CN116036236B - Application of biological factor Sparcl1 in drugs to promote inner ear hair cell regeneration - Google Patents

Abstract

The application discloses an application of a biological factor Sparcl in a medicine for promoting inner ear hair cell regeneration, which belongs to the technical field of genes for promoting inner ear cell regeneration, wherein the biological factor Sparcl1 is used for promoting inner ear stem cell regeneration into hair cells, the amino acid sequence of the biological factor Sparcl1 is shown as SEQNO.1, compared with the prior art, the application explores the regeneration effect of Sparcl genes on inner ear hair cells by combining gene regulation and inner ear stem cell recovery with the function of hearing, and the application adopts AAV-ie as a viral vector for delivering target genes and then carries out injection through neonatal mouse ear round window, so that the method has the highest delivery efficiency, small cochlear injury and fundamentally avoids the problems of low vector transfection efficiency and delivery mode selection. Secondly, the patent uses the human recombinant Sparcl protein which can directly act on human, thus having important value and effect on regeneration of clinical inner ear hair cells and recovery of hearing loss.

Description

Application of biological factor Sparcl1 in medicine for promoting regeneration of inner ear hair cells

Technical Field

The invention relates to the technical field of genes for inner ear cell regeneration, in particular to application of a biological factor Sparcl in a medicine for promoting inner ear hair cell regeneration.

Background

Hair cells in the basilar membrane of the inner ear cochlea are known to be mechanoreceptors of the auditory system, including outer hair cells and inner hair cells. These hair cells convert acoustic vibrations into electrical signals, which are then transmitted through neurons to the brain. In fact, hearing disorders are mostly caused by hair cell damage. But hair cells cannot regenerate themselves in mammals, so hearing loss due to hair cell damage is permanent, and the cochlea of a neonatal mouse contains some progenitor cells, which have limited ability to produce new HCs and lose that ability in the cochlea of an adult mouse. Therefore, finding a suitable gene to regulate the re-entry of inner ear progenitor cells into the cell cycle to regenerate hair cells is important for hearing recovery.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides application of a biological factor Sparcl1 in a medicament for promoting regeneration of inner ear hair cells.

The aim of the invention can be achieved by the following technical scheme:

the application of the biological factor Sparcl1 or a medicinal derivative thereof in preparing a medicament for promoting the regeneration of inner ear stem cells into hair cells is characterized in that the biological factor Sparcl1 is used for promoting the regeneration of inner ear stem cells into hair cells, and the amino acid sequence of the biological factor Sparcl1 is shown as SEQ NO. 1.

Further, the inner ear stem cells are precursor cells having proliferation and differentiation potential in the inner ear, and can be differentiated into hair cells through mitosis or transdifferentiation.

Further, the medicine of the invention is added with conventional auxiliary materials, and can be prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral liquid, troches, injections, ointments, granules or various sustained and controlled release preparations and the like according to the conventional process.

The carriers of the medicaments of the present invention are of the usual type available in the pharmaceutical arts and include binders, lubricants, disintegrants, co-solvents, diluents, stabilizers, suspending agents or matrices and the like.

Further, the medicament is used in a dosage form suitable for degrading the biological factor Sparcl1 or a pharmaceutically acceptable derivative thereof in the inner ear of a user, wherein the dosage form is an injection.

On the other hand, the application of the nucleic acid molecule in preparing the medicine for promoting the regeneration of the inner ear stem cells into the hair cells is also provided, and the nucleotide sequence of the nucleic acid molecule for encoding the biological factor Sparcl1 is shown as SEQ NO. 2.

On the other hand, the application of the recombinant plasmid in preparing the medicine for promoting the regeneration of the inner ear stem cells into the hair cells is also provided, and the recombinant plasmid is characterized in that the nucleotide sequence for encoding the biological factor Sparcl is integrated.

