Enrichment method of small extracellular vesicles of cochlea tissue

Abstract

The invention discloses a method for enriching small extracellular vesicles of cochlea tissue, and belongs to the field of biology. The method comprises the following steps: putting a basilar membrane, a spiral ligament and a cochlea shaft of the cochlea tissue into a culture dish for culture, and cutting into blocks; adding enzyme for digestion treatment; treating the digested cochlea by an ultracentrifugation method, a sucrose density gradient method or a size exclusion method, and separating to obtain the small extracellular vesicles of the cochlea tissue. The invention aims to solve the technical problem of extracting and enriching sEV in inner ear tissues, provides a method for extracting and enriching sEV in inner ear tissues, and provides a method for separating and purifying sEV for deeply researching the action of sEV in an auditory system. According to the method, the optimal extraction mode suitable for the inner ear tissue sEVs is obtained by changing the extraction and enrichment mode of other tissue sEVs in literatures. According to the method, the research of sEVs in the hearing field is promoted to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular to a method for enriching small extracellular vesicles of cochlear tissue. Background Art

[0002] Small extracellular vesicles (sEV) are secreted by various cells with a diameter of 30-200nm. Small extracellular vesicles contain a variety of biologically active substances such as proteins, nucleic acids and lipids, and their phospholipid bilayer structure can protect the contents from being degraded by proteases in the body. The contents of sEV can be transferred from donor cells to recipient cells to participate in intercellular signaling. As an intracellular medium, it can participate in various physiological and pathological processes such as cell proliferation, immune regulation, and tissue repair.

[0003] At first, the research on sEV mainly focused on the research in in vitro culture system. With the further in-depth research on sEV, it was found that the function of sEV in in vitro system could not replace the function of sEV in the real environment in vivo. Because the single environment in vitro is an idealized situation, while the presence of multiple cells in the body is a complex and diversified communication environment. Therefore, the study of tissue sEV can better reflect the biological function of sEV in the real environment. At present, the research on tissue sEV is mainly in larger tissues, such as brain, adipose tissue, and cancerous tissue. The method used to separate sEV from tissue is mainly to use enzyme digestion of tissue and then use ultracentrifugation, size exclusion and density gradient to separate sEV. However, the types of enzymes and separation methods used for different tissues are not the same. As a biological organ, the inner ear is very small compared to other tissues and organs. Therefore, in the process of obtaining sEV, it will face the problem of sample collection difficulties. Therefore, it is very important to obtain an efficient separation and purification method for extracting sEV from inner ear tissue. It is precisely because the inner ear tissue is very small that it is not easy to obtain sEV in both in vitro culture system and in vivo system.

[0004] The inventors found in previous studies that in the extraction of sEV from cochlear tissue, the extracted sEVs are of mixed origin because the entire cochlear tissue contains cellular components such as bone. In addition, the method of grinding tissue will introduce intracellular vesicle contamination. In the present invention, the tissue is further refined and the extraction method is optimized to obtain sEVs that are more representative of the secretion of inner ear tissue cells.

[0005] Therefore, the present invention proposes a method for efficiently extracting exosomes from inner ear tissue, which can fill the gap in the extraction of sEVs from inner ear tissue and further promote the research of sEVs in the field of hearing. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention proposes a method for enriching small extracellular vesicles from cochlear tissue.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The first aspect of the present invention relates to a method for enriching small extracellular vesicles in cochlear tissue, comprising the following steps:

[0009] placing the basement membrane, spiral ligament and modiolus of the cochlear tissue in a culture dish for culture, and cutting into pieces;

[0010] Add enzymes for digestion;

[0011] The digested cochlea is processed by ultracentrifugation, sucrose density gradient method or size exclusion method to isolate small extracellular vesicles from cochlear tissue.

[0012] Optionally, the ultracentrifugation method includes the following steps: centrifugation at 300g for 10 min to remove large debris, taking the supernatant and centrifuging at 2000g for 20 min to remove large cell debris; taking the supernatant and filtering it with 0.22μm; collecting the filtrate and centrifuging it at 16500g for 20 min to remove large vesicles; taking the supernatant and ultracentrifuging it at 110000g for 2h; removing the supernatant and resuspending sEVs with DPBS.

[0013] Optionally, the sucrose density gradient method comprises the following steps:

[0014] The cochlear tissue was separated by differential centrifugation;

[0015] Add sucrose solutions with concentrations from low to high in sequence to prepare a sucrose density gradient solution;

[0016] Then, the cochlear tissue was added to a sucrose density gradient solution and centrifuged;

[0017] The supernatant was taken and resuspended to obtain the small extracellular vesicles in the cochlear tissue.

