Preparation method of vascular tissue-derived extracellular vesicles

Abstract

The invention discloses a preparation method of vascular tissue-derived extracellular vesicles, and belongs to the field of cytobiology. According to the preparation method of the extracellular vesicles, the artery blood vessel tissue is directly subjected to enzymolysis through collagenase, the extracellular vesicles are obtained by combining low-speed centrifugation and ultra-speed centrifugation methods and utilizing a differential centrifugation mode, and the technical blank of obtaining the extracellular vesicles from the blood vessel tissue is filled. Experimental results show that the extracellular vesicles can be successfully prepared from collagenase I, collagenase II, collagenase IV, collagenase V or collagenase IV combined with elastin, the prepared extracellular vesicles are different in particle size, form, yield, protein quantity, purity and the like, and the quality of the extracellular vesicles obtained by enzymolysis of collagenase IV combined with elastin is optimal. The extracellular vesicles are extracted from vascular tissues with normal physiological characteristics for the first time, a new source is provided for preparation of the extracellular vesicles, and the extracellular vesicles have great significance in expanding application of the extracellular vesicles.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology, in particular to a preparation method of vascular tissue-derived extracellular vesicles. BACKGROUND

[0002] Extracellular vesicles are lipid bilayer membrane structures secreted by most cell types. Extracellular vesicles act as messengers for horizontal transfer of lipids, proteins, and nucleic acids, and affect various pathophysiological processes of parent cells and recipient cells.

[0003] Currently, there are mature technologies for extracting extracellular vesicles from cell culture medium and body fluids: ultracentrifugation, size exclusion chromatography, density gradient centrifugation, immunoaffinity capture, microfluidic technology.

[0004] At present, a large number of studies are to extract extracellular vesicles from cell culture supernatant, body fluid and the like, and fewer studies are to extract extracellular vesicles from tissues. Some studies extract extracellular vesicles from cancer tissues, adipose tissues, brain tissues, and liver tissues. Compared with extracellular vesicles obtained from body fluids or cell culture supernatants, EVs isolated directly from tissues have many advantages, including tissue specificity and accurate reflection of tissue microenvironment, so attention should be paid to tissue-derived extracellular vesicles.

[0005] So far, only some studies have isolated and obtained extracellular vesicles from pathological vascular tissues (arterial plaques, valves, etc.), and no studies have been reported on extracting extracellular vesicles from vascular tissues with normal physiological characteristics. SUMMARY

[0006] The purpose of the present application is to provide a preparation method of vascular tissue-derived extracellular vesicles to solve the problems existing in the prior art. The present application first extracts extracellular vesicles from vascular tissues with normal physiological characteristics, providing a new source for the preparation of extracellular vesicles, which has great significance for expanding the application of extracellular vesicles.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] The present application provides a preparation method of vascular tissue-derived extracellular vesicles, comprising the following steps:

[0009] Take an arterial blood vessel, cut it into pieces, wash it, and then perform enzymatic digestion. After enzymatic digestion, centrifuge, collect the supernatant, filter, and obtain the filtrate.

[0010] Ultracentrifuge the filtrate, collect the precipitate and the top oil film, resuspend, ultracentrifuge again after resuspension, collect the precipitate, and obtain the extracellular vesicles.

[0011] Further, the enzyme used for enzymatic digestion includes collagenase or elastase.

[0012] Further, the collagenase is collagenase I, collagenase II, collagenase IV or collagenase V.

[0013] Further, the enzymolysis temperature is 37℃, the rotation speed is 300rpm, and the time is 6h.

[0014] Further, the post-enzymolysis centrifugation comprises a first centrifugation and a second centrifugation.

[0015] The first centrifugation temperature is 4℃, the relative centrifugal force is 500xg, and the time is 10min.

[0016] The second centrifugation temperature is 4℃, the relative centrifugal force is 4000xg, and the time is 60min.

[0017] Further, the ultracentrifugation temperature is 4℃, the relative centrifugal force is 120000xg, and the time is 70min.

[0018] The application further provides an extracellular vesicle obtained by the preparation method.

[0019] The application discloses the following technical effects:

[0020] The preparation method of the extracellular vesicle provided by the application is to directly enzymolyze the arterial blood vessel tissue by using collagenase, combine low-speed centrifugation and ultracentrifugation, and obtain the extracellular vesicle by using the differential centrifugation method, thereby filling the technical blank of obtaining the extracellular vesicle from the blood vessel tissue. The experimental results show that the collagenase I, collagenase II, collagenase IV, collagenase V or the combination of the collagenase V and elastase can be used to successfully prepare the extracellular vesicle, and the prepared extracellular vesicles are different in particle size, morphology, yield, protein amount and purity. The extracellular vesicle obtained by using the collagenase IV combined with the elastase has the best quality, and under the condition of single enzyme, the extracellular vesicle obtained by using the collagenase I and II has the best quality. The application first extracts the extracellular vesicle from the blood vessel tissue with normal physiological characteristics, provides a new source for preparing the extracellular vesicle, and has great significance for expanding the application of the extracellular vesicle. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0022] Figure 1Schematic diagram of the process for extracting extracellular vesicles from blood vessel tissue using five different collagenases for enzymatic digestion;

