CN117384822A - Preparation method and application of apoptotic vesicles derived from gingival tissues of rats - Google Patents
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Abstract
The invention provides a preparation method of an apoptosis vesicle from animal tissue, which comprises the following steps: separating animal tissue, shearing, inducing apoptosis in culture medium containing staurosporine, and separating by gradient centrifugation to obtain tissue-derived apoptotic vesicles. The invention prepares the apoptosis vesicle by taking animal tissues as the starting materials for the first time, and has the advantages of simple preparation method, short time, high yield and low cost. The apoptosis vesicle prepared by the method has the effects of regulating and controlling the activity of the rat bone marrow mesenchymal stem cells, promoting the bone marrow mesenchymal stem cells to be osteogenic differentiated, promoting the fibroblast migration and the like, and has wide application prospect in the treatment of related diseases.
Description
The application is a divisional application of 2022, 12 months and 12 days, the application number is 202211592026.0, and the name is 'a preparation method of apoptosis vesicles derived from animal tissues and application thereof'.
Technical Field
The invention relates to the technical field of biological tissue engineering, in particular to a preparation method and application of an animal tissue-derived apoptosis vesicle.
Background
Extracellular Vesicles (EVs) are vesicle-like bodies with a bilayer membrane structure secreted by cells, which contain abundant proteins, RNAs and lipids, are key communication media for cells or tissues, and play an important role in various physiological processes. Apoptotic vesicles (apoptotic vesicles, apoVs) are extracellular vesicles released after apoptosis, and are key communication media for cells or tissues due to the abundant proteins, RNAs and lipids contained therein, and are also an emerging field of extracellular vesicle research. Apoptotic vesicles can play an important therapeutic role in a variety of diseases, such as diabetes, hemophilia, osteoporosis, and others.
Cells are the basic units that make up the structure and function of the human body, and tissues are composed of a large number of cells of different structures and functions, and vesicles derived from tissues have certain tissue specificities and functions and are likely to play an important role in different diseases. At present, the extraction of apoptotic vesicles mainly comes from cultured cells or body fluids, but the extraction of apoptotic vesicles from cells requires the cultivation of a large number of cells, which is time-consuming, costly and has a small extraction of apoptotic vesicles from body fluids, thus greatly limiting the application of apoptotic vesicles in the biomedical field. Therefore, it is important to find an extraction method of apoptotic vesicles with large extraction amount and low cost.
Disclosure of Invention
Aiming at the defects of the existing extracellular vesicle extraction method, the invention aims to provide a preparation method and application of apoptotic vesicles (apoVs) derived from animal tissues.
The invention solves the technical problems by the following technical proposal:
a method for preparing apoptotic vesicles derived from animal tissue, comprising the steps of;
(1) Separating and shearing animal tissue;
(2) Inducing apoptosis in the tissue sheared in step (1) in a staurosporine-containing medium;
(3) Collecting the tissue supernatant induced in the step (2), and separating by a gradient centrifugation method to obtain the tissue-derived apoptotic vesicles.
Specifically, the animal is a mammal, including a human, rat, mouse, monkey, dog, cat, cow, rabbit, horse, or pig;
more specifically, the mammal is a human, rat, mouse.
Specifically, the tissue includes adipose tissue, alveolar bone tissue, gingival tissue;
more specifically, the tissue is adipose tissue, alveolar bone tissue, gingival tissue.
Specifically, the apoptotic vesicles are apoptotic microvesicles having a particle size of less than 1 μm.
Specifically, the culture medium in the step (2) is an alpha-MEM culture medium containing staurosporine, and the apoptosis induction time is 12 hours.