[0018] Optionally, the size exclusion method comprises the following steps:

[0019] The cochlear tissue was separated by differential centrifugation;

[0020] The separated solution was concentrated and then added to the SEC-qEV column;

[0021] Then, PBS solution was added, and the liquid flowing out from the lower end of the SEC-qEV column was collected in groups to obtain the cochlear tissue small extracellular vesicles.

[0022] Optionally, the sucrose solutions with concentrations from low to high are 0.6 Mol / L, 1.3 Mol / L, and 2.5 Mol / L sucrose solutions, respectively.

[0023] Optionally, the enzyme used for the digestion is type 3 collagenase, papain, or type D collagenase combined with DNase1.

[0024] Optionally, the digestion treatment comprises the following steps: adding type 3 collagenase to the cochlear tissue for digestion, placing the cochlear tissue on ice, and adding a protease inhibitor and a phosphatase inhibitor to terminate the digestion.

[0025] Optionally, the digestion treatment comprises the following steps: adding type D collagenase and DNase1 to the cochlear tissue for digestion, and then placing the cochlear tissue on ice to terminate the digestion.

[0026] Optionally, the digestion treatment comprises the following steps: adding papain to the cochlear tissue for digestion, placing the cochlear tissue on ice, and adding a protease inhibitor to terminate the digestion.

[0027] The second aspect of the present invention relates to the application of the above-mentioned method for enriching small extracellular vesicles of cochlear tissue in auditory research.

[0028] Beneficial effects of the present invention:

[0029] The present invention combines and improves the existing sEV extraction technology to obtain the most suitable extraction method for sEVs in inner ear tissue. The present invention will promote the research of sEVs in the field of hearing to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 This is an experimental flow chart of the extraction method of this application; the first step is to separate different tissues from the mouse cochlea and cut them into tissue fragments; the second step is to digest the tissues using different enzymes; the third step is to separate sEVs using different separation methods.

[0032] Figure 2 Comparison of sEVs extracted by UC after different digestion methods in this application; (A, B) Collagenase D and DNase1, collagenase 3 (A), papain (B) were used to digest tissue and extract sEVs, and the marker proteins of different sEVs (CD63, Tsg101, CD9, Flotillin-1), as well as the marker proteins of other vesicles (endoplasmic reticulum, lysosome) were used as negative controls (Calnexin, Rab7).

[0033] Figure 3Sucrose density gradient was used to extract sEVs in this application. (A) Protein quantification results of different components. (B) Representative graph of particle number of different components. (C) Marker proteins of sEVs of different components (CD63, Tsg101, CD9), and marker proteins of other vesicles as negative controls (Calnexin, Rab7). (D) F2 and F3 TEM detection of sEV morphology.

[0034] Figure 4 The size exclusion method was used to extract sEVs in this application. (A) Protein quantification results of different components. (B) Representative graph of particle number of different components. (C) Marker proteins of sEVs of different components (CD63, Tsg101, CD9), and marker proteins of other vesicles as negative controls (Calnexin, Rab7). (D) TEM detection of sEV morphology by F1, F2 and F3.

[0035] Figure 5 Comparison of different extraction methods for sEVs in this application. (A) Comparison of protein amount after sEV extraction by UC, SEC and SDGU. (B) Comparison of particle number after sEV extraction by UC, SEC and SDGU. (C) Comparison of the ratio of particle number to protein amount after sEV extraction by UC, SEC and SDGU. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] like Figure 1 As shown, in some embodiments of the present invention, a method for enriching small extracellular vesicles in cochlear tissue is disclosed, which specifically comprises the following steps:

[0038] 1. Methods for digestion and dissociation of cochlear basilar membrane tissue

[0039] Tissue dissection: 45 P3 mice were divided into three groups. The mouse cochlea was dissected in cold PBS. The separated basilar membrane, spiral ligament and modiolus were placed in a culture dish containing 1 ml of cold culture medium, and the tissue was cut into small pieces with a scalpel.

[0040] This example provides three enzyme digestion methods:

[0041] ① Evenly add 375U / ml type 3 collagenase (collagenase 3) to the treated tissue, digest at 37℃ for 30min, then immediately place the tissue on ice and add Complete Protease Inhibitor (Complete Protease Inhibitor) and Phosphatase inhibitor (PhosSTOP) to terminate digestion.

[0042] ② Evenly add 3U collagenase D and 40U / ml DNase1 to the treated tissue, digest at 37℃ for 45min, and immediately place the tissue on ice to terminate digestion.