[0023] Figure 2 Results chart for measuring the particle size of blood vessel tissue extracellular vesicles obtained using nanoparticle tracking technology with five different collagenases;

[0024] Figure 3 Results chart for the number of extracellular vesicle particles and protein amount isolated from blood vessel tissue using five collagenases for enzymatic digestion;

[0025] Figure 4 Results chart for comparing the yield of extracellular vesicles obtained from 1 g of blood vessel tissue using five different collagenases for enzymatic digestion;

[0026] Figure 5 Results chart for comparing the purity of extracellular vesicles obtained from blood vessel tissue using five different collagenases for enzymatic digestion;

[0027] Figure 6 Observation chart of the morphology of extracellular vesicles from blood vessel tissue under electron transmission microscopy. DETAILED DESCRIPTION

[0028] Various illustrative embodiments of the present application are now described in detail below. The following description includes specific details for the purpose of providing a thorough understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced without these specific details. In other instances, well-known structures and functions have not been described in detail in order to avoid obscuring the understanding of the present application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, as well as any other stated or intervening value in that stated range is encompassed. In addition, any combination of the above ranges, as well as any other stated or intervening value in that stated range, is encompassed. All ranges are inclusive of the endpoints.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the event of conflict between the present specification and any document incorporated by reference, the present specification controls.

[0031] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from this specification, which is to be regarded in an illustrative manner. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the embodiments disclosed herein. The specification and examples given are exemplary only.

[0032] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms, i.e., are intended to permit but not exclude any additional item, limitation, etc.

[0033] The purpose of the present application is to provide a method capable of obtaining extracellular vesicles from normal physiological vascular tissue to solve the problems existing in the prior art, to further explore the extracellular vesicles of high yield and high purity of vascular tissue, and to evaluate the extracellular vesicles from the determination of concentration index and morphological index, so as to select extracellular vesicles with higher purity, and to provide a theoretical basis for further research on the biological activity of extracellular vesicles derived from vascular tissue.

[0034] The present application uses collagenase I, collagenase II, collagenase IV, collagenase V or collagenase IV combined with elastase to enzymatically hydrolyze vascular tissue to separate extracellular vesicles, and identifies the extracellular vesicles extracted from the vascular tissue by enzymatic hydrolysis of the five different enzymes.

[0035] Example 1

[0036] 1. Extract extracellular vesicles (VEVs) from vascular tissue by enzymatic hydrolysis of five different collagenases

[0037] 1.1 Experimental materials

[0038] 1.1.1 Reagents

[0039] Collagenase I, II, IV and V were purchased from Beijing Solabio Technology Co., Ltd.; elastase was purchased from MedChemExpress; Ham's F-12k (Kaighn's) medium; phosphate-buffered saline (PBS) (1x) was purchased from Wuhan Sevier Biological Technology Co., Ltd.; BCA kit was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.

[0040] 1.1.2 Consumables / instruments

[0041] Heareus Pico 17 centrifuge was purchased from Thermo Fisher Scientific, USA; Optima L 100-XP ultracentrifuge was purchased from Beckman, USA; multifunctional enzyme marker was purchased from Wuhan Sevier Biological Technology Co., Ltd.

[0042] 1.2 Experimental method

[0043] 1.2.1 Extraction of vascular tissue-derived extracellular vesicles

[0044] The schematic diagram of the extracellular vesicle extraction method of the present application is shown in Figure 1

[0045] 1.2.1.1 0.5 g of aortic blood vessels of healthy, non-cardiovascular disease pigs were taken into a centrifuge tube, and an appropriate amount of PBS was added. The blood vessels were cut into 1 mm x 1 mm fragments. After cutting, the blood vessel tissue was washed with PBS by centrifugation (4°C, 500 x g, 5 min) and repeated twice. 10 mL of different enzymes (collagenase I, II, IV, V, collagenase IV combined with elastase) dissolved in Ham's F-12k (Kaighn's) medium at a concentration of 2 mg / mL were added to each 0.5 g of tissue sample, and the enzyme reaction was carried out on a shaker (37°C, 300 rpm), and the start time of the reaction was recorded. The enzyme reaction was observed every 2 h, and the time was marked. The enzyme reaction was completed after 6 h.

[0046] 1.2.1.2 The enzyme-degraded sample was centrifuged for 10 min (4°C, 500 x g), and the supernatant was taken and centrifuged again for 60 min (4°C, 4000 x g). After centrifugation, the supernatant was filtered with a 0.22 μm filter. The sample was stored in a 4°C refrigerator.

[0047] 1.2.1.3 The sample was moved to an ultracentrifuge tube and was adjusted to the same volume with PBS (with an error of within 10 mg). The first centrifugation was started for 70 min (4°C, 120000 x g, SW32Ti). After centrifugation, the supernatant was removed by vacuum, and the oil film and the precipitate at the top were retained. The bottom precipitate was blown and mixed evenly with PBS, and 10 mL of PBS was added to adjust the volume. The second centrifugation was carried out for 70 min (4°C, 120000 x g, SW41Ti). After centrifugation, the supernatant was discarded and the precipitate was blown and mixed evenly. The sample was moved to a 1.5 mL centrifuge tube and stored in a -80°C freezer.