On the other hand, the application of the recombinant vector in preparing medicaments for promoting the regeneration of inner ear stem cells into hair cells is also provided, and the recombinant vector is characterized in that the recombinant vector is used for transforming the recombinant plasmid.

In another aspect, there is also provided a medicament for promoting regeneration of inner ear stem cells into hair cells, comprising a nucleic acid molecule as described above, a recombinant plasmid as described above, or any one of the recombinant vectors as described above.

The invention has the beneficial effects that:

The patent explores the regeneration effect of Sparcl genes on inner ear hair cells by combining gene regulation and inner ear stem cells to restore hearing functions. The difficulty of gene therapy is generally that the transfection efficiency of the vector is low and whether the delivery mode damages model animals or not, while the AAV-ie is used as a viral vector for delivering target genes, and then the AAV-ie is injected through the round window of the neonatal mouse, so that the method has the highest delivery efficiency and less damage to cochlea, and the problems of low transfection efficiency of the vector and selection of the delivery mode are fundamentally avoided. Secondly, the patent uses the human recombinant Sparcl protein which can directly act on human, thus having important value and effect on regeneration of clinical inner ear hair cells and recovery of hearing loss.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 is a schematic flow chart of the present application;

FIG. 2 is a graph showing RNA expression levels of Sparcl1 of the present application at various stages of age;

FIG. 3 is a graph showing protein expression levels of Sparcl1 of the present application at various age stages;

FIG. 4 is a graph showing the increase in number and diameter of stem cell pellets promoted in vitro by Sparcl1 of the present application;

FIG. 5 is a graph showing the increase in EdU of Sparcl of the present application in promoting stem cell pellet in vitro;

FIG. 6 is a diagram showing the promotion of hair cell differentiation in vivo by Sparcl A of the present application;

FIG. 7 is a diagram showing the promotion of hair cell differentiation in vivo by recombinant human Sparcl protein of the present application.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The invention uses the following steps to verify SPARCL1 effect on hair cell regeneration, see figure 1

EXAMPLE 1 expression of Sparcl1 in cochlea of wild-type mice of different age groups

1.1 Expression of Sparcl1 at RNA and protein levels of inner ear of mice at different age stages such as P0, P7, P14, P21, P30 was detected by extracting RNA and total protein of cochlea of FVB wild type mice at different age stages such as P0, P7, P14, P21, P30, etc., by real-time quantitative PCR (qPCR) and experimental method of immunoblotting (Western Blot).

1.1.1QPCR experiments

(1) RNA extraction:

a) Preparing required reagent and consumable materials including Trizol, chloroform, isopropanol, DEPC water, rnase-free different specification gun heads, 1.5mLRnase-freep EP tube

B) The method comprises the following steps of dissecting wild-type cochlea of a mouse at different age stages under a microscope, placing the cochlea of the wild-type mouse into EP pipes, adding 1ml of Trizol into each EP pipe, adding steel balls into the EP pipes, grinding the mixture by using an automatic grinding instrument, taking 0.9ml of supernatant, placing the supernatant into a new EP pipe, pre-cooling the mixture to 4 ℃, adding 0.2ml of chloroform into each EP pipe, swirling the mixture for 15s at room temperature, standing the mixture for 5 minutes at room temperature, setting the centrifugation condition to 12000rcf for 10 minutes, centrifuging the mixture for three layers after centrifugation, taking out the supernatant to colorless aqueous RNA, taking out the supernatant and the lower layer to be an organic phenol chloroform layer, carefully taking out the EP pipes, vertically sucking 0.4ml of supernatant, adding 0.4ml of isopropanol, reversing the mixture for ten times, standing the mixture at room temperature for 10 minutes, setting the centrifugation condition to 12000rcf for 10 minutes, then discarding the supernatant, adding 75% ethanol configured by DEPC water into each EP pipe, sufficiently rinsing white RNA precipitate at the bottom, setting the centrifugation condition to 7500rcf for 5 minutes, taking out the supernatant, removing the supernatant, drying the supernatant, adding the supernatant to 20 to the concentration of the supernatant, and then measuring the supernatant by using a filter paper for 20 ng, and then using a photometer to dissolve the PC for 2 ng.