Specifically, the gradient centrifugation method in step (3) comprises the following steps:
(1) Centrifuging at 4deg.C for 10min at 800g, and collecting supernatant;
(2) Centrifuging the supernatant obtained in the step (1) at 2000g and 4 ℃ for 10min, and collecting the supernatant;
(3) Centrifuging the supernatant obtained in the step (2) at 16000g and 4 ℃ for 30min, and collecting the precipitate;
(4) Washing the precipitate obtained in the step (3) with sterile PBS, and centrifuging at 16000g and 4deg.C for 30min to obtain tissue-derived apoptotic vesicles.
The invention also provides application of the method in preparing a medicament for regulating and controlling the activity of the mesenchymal stem cells;
specifically, the bone marrow mesenchymal stem cells are derived from human, rat, mouse, monkey, dog, cat, cow, rabbit, horse or pig;
in particular, the dose of apoptotic vesicles in the application is 0.01-0.1ug/mL.
The invention also provides application of the method in preparing a medicament for promoting bone marrow mesenchymal stem cell osteogenic differentiation;
specifically, the bone marrow mesenchymal stem cells are derived from human, rat, mouse, monkey, dog, cat, cow, rabbit, horse or pig;
in particular, the dose of apoptotic vesicles in the application is 1-100ng/mL.
The invention also provides application of the method in preparing a medicament for promoting migration of human skin fibroblasts;
in particular, the dose of apoptotic vesicles in the application is 1-100ng/mL.
Compared with the prior art, the invention has the beneficial effects that:
(1) Compared with the apoptotic vesicles from cells, the apoptotic vesicles from tissue prepared by the method do not need to be subjected to cell culture, and the preparation method is simple and low in cost.
(2) Compared with apoptotic vesicles derived from body fluids, the tissue-derived apoptotic vesicles prepared by the method have higher yield and certain tissue specificity.
(3) The apoptosis vesicle prepared by the method has various biological activities of improving the cell activity, promoting the osteogenic differentiation of the mesenchymal stem cells, promoting the migration of the fibroblasts and the like, and is likely to play an important role in the treatment of related diseases.
Drawings
FIG. 1 is a flow chart of extraction of apoptotic vesicles from animal tissue.
Fig. 2 is a schematic representation of the extraction of apoptotic vesicles from animal tissue sources.
FIG. 3 is a transmission electron microscope view of apoptotic vesicles of different tissue origin; fat apoVs represent apoptotic vesicles of adipose tissue origin, alveolar bone apoVs represent apoptotic vesicles of alveolar bone tissue origin, and gingival apoVs represent apoptotic vesicles of gingival tissue origin.
FIG. 4 is a graph of nanoparticle trace analysis detection results of apoptotic vesicles of different tissue origin; fat apoVs represent apoptotic vesicles of adipose tissue origin, alveolar bone apoVs represent apoptotic vesicles of alveolar bone tissue origin, and gingival apoVs represent apoptotic vesicles of gingival tissue origin.
Fig. 5 shows the results of a vesicle marker western blot of apoptotic vesicles of different tissue origin, fig. 5A shows the electrophoresis pattern of vesicle markers CD9 and CD81 of adipose tissue and adipose apoVs (apoptotic vesicles of adipose tissue origin), fig. 5B shows the electrophoresis pattern of vesicle markers CD9 and CD81 of alveolar bone tissue and alveolar bone apoVs (apoptotic vesicles of alveolar bone origin), and fig. 5C shows the electrophoresis pattern of vesicle markers CD9 and CD81 of gingival tissue and gingival apoVs (apoptotic vesicles of gingival tissue origin).
FIG. 6 is a graph showing the results of CCK8 experiments to detect the effect of apoptotic vesicles of different tissue sources on the activity of rat bone marrow mesenchymal stem cells; FIG. 6A is the effect of varying concentrations (0/0.01/0.1/1/10. Mu.g/mL) of fatty apoVs (adipose tissue-derived apoptotic vesicles) on rat bone marrow mesenchymal stem cell activity over time; FIG. 6B is the effect of varying concentrations (0/0.01/0.1/1/10. Mu.g/mL) of alveolar bone apoVs (apoptotic vesicles derived from alveolar bone tissue) on rat bone marrow mesenchymal stem cell activity over time; FIG. 6C is the effect of different concentrations (0/0.01/0.1/1/10. Mu.g/mL) of gingival apoVs (apoptotic vesicles derived from gingival tissue) on the activity of rat bone marrow mesenchymal stem cells over time.
