 2 of claim 1 is set forth in SEQ No. 2.

3. A recombinant plasmid incorporating the nucleotide sequence encoding synaptic activity domain protein binding protein 2 of claim 2.

4. A recombinant vector transformed with the recombinant plasmid of claim 3.

5. Use of the biological factor RIMBP2 according to claim 1 in the preparation of related products for treating RIMBP2 gene deficiency diseases.

6. A gene-targeted drug, wherein the active ingredient of the gene-targeted drug comprises any one of the nucleic acid molecule of claim 2, the recombinant plasmid of claim 3, or the recombinant vector of claim 4.

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