(2) Reverse transcription PCR was performed on the extracted and leveled RNA using reverse transcription kit (REVERTAID FIRST STRAND CDNA SYNTHESIS KIT, thermo, K1622).

(3) Real-time quantitative PCR (qPCR) cDNA obtained by the previous step was diluted ten times with RNase-free water, and the following system was used to ensure reproducibility of three duplicate wells per group:

2×ChamQ SYBR qPCR Master Mix	10.0μl
		Primer 1(10μM)	0.4μl
Primer 2(10μM)	0.4μl
		50×ROX Reference Dye 1	0.4μl
Template DNA/cDNA	2.0μl
		ddH2O	To 20.0μl

the qPCR primer sequences were as follows:

sparcl1-Q-F	GCAGACAACCAAGAGGCCAA
		sparcl1-Q-R	GGTTTCCCTTGTGGATCGGT
GAPDH-Q-F	GGAGCCAAACGGGTCATCAT
		GAPDH-Q-R	TCACGCCACATCTTTCCAGA

After the qPCR system is configured, a Bio-Rad IQ5 Real-Time PCR instrument is used for reaction, and corresponding Ct value data are analyzed later (figure 2).

1.1.2Western Blot experiment

(1) Protein extraction experiment:

a) Preparing required reagent and consumable Ripa protein lysate, cocktail protease inhibitor, sterilized 1.5mlEP tube

B) The specific operation comprises cutting cochlea of different ages under microscope, adding Ripa protein lysate containing 1% Cocktail 0.2ml, adding steel ball, grinding on grinder, setting centrifugation condition at 12000rpm,4 deg.C for 20min, and carefully sucking 0.15ml supernatant to obtain the desired protein.

(2) Protein concentration determination

The following assays were performed using BCA protein concentration assay kit (enhanced) (bi yun day; P0010S):

a) Standard substances are added into standard substance holes of a 96-well plate according to 0,1, 2,4, 8, 12, 16 and 20 mul, and standard substance diluent is added to be 20 mul, which is equivalent to the concentration of the standard substances of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml respectively.

B) An appropriate volume of sample is added to the sample wells of a 96-well plate. If the sample is less than 20. Mu.l, the standard diluent is added to make up to 20. Mu.l. Note that the sample volume was recorded.

C) 200 μl BCA working fluid was added to each well and left at 37deg.C for 20-30 min.

Note that the reaction mixture may be left at room temperature for 2 hours or at 60℃for 30 minutes. When the BCA method is used to measure protein concentration, the color will increase over time. And the color reaction is accelerated by the temperature rise. If the concentration is lower, it is suitable to incubate at a higher temperature, or to extend the incubation time appropriately.

D) Absorbance at wavelengths between a562, or 540-595nm, was measured with a microplate reader.

E) The protein concentration of the sample was calculated from the standard curve and the sample volume used.

F) After leveling with the previously prepared protein lysate according to the calculated protein concentration, 5xSDS was added. Boiling for 7 min at 100deg.C in metal bath before electrophoresis.

(3) Immunoblotting experiments

A) Gel preparation-preparation of kit using 12.5% PAGE gel (Yase; PG 113) 12.5% SDS-PAGE gel and separation gel were prepared:

1. Taking equal volumes of lower layer glue solution and lower layer glue buffer solution, respectively, uniformly mixing 2.0mL, adding 40 mu L of improved coagulant, uniformly stirring, injecting into a glue-making glass plate, enabling the distance between the liquid surface and the upper edge of the short glass plate to be 0.5cm longer than that of the comb teeth, adding a proper amount of water to cover the lower layer glue, waiting for 30 minutes to solidify, and pouring out upper layer water;

2. Taking 0.5mL of each of the equal volume of the upper glue solution and the color upper glue buffer solution, uniformly mixing, adding 10 mu L of the improved coagulant, injecting the mixed solution into a glue-making glass plate, and inserting comb teeth;

3. After waiting for 15 minutes, the upper layer is gelled and fixed, and the comb teeth are pulled out for electrophoresis.