FIG. 7 is a graph of ALP staining and ARS staining results of the effects of apoptotic vesicles of different tissue origin on the osteogenic differentiation of rat bone marrow mesenchymal stem cells; FIG. 7A is an ALP staining and ARS staining for the detection of the effects of different concentrations (1/10/100 ng/mL) of fatty apoVs (adipose tissue-derived apoptotic vesicles) on bone marrow mesenchymal stem cell osteodifferentiation in rats; FIG. 7B is an ALP staining and ARS staining for the detection of the effects of different concentrations (1/10/100 ng/mL) of alveolar bone apoVs (apoptotic vesicles derived from alveolar bone tissue) on bone differentiation of rat bone marrow mesenchymal stem cells; FIG. 7C is an ALP staining and ARS staining of different concentrations (1/10/100 ng/mL) of gingival apoVs (apoptotic vesicles derived from gingival tissue) on the osteogenic differentiation of rat bone marrow mesenchymal stem cells.
FIG. 8 shows the results of a cell scoring experiment for detecting apoptotic vesicles of different tissue origin on skin fibroblasts; FIG. 8A is the effect of varying concentrations (0/1/10/100 ng/mL) of fatty apoVs (adipose tissue-derived apoptotic vesicles) on skin fibroblast migration rate over time; FIG. 8B is the effect of varying concentrations (0/1/10/100 ng/mL) of alveolar bone apoVs (apoptotic vesicles derived from alveolar bone tissue) on the migration rate of dermal fibroblasts over time; FIG. 8C is the effect of varying concentrations (0/1/10/100 ng/mL) of gingival apoVs (apoptotic vesicles from gingival tissue) on the migration rate of dermal fibroblasts over time.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent manufacturers.
Example 1 extraction procedure and characterization of tissue-derived apoptotic vesicles
As shown in the flow chart of fig. 1-2, the steps for extracting tissue-derived apoptotic vesicles are as follows: after the rat was sacrificed, rat tissues (such as abdominal adipose tissue, gingival tissue, alveolar bone tissue, etc.) were removed using scissors, washed with sterile PBS multiple times, the tissues were minced and transferred to a petri dish or flask, and apoptosis induction was performed in a cell incubator by adding a-MEM medium (Aqlabtech, AQ 12571) containing Staurosporine (STS, apex bio, a 8192) to the culture dish or flask, and adding a volume ratio of tissue to medium of 1:10-1:20. after 12 hours of induction, the tissue supernatant was collected by filtration using a 40 μm cell filter. The tissue-derived apoptotic vesicles were then obtained by gradient centrifugation.
The gradient centrifugation method comprises the following steps: centrifuging the supernatant collected after filtration at 800g and 4deg.C for 10min (first centrifugation), and collecting supernatant; centrifuging the supernatant collected after the first centrifugation at 2000g at 4deg.C for 10min (second centrifugation), and collecting the supernatant; centrifuging the supernatant collected after the second centrifugation at 16000g and 4deg.C for 30min (third centrifugation), and collecting precipitate; and washing the precipitate collected after the third centrifugation with sterile PBS, and centrifuging at 16000g and 4 ℃ for 30min to obtain the tissue-derived apoptotic vesicles.
The morphology and particle size of the tissue-derived apoptotic vesicles were observed by transmission electron microscopy, the concentration was detected by nanoparticle tracking analysis, and the expression of vesicle markers CD9 and CD81 was detected by western blot.
As shown in fig. 3, apoptotic vesicles of three different tissue sources (fat, alveolar bone, gum) all had a biconcave discoid shape.