[0043] ③ Add 20U / ml papain evenly to the treated tissue, digest at 37℃ for 30min, then immediately place the tissue on ice and add protease inhibitors to terminate digestion.

[0044] like Figure 2 As shown, after the enrichment obtained by the three extraction methods provided in this example, the characteristic protein characterization results of protein immunoblotting showed that there was less contamination of foreign proteins when the D-type collagenase and DNase 1 were co-digested ( Figure 2 ), but papain digestion could not detect the marker proteins of sEVs. In summary, the purity of sEVs obtained by type D collagenase and DNase1 digestion was better.

[0045] 2. Method of isolating sEVs from cochlear tissue by ultracentrifugation (UC)

[0046] ① Filter the digested tissue through a 40μm cell sieve to remove large tissue pieces;

[0047] ② Centrifuge at 300g for 10 min to remove large debris, take the supernatant and centrifuge at 2000g for 20 min to remove large cell debris;

[0048] ③ Take the supernatant and filter it through 0.22μm;

[0049] ④ After collecting the filtrate, centrifuge at 16500g for 20 min to remove large vesicles;

[0050] ⑤ Take the supernatant and perform ultracentrifugation at 110,000 g for 2 h;

[0051] ⑥ Remove the supernatant and resuspend sEVs in 150ul DPBS.

[0052] 3. Sucrose density gradient method (SDGU) for the isolation of sEVs from cochlear tissue

[0053] First, the sample was pre-processed. After the digested sample was subjected to 1-4 steps of differential centrifugation in UC centrifugation, 0.6M, 1.3M and 2.5M sucrose solutions were prepared. 1.2ml 0.6M, 1.2ml 1.3M, and 1ml 2.5M sucrose solutions were added to the ultracentrifuge tube to form a sucrose density gradient solution. Then, the sample was added and the total liquid on the sucrose density was filled to 7.5ml with DPBS. Subsequently, centrifugation was performed at 180000g for 3h. After the end, the 6.3ml liquid above was removed and the sample was collected. 1.2ml was taken as F (fraction) 1, 1.2ml as F2, 1.2ml as F3, and 1ml as F4. Each collected component was then centrifuged at 110000g for 70min, the supernatant was removed, and sEVs were resuspended with 150ul DPBS.

[0054] like Figure 3 As shown in the figure, protein quantification, particle size and concentration determination, and Western blot detection of marker proteins and negative control proteins were performed on the four components F1 to F4. TEM images also showed that sEVs in the F2 and F3 components showed obvious cup-shaped structures. The results showed that sEVs were mainly enriched in the F2 and F3 components.

[0055] 4. Method for isolating sEVs from cochlear tissue using size exclusion method (SEC)

[0056] First, the sample was pre-processed. After the digested sample was subjected to steps 1-4 of UC centrifugation, the sample was concentrated to 500ul using a 100KDMWCO ultrafiltration tube, and then the operation was performed according to the SEC-qEV (Huaying Bio) column manual. In short, the sample was added to the column, and PBS was added to the column after the sample entered the sieve plate of the column. Then, the first 3ml of liquid flowing down from the bottom of the column was removed, and then each 500ul was collected in a 1.5ml EP tube as a component, and 6 components (F1-6) were collected continuously.

[0057] like Figure 4 As shown in the figure, protein quantification, particle size and concentration determination, and Western blot detection of marker proteins and negative control proteins were performed on the six components. TEM was also used to detect the morphology of F1-F3 sEVs. The results showed that sEVs were mainly enriched in F1-F3 components.

[0058] 5. Characterization methods of sEVs

[0059] ⑴ Protein immunoblotting

[0060] The extracted sEVs samples were lysed with RIPA solution, and then the protein concentration was quantified using a BCA kit. SDS was added to the protein sample and heated at 95°C for 15 min. The treated protein samples were electrophoresed with SDS-PAGE gel and then transferred to the membrane. After the transfer, 5% BSA was used for blocking for 2 hours. After blocking, the primary antibody was added and incubated overnight at 4°C.

[0061] The next day, the primary antibody was removed, and the sections were washed three times with TBST for 10 min each time, followed by the addition of the secondary antibody. Finally, the protein bands were developed using ECL chemiluminescent solution.

[0062] ⑵Nanoparticle tracking (NTA)

[0063] The size and concentration of sEVs were identified using the Zata view instrument. 1 ml of sample was injected into the detection port, and 5 60-second videos were taken for each sample. The scattered light signals of sEVs were collected using an optical microscope, and then the size and concentration of sEVs in the 5 videos were statistically analyzed based on the Brownian motion of the particles.