B) Electrophoresis:

1. Placing the solidified rubber plate into an electrophoresis tank, and adding enough 1xRunning buffer;

2. mixing the boiled protein sample by vortex, and respectively adding 10 mu L of molecular weight protein standard sample and 15 mu L of protein sample into sample application holes after instantaneous separation;

3. setting electrophoresis conditions of constant pressure 80v for 30 min, followed by 100v for 60 min

C) Transferring:

1. cutting PVDF film with corresponding size according to the target strip, and waking up with methanol for 10 minutes;

2. after electrophoresis, using a glue opener to pry the glass plate, cutting off the separating glue, and placing the separating glue in a transfer film buffer solution;

3. The sandwich method is adopted, namely, a positive electrode (white) -a foam-rubber cushion, three layers of filter paper, PVDF membrane, separation gel, three layers of filter paper, foam-rubber cushion and a negative electrode (black), and the whole process is operated in a membrane transferring liquid, and the foam is removed by lightly rolling layer by layer.

4. Setting film transferring conditions, namely constant current 280mA for 60 minutes, and placing the film transferring groove in a low-temperature environment because heat is generated in the film transferring process.

D) Closing:

1. After the transfer of the membrane is finished, taking out the corresponding PVDF membrane, dyeing the membrane for 2-3min with ponceau, cutting the corresponding target protein band after obvious protein bands appear on the membrane, then washing with distilled water for 2-3 times, and washing off ponceau;

2. The membrane was placed in a previously prepared blocking solution (2.5 g of skimmed milk powder+50 ml of 1 XTBST) and blocked for 1 hour on a shaking table at normal temperature.

F) Immune response:

1. slowly washing off the surface sealing liquid by using 1 xTBST;

2. Primary antibody (5 mu l Sparcl1 primary antibody +10ml1xtbst;5 mu lGapdh primary antibody +10ml1 xTBST) was formulated and added to the incubation cassette so that the membrane could be gently shaken in the cassette with a shaker overnight at 4 ℃.

3. Recovering the primary antibody, washing the membrane for 3 times by using 1xTBST, incubating the secondary antibody at normal temperature after 7 minutes each time for 60 minutes, recovering the secondary antibody, and washing the membrane for 3 times by using 1xTBST each time for 7 minutes. The membrane was immersed in 1xTBST solution for the next step.

G) Color reaction

1. Opening Tanon5200 full-automatic luminous imaging analysis system, and pre-cooling for 20 minutes;

2. And uniformly mixing the luminescent liquid A and the luminescent liquid B according to the ratio of 1:1, uniformly spreading the luminescent liquid A and the luminescent liquid B on a film by a liquid transfer device, and exposing and photographing.

The results indicate that Sparcl1 reached the highest corresponding mRNA and protein levels at P0-P7, followed by a decrease.

EXAMPLE 2 Sparcl1 promotes capillary proliferation in vitro

2.1 Culturing inner ear stem cells in vitro 3D, adding AAV-ie virus which over expresses Sparcl and controls, collecting samples on the tenth day of proliferation, treating with EdU for 60 minutes before collecting samples, further characterizing proliferation indexes by calculating the number and diameter of stem cell balls and checking the EdU mark number through immunofluorescence, and carrying out data and image processing analysis by combining software such as image j, prism8, ZEN and the like.

2.1.1 Viral packaging and purification

(1) HEK293T cells are co-transfected by pAAV-ie-Sparcl1, pAAV-helper and pAAV-ie plasmids, wherein the nucleotide sequence of pAAV-ie-Sparcl1 for encoding the biological factor Sparcl is shown as SEQ NO. 2.