As shown in fig. 4, the apoptotic vesicles of adipose tissue origin had a particle size of 218.8+/-2.8nm; the particle size of the apoptotic vesicles derived from alveolar bone tissue is 201.0+/-10.9nm; the particle size of apoptotic vesicles derived from gingival tissue was 181.1+/-7.4nm.
As shown in fig. 5, the vesicle markers CD9 and CD81 were expressed in low amounts in adipose tissue, alveolar bone tissue, and gingival tissue, while they were expressed in high amounts in apoptotic vesicles extracted from all three tissues, indicating that this example successfully extracted apoptotic vesicles from adipose tissue, alveolar bone tissue, and gingival tissue.
Example 2 in vitro detection of the modulation of tissue-derived apoptotic vesicles on rat bone marrow mesenchymal Stem cell Activity
By BCA kit (Pierce) TM BCA Protein Assay, thermo scientific, 23228) detects the concentration of apoptotic vesicles of different tissue origin and sets up different concentration groups, in particular 6 concentration groups: 0 mug/mL,Cell proliferation assay was performed on rat bone marrow mesenchymal stem cells at 0.01. Mu.g/mL, 0.1. Mu.g/mL, 1. Mu.g/mL, 10. Mu.g/mL, and cell activity was assayed by CCK8 assay 1 to 7 days after inoculation.
As shown in FIG. 6, over time, the apoptotic vesicles derived from adipose tissue had no significant effect on cell activity at concentrations ranging from 0.01 to 1. Mu.g/mL, while the cell activity was significantly inhibited at concentrations up to 10. Mu.g/mL. The apoptosis vesicle of alveolar bone source has no obvious effect on cell activity in the concentration range of 0.01-0.1 mug/mL, and when the concentration exceeds 1 mug/mL, the apoptosis vesicle has obvious inhibition on cell activity. The apoptosis vesicle of gum source has no obvious effect on cell activity in the concentration range of 0.01-1 mug/mL, and has obvious inhibition on cell activity in the concentration of 10 mug/mL.
Example 3 in vitro detection of the modulation of osteogenic differentiation of tissue-derived apoptotic vesicles on rat bone marrow mesenchymal Stem cells
After the primary bone marrow stem cells of the rat were extracted, the rat was sacrificed by anesthesia, the femur was isolated, the bone marrow cavity was flushed out using a medium after cutting, after culture passage, the bone marrow stem cells of the rat were inoculated into an orifice plate, and normal proliferation culture (PM, a-MEM medium containing 10% FBS and 1% neo-streptomycin diabody), osteoinduction culture (OM, a-MEM medium containing 10% FBS, 1% neo-streptomycin diabody, 10nM dexamethasone, 0.2mM ascorbic acid, 10mM beta-sodium glycerophosphate) and tissue-derived apoptotic vesicles (OM+apo) were added under osteoinduction culture conditions (Vs) were set at three concentrations of 1ng/mL, 10ng/mL, 100ng/mL, and alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining were performed after 7 days and 14 days, respectively.
As shown in fig. 7, the ALP staining results showed that the ALP staining of the mesenchymal stem cells of the rat was deepened compared to the control group after the apoptotic vesicles derived from the adipose tissue, the alveolar bone and the gum tissue were added, the ARS staining results showed that the ARS staining degree of the mesenchymal stem cells of the rat was significantly deepened compared to the control group after the apoptotic vesicles derived from the adipose tissue, the alveolar bone and the gum tissue were added, indicating that the apoptotic vesicles derived from the adipose tissue, the alveolar bone and the gum had an accelerating effect on the osteogenic differentiation of the mesenchymal stem cells of the rat bone marrow in a safe concentration range of 1 to 100ng/mL.