[0064] ⑶ Transmission electron microscopy (TEM)

[0065] The sEVs sample was dropped onto a copper grid and then stained with 1.5% phosphotungstic acid (pH 7.4) at room temperature for 5 minutes. After the sample was dried, it was photographed using a TEM (JEM-2100) at an accelerating voltage of 200 kV.

[0066] The statistical results of the above characterization methods are as follows Figure 5 As shown in the figure, it can be seen that the UC method obtains the most protein, but the number of particles and the ratio of the number of particles to the amount of protein are both higher in SDGU. The above results show that the UC separation method may collect more contaminant proteins during the extraction process, while the SDGU separation method can help obtain sEVs with higher purity.

[0067] Based on the above experimental data, Figure 2 This indicates that type D collagenase and DNase 1 are more effective than the other two digestive enzymes. Figure 3 to Figure 5 This indicates that the SDGU method is the best way to extract sEVs compared to UC and SEC. Therefore, digesting cochlear tissue with type D collagenase and DNase 1 and then extracting sEVs with SDGU is the best way to obtain sEVs from cochlear tissue.

[0068] It should be noted that the method for enriching small extracellular vesicles of cochlear tissue of the present application is mainly used in the research of auditory field and related tissue cells, etc., and may also be used in diagnosis using sEV as a target. However, the method of the present application is only an enrichment and extraction method, and does not have a diagnostic function in itself. The method for enriching small extracellular vesicles of cochlear tissue of the present application cannot be used alone for diagnostic purposes.

[0069] In addition, the main technical solution of this application is the separation and extraction of small extracellular vesicles in cochlear tissue. Although the embodiments of this application mention the extraction of cochlear tissue, the improvement of the method of this application does not lie in the means of surgical separation of cochlear tissue. Therefore, the improved technical solution of this application can be replicated in the industry and stably obtain beneficial effects.

[0070] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0071] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for enriching small extracellular vesicles from cochlear tissue, characterized in that: The following steps are involved: placing the basement membrane, spiral ligament and modiolus of the cochlear tissue in a culture dish for culture, and cutting into pieces; Add enzymes for digestion; The digested cochlea is processed by ultracentrifugation, sucrose density gradient method or size exclusion method to isolate small extracellular vesicles from cochlear tissue.

2. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The ultracentrifugation method includes the following steps: centrifugation at 300g for 10 minutes to remove large debris, taking the supernatant and centrifuging at 2000g for 20 minutes to remove large cell debris; taking the supernatant and filtering it with 0.22μm; collecting the filtrate and centrifuging it at 16500g for 20 minutes to remove large vesicles; taking the supernatant and ultracentrifuging it at 110000g for 2 hours; removing the supernatant and resuspending sEVs with DPBS.

3. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The sucrose density gradient method comprises the following steps: The cochlear tissue was separated by differential centrifugation; Add sucrose solutions with concentrations from low to high in sequence to prepare a sucrose density gradient solution; Then, the cochlear tissue was added to a sucrose density gradient solution and centrifuged; The supernatant was taken and resuspended to obtain the small extracellular vesicles in the cochlear tissue.

4. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The size exclusion method comprises the following steps: The cochlear tissue was separated by differential centrifugation; The separated solution was concentrated and then added to the SEC-qEV column; Then, PBS solution was added, and the liquid flowing out from the lower end of the SEC-qEV column was collected in groups to obtain the cochlear tissue small extracellular vesicles.

5. The method for enriching small extracellular vesicles from cochlear tissue according to claim 3, characterized in that: The sucrose solutions with concentrations from low to high are 0.6 Mol / L, 1.3 Mol / L, and 2.5 Mol / L sucrose solutions respectively.

6. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The enzyme used for the digestion is type 3 collagenase, papain, or type D collagenase combined with DNase 1.

7. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The digestion treatment comprises the following steps: adding type 3 collagenase to the cochlear tissue for digestion, placing the cochlear tissue on ice, and adding a protease inhibitor and a phosphatase inhibitor to terminate the digestion.

8. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The digestion treatment comprises the following steps: adding type D collagenase and DNase1 to the cochlear tissue for digestion, and then placing the cochlear tissue on ice to terminate the digestion.

9. The method for enriching small extracellular vesicles from cochlear tissue according to claim 1, characterized in that: The digestion treatment comprises the following steps: adding papain to the cochlear tissue for digestion, placing the cochlear tissue on ice, and adding a protease inhibitor to terminate the digestion.

10. Use of the method for enriching small extracellular vesicles from cochlear tissue according to any one of claims 1 to 9 in auditory research.

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