A) Recovering HEK293T cells, carrying out transfection after passage for 3-4 generations, and packaging viruses after at least meeting the requirement of eight dishes of 15cm culture dish.

B) The transfection preparation A and B are as follows:

A		B
				DMEM	471μL	DMEM	413μL
pAAV-ie-Sparcl1(1ng/μL)	7μL	PEI	87μL
				pAAV-helper(1ng/μL)	14μL
pAAV-ie(1ng/μL)	9μL
				Total	500μL	Total	500μL

the solution B was slowly added dropwise to the solution A, gently mixed and allowed to stand for 20 minutes, and 1ml of the solution was added to the mixture.

C) After 12 hours of transfection, the culture was changed to DMEM medium containing 1% FBS 1. After 48 hours of transfection, the supernatant from the dishes was harvested and replaced with fresh DMEM medium containing 1% fbs. After 96 hours, the supernatant and cells were collected together.

(2) Virus purification

A) Centrifuging the collected 48h supernatant and 96h supernatant with cells under 11000rcf for 7 minutes, and uniformly dividing the final cell sediment into three centrifuge tubes, and pouring the supernatant into a bottle for storage;

b) The pellet of cells was resuspended to 27ml per tube using a small amount of supernatant, then 3.5ml chloroform was added per tube to lyse the cells, and the cells were placed on a shaker at 37℃for 1.5 hours at 220rcf to ensure that the cells were sufficiently lysed;

c) After completion of lysis, centrifugation conditions were 11000rcf for 22 minutes, and the supernatant was carefully aspirated into the supernatant stored in the first section for volume measurement.

D) After the volume of the total supernatant is calculated, adding PEG8000 and NaCl powder, mixing well, and standing at 4 ℃ overnight;

PEG 8000 used amount (g) =supernatant volume (ml)/(10)

NaCl usage (g) =total supernatant volume (ml)/(1000×58.4)

E) Taking out the supernatant of the previous day, centrifuging for a plurality of times, collecting the precipitate in a 2ml EP tube, wherein the centrifuging condition is 12000rcf for 22 minutes;

f) Adding enzymolysis liquid into the collected precipitate, and placing in a 37 ℃ water bath for 2 hours, and continuously blowing until the precipitate is dispersed;

g) Adding chloroform with the same volume as the enzymolysis liquid, the centrifugation conditions were 12000rcf for 7 minutes. The supernatant was carefully aspirated into 1.5ml EP tubes, and an aliquot of an analytical solution such as PEG8000+f68 was added to each tube in an equal volume as the supernatant, and allowed to stand at 4℃overnight.

H) Taking out the virus liquid treated in the previous day, centrifuging for collecting the precipitate for multiple times in a 1.5ml EP tube under the centrifugation condition of 12000rcf for 7 minutes, and collecting the precipitate in one tube;

i) Adding 15-25 mu L of enzymolysis liquid into the collected precipitate, blowing and mixing until the precipitate is dissolved, adding equal volume of chloroform, centrifuging for 12000rcf for 7 minutes, and sucking the supernatant to obtain AAV solution.

(3) Virus titer assay

A) The PCR system was prepared as follows:

AAV solutions	1μL
		DNase I Buffer with MgCl2	1μL
DNase I	1μL
		ddH2O	7μL
Total	10μL

PCR procedure 1:37℃for 20min, 95℃for 10 min.

After the end, 10. Mu.l of Lysis and 2. Mu.l of proteinase K were added.

PCR procedure 2:55℃for 30min, 95℃for 10 min;

AAV lysate was obtained and subjected to qPCR experiments after 100-fold dilution.