Example 4 in vitro detection of the modulation of cell migration of skin fibroblasts by tissue-derived apoptotic vesicles
Human skin fibroblasts (scientific) were seeded in well plates, when cell density reached 100%, scratches were made, and cells were continued to be cultured using serum-free or low serum (FBS < 2%) medium (α -MEM medium containing no FBS or < 2% FBS and 1% neo-streptomycin diab). Different concentrations (0 ng/mL, 1ng/mL, 10ng/mL, 100 ng/mL) of apoptotic vesicles derived from adipose tissue, alveolar bone and gum were added to the culture medium, cells were taken out after 0h, 24h and 48h, photographed under a microscope, and the cell migration rate was calculated according to the width of the scratch.
As shown in fig. 8, the apoptotic vesicles derived from adipose tissue, alveolar bone and gum all promote the migration ability of skin fibroblasts within a certain concentration range after adding the vesicles derived from three tissues at different concentrations. The apoptotic vesicles from adipose tissue can promote the migration capacity of skin fibroblasts at the concentration of 1ng/mL to 100ng/mL, wherein the concentration promotion effect of 1ng/mL is more obvious; the apoptotic vesicles from the alveolar bone can promote the migration capacity of skin fibroblasts at the concentration of 1-100ng/mL, wherein the concentration promotion effect of 1ng/mL is more remarkable; the apoptotic vesicles from gum source can promote the migration capacity of skin fibroblasts at the concentration of 1-100ng/mL, wherein the concentration of 10ng/mL has more remarkable promotion effect.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (3)
1. An application of an apoptosis vesicle derived from animal tissues in preparing a medicament for promoting bone marrow mesenchymal stem cell osteogenic differentiation, which is characterized in that the preparation method of the apoptosis vesicle comprises the following steps of;
(1) Separating and shearing animal tissue;
(2) Inducing apoptosis in the tissue sheared in step (1) in a staurosporine-containing medium;
(3) Collecting the tissue supernatant induced in the step (2), separating by a gradient centrifugation method to obtain the apoptotic vesicles from the tissue,
wherein the animal tissue is gingival tissue of a rat, and the apoptosis vesicle is an apoptosis microvesicle with the particle size smaller than 1 mu m.
2. The use according to claim 1, wherein the medium in step (2) is an alpha-MEM medium containing staurosporine and the time to induce apoptosis is 12h.
3. The use according to claim 1, wherein the gradient centrifugation in step (3) is performed as follows:
(1) Centrifuging at 4deg.C for 10min at 800g, and collecting supernatant;
(2) Centrifuging the supernatant obtained in the step (1) at 2000g and 4 ℃ for 10min, and collecting the supernatant;
(3) Centrifuging the supernatant obtained in the step (2) at 16000g and 4 ℃ for 30min, and collecting the precipitate;
(4) Washing the precipitate obtained in the step (3) with sterile PBS, and centrifuging at 16000g and 4deg.C for 30min to obtain tissue-derived apoptotic vesicles.
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| JP7340240B2 (en) * | 2019-08-26 | 2023-09-07 | 学校法人関西医科大学 | Method of inducing apoptosis in cells or tissues |
| CN117965431A (en) * | 2020-01-20 | 2024-05-03 | 医微细胞生物技术(广州)有限公司 | Vesicle and application thereof |
| CN113952362A (en) * | 2020-07-06 | 2022-01-21 | 医微细胞生物技术(广州)有限公司 | Use of induced extracellular vesicles for the preparation of a preparation for prolonging the lifespan of a mammal or for treating or preventing aging |
| CN113969304A (en) * | 2020-07-24 | 2022-01-25 | 医微细胞生物技术(广州)有限公司 | Use of extracellular vesicles in the manufacture of a medicament for the treatment or prevention of metabolic inflammatory syndrome |
| AU2022260542A1 (en) * | 2021-04-23 | 2023-10-19 | Avulotion Ab | Extracellular vesicles from mesenchymal stromal cells for treatment of diseases |
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