[0048] 2.5 Identification of extracellular vesicles derived from vascular tissue by different collagenases

[0049] 2.1 Experimental materials

[0050] The extracted vascular tissue-derived extracellular vesicles, PBS, and 1.5 mL EP tubes.

[0051] 2.2 Experimental method

[0052] 2.2.1 BCA kit for determining protein concentration

[0053] ​A standard curve was made, two parallel holes were set, 0, 1, 2, 4, 8, 12, 16, 20 μL of standard was added in turn; 20, 19, 18, 16, 12, 8, 4, 0 μL of water was added in turn, and the concentration was 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL in turn. The vesicles (5 μL) were diluted ten times, and two parallel holes were also set, 20 μL of sample was added to each hole (to avoid bubbles). 200 μL of BCA working solution (A solution: B solution = 50:1) was added to each hole, and the hole plate was covered with tin paper after mixing, and incubated at 37°C for 30 min. The absorbance value at 562 nm was detected by using the microplate reader.

[0054] 2.2.2 Nanoparticle tracking technology

[0055] 1 μl of the obtained vesicles was taken in 999 μL PBS, i.e. diluted 1000 times, and placed in a 1.5 mL EP tube, and the concentration, yield, particle size, etc. were analyzed by using a nanoparticle analyzer.

[0056] 2.2.3 Morphological observation

[0057] Transmission electron microscopy was used to observe the morphology of the extracellular vesicles from the collagenase enzymolysis of vascular tissue-derived cells.

[0058] 2.3 Experimental results

[0059] The results are shown in Table 1. Figures 2-6 The protein concentration of the extracellular vesicles enzymolyzed by collagenase I was 0.7334 mg / ml, the protein amount was 0.3667 mg, the particle number was 7.75 x 10 11 particles, the particle number per gram was 1.4192 x 10 12 Particles / g, the purity was 2.1134 x 10 9 particles / μg, and the particle size was 130.4 nm; the protein concentration of the extracellular vesicles enzymolyzed by collagenase II was 0.6722 mg / mL, the protein amount was 0.4033 mg, the particle number was 8.28 x 10 11 particles, the particle number per gram was 1.6370 x 10 12 Particles / g, the purity was 2.0531 x 10 9 particles / μg, and the particle size was 118.2 nm; the protein concentration of the extracellular vesicles enzymolyzed by collagenase IV was 1.0526 mg / ml, the protein amount was 0.5263 mg, the particle number was 4.265 x 10 11 particles, the particle number per gram was 7.9290 x 10 11 Particles / g tissue, the purity was 1.6207 x 109 particles / ug, the particle size was 115.8 nm; the protein concentration of the extracellular vesicles digested by collagenase V was 0.9608 mg / ml, the protein amount was 0.5765 mg, and the particle number was 6.72 x 10 11 particles, the particle number per gram was 1.2923 x 10 12 Particles / g tissue, the purity was 1.9428 x 10 9 particles / ug, the particle size was 115.8 nm; the protein concentration of the extracellular vesicles digested by collagenase V was 0.9608 mg / ml, the protein amount was 0.5765 mg, and the particle number was 6.72 x 10 11 particles, the particle number per gram was 1.75 x 10 12 Particles / g tissue, the purity was 4.1836 x 10 9 particles / ug, the particle size was 115.8 nm; the protein concentration of the extracellular vesicles digested by collagenase V was 0.9608 mg / ml, the protein amount was 0.5765 mg, and the particle number was 6.72 x 10

[0060] The above-described embodiments are only to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing a vascular tissue-derived extracellular vesicle, characterized by, The method comprises the following steps: taking an artery blood vessel, cutting and crushing, washing, and then performing enzymolysis, centrifuging after the enzymolysis, collecting supernatant, filtering, and obtaining a filtrate; ultracentrifuging the filtrate, collecting a precipitate and a top oil film, resuspending, ultracentrifuging again after the resuspending, and collecting the precipitate to obtain the extracellular vesicles.

2. The production method according to claim 1, wherein The enzyme used in the enzymolysis comprises collagenase and elastase.

3. The production method according to claim 2, wherein The collagenase is collagenase I, collagenase II, collagenase IV or collagenase V.

4. The production method according to claim 1, wherein The temperature of the enzymolysis is 37℃, the rotation speed is 300 rpm, and the time is 6 h.

5. The production method according to claim 1, wherein The centrifuging after the enzymolysis comprises first centrifuging and second centrifuging. The temperature of the first centrifuging is 4℃, the relative centrifugal force is 500×g, and the time is 10 min. The temperature of the second centrifuging is 4℃, the relative centrifugal force is 4000×g, and the time is 60 min.

6. The production method according to claim 1, wherein The temperature of the ultracentrifuging is 4℃, the relative centrifugal force is 120000×g, and the time is 70 min.

7. Extracellular vesicles obtained by the preparation method according to any one of claims 1-6.

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