B) The qPCR system was formulated as follows:

AAV cleavage dilutions	2μL
		SYBR	10μL
ROX Dye 2	0.4μL
		WPRE-F	0.5μL
WPRE-R	0.5μL
		ddH2O	6.6μL
Total	20μL

And (5) performing qPCR on-machine detection, analyzing the obtained CT value, and calculating the corresponding virus titer.

2.1.2 3D culture of Stem cells

The P1 neonatal rat basement membrane was dissected under a microscope, digested with 0.25% pancreatin at 37℃for 6 minutes, then terminated with DMEM-F12, blown with 200. Mu.L of a gun head, after which undigested tissue was filtered off, centrifugation conditions were set to 2500rcf,3 minutes, resuspended with DMEM-F12 and then added with 30% Matrigel, mixed and seeded into 24-well dishes with round slides (for convenient subsequent collection) for proliferation culture.

The conditions of the proliferation culture solution were as follows:

DMEM/F12	50mL
		N2	500uL
B27	1000uL
		EGF	10uL
IGF	25uL
		FGF	20uL
VPA	120uL
		Ampicillin	60uL

2.1.3 immunofluorescent staining

(1) Incubation with primary antibody

Collecting stem cell pellet with proliferation time of 10 days, fixing 4% PFA at room temperature for 1 hr, rinsing with PBS 3 times for 5min each time, blocking with Blocking medium (1 ml donkey serum+9 ml5/1000 Tritonx-100) at room temperature for 1 hr, staining with cell proliferation marker EdU with EdU Cell Proliferation Kit (thermo) for one hr, blocking for 1 hr, adding supporting cell marker sox2 diluted with PBT (0.1 ml donkey serum+990 ml5/1000 Tritonx-100) at 1:200, and incubating at 4deg.C overnight.

(2) Second antibody incubation

Rinsing with PBS for 3 times and 5min each time, diluting the corresponding secondary antibody with PBT at 1:400 (further diluting DAPI at 1:1000), incubating at room temperature for 1h, rinsing with PBS for 3 times and 5min each time, adding DAKO, back-fastening on glass slide, sealing the glass slide with nail oil, and collecting picture with confocal microscope.

The results showed that Sparcl virus treated groups were higher in number and diameter than control groups, and immunofluorescence results showed that the number of EdU-labeled cells in spheres of Sparcl virus treated groups was significantly greater than control groups.

EXAMPLE 3 Sparcl1 promotes hair cell differentiation in vivo

3.1 Mice were virus injected by injecting p1.5 neonatal mice with AAV-ie virus overexpressing Sparcl and Control through the ear round window, and virus injected mice were harvested on the seventh day, and subsequently were subjected to ear round window injection of exogenous recombinant human Sparcl1 protein, as this protein was secreted, and their hair cell marker Myosin7a was viewed by immunofluorescence. And carrying out processing analysis of data, pictures and the like by combining software such as image j, prism8 and ZEN and the like.

The results show that Sparcl virus treated groups have more xenobiotic hair cells than control groups, and the xenobiotic hair cell phenomenon of mice injected with exogenous recombinant human Sparcl protein is very obvious.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.

Claims (5)

1. The application of the biological factor Sparcl 1 in preparing the hearing repair medicament is characterized in that the amino acid sequence of the biological factor Sparcl 1 is shown in SEQ NO. 1.

2. The use according to claim 1, wherein the pharmaceutical dosage form is a tablet, capsule, liquid, aerosol, injection, ointment, granule.

3. The application of the nucleic acid molecule in preparing the hearing repair medicine is characterized in that the nucleotide sequence of the nucleic acid molecule encoding the biological factor Sparcl is shown as SEQ NO. 2.

4. Use of a recombinant plasmid for the preparation of a hearing repair drug, characterized in that the recombinant plasmid incorporates the nucleotide sequence encoding the biological factor Sparcl according to claim 3.

5. A hearing repair drug comprising the nucleic acid molecule of claim 3 or the recombinant plasmid of claim 4.