CN115948329A - Extraction method and application of exosome of myocardial cells and myocardial inflammatory cells - Google Patents
Extraction method and application of exosome of myocardial cells and myocardial inflammatory cells Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to a method for extracting exosomes of myocardial cells and myocardial inflammatory cells and application thereof. The method adopts a mode of applying lipopolysaccharide (or lipopolysaccharide combined with adiponectin) and screens and optimizes induction conditions, so that myocardial cells are successfully induced to be converted into myocardial inflammatory cells, and exosomes in cell culture supernatant are extracted by adopting a mode of combining a differential centrifugation method and an ultrafiltration method, so that the obtained exosome extract has the advantages of high yield, high purity and the like. Meanwhile, the technical scheme further cultures the myocardial cells and myocardial inflammatory cells through optimized adiponectin addition concentration and addition time, so that the yield and purity of exosomes can be obviously improved. Therefore, subsequent research on the content and the function of the exosome is facilitated, a corresponding exosome identification method is provided, a methodological basis is provided for the subsequent research on the action mechanism of the exosome in myocardial inflammation, and therefore the exosome identification method has good value of practical application.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a method for extracting exosomes of myocardial cells and myocardial inflammatory cells and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The exosome is an extracellular vesicle with the diameter of 40-160nm and a lipid bilayer membrane structure, which is produced by living cells and can be widely present in various body fluids and cell culture supernatants. Exosomes contain various bioactive substances such as nucleic acids, proteins, lipids, and the like, and play an important role in intercellular communication. It has been shown that in the pathophysiological processes of various diseases, the content of exosomes and their contents are altered to promote or inhibit disease progression. In recent years, with the intensive research on exosomes, the roles of exosomes in resisting inflammation, improving ventricular remodeling, promoting angiogenesis and the like in cardiovascular diseases such as atherosclerosis, acute myocardial infarction, heart failure and the like are receiving more and more attention from students.
Myocarditis is life-threatening myocarditis caused by various causes and can cause serious consequences such as cardiac dysfunction and arrhythmia, but the pathogenesis of myocarditis is still unclear at present, and whether exosomes play a role in myocarditis and how the exosomes play a role are needed to be researched.
The appropriate purification methods such as exosome extraction play an important role in subsequent content analysis and function research, and scholars have studied the collection and purification methods of cardiovascular exosomes such as endothelial cells and cardiac progenitor cells, but the inventor finds that the extraction and purification methods of exosomes derived from human cardiomyocytes and myocardial inflammatory cells are not reported yet.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor provides a method for extracting exosomes of myocardial cells and myocardial inflammatory cells and application thereof through long-term technical and practical exploration. The method generates myocardial inflammatory cells through lipopolysaccharide induction, extracts exosomes in cell culture supernatant by adopting a mode of combining a differential centrifugation method and an ultrafiltration method, and provides a methodological basis for the subsequent research of an action mechanism of the exosomes in myocardial inflammation. The present invention has been completed based on the above results.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for extracting exosomes of cardiomyocytes and cardiomyocyte inflammatory cells, the method comprising:
s1, culturing cardiac muscle cells and inducing the generation of cardiac muscle inflammatory cells;
s2, inducing successful myocardial inflammatory cells to continue culturing, collecting a culture medium, and centrifuging to obtain a supernatant I;
s3, performing high-speed centrifugation on the supernatant I obtained in the step S2, and collecting a supernatant II;
and S4, filtering the supernatant II collected in the S3 into an ultracentrifuge tube through a filter, ultracentrifuging, and obtaining a precipitate which is an exosome after centrifuging.
The exosome in the cell culture supernatant is extracted by adopting a mode of combining a differential centrifugation method and an ultrafiltration method, the obtained exosome has high purity and less impurities, and the subsequent exosome content and function research is facilitated.
In addition, although the extraction of exosome cells has been specifically described in the above extraction method taking myocardial inflammatory cells obtained by lipopolysaccharide induction as an example, the above extraction method is also applicable to extraction of exosomes of normal myocardial cells, and the difference is only that the step of using myocardial inflammatory cells obtained by lipopolysaccharide induction is not used.
In a second aspect of the present invention, there is provided exosomes extracted by the above method for extracting exosomes of cardiomyocytes and cardiomyocytes.
In a third aspect of the invention, the exosome is provided for application in basic research and clinical diagnosis and treatment of cardiovascular related diseases.
Compared with the prior art, one or more technical schemes have the following beneficial effects:
the technical scheme reports that the method for extracting the exosomes of the myocardial cells and the myocardial inflammatory cells successfully induces the myocardial cells to be converted into the myocardial inflammatory cells by adopting a mode of applying lipopolysaccharide (or lipopolysaccharide combined with adiponectin) and screening and optimizing induction conditions, and the exosomes in cell culture supernatant are extracted by adopting a mode of combining a differential centrifugation method and an ultrafiltration method, so that the obtained exosome extract has the advantages of high yield, high purity and the like.
The technical scheme further cultures the myocardial cells and myocardial inflammatory cells through optimized adiponectin addition concentration and addition time, so that the yield and purity of exosomes can be obviously improved. Therefore, subsequent research on the content and the function of the exosome is facilitated, a corresponding exosome identification method is provided, a methodological basis is provided for the subsequent research on the action mechanism of the exosome in myocardial inflammation, and therefore the exosome identification method has good value of practical application.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph of exosome particle concentration under each stimulation condition in example 1 of the present invention.
FIG. 2 is a graph showing the observation of the culture of cardiomyocytes and cardiomyocytes in example 1 of the present invention; wherein A is an observation picture of growth of the myocardial cells before starvation treatment; b is the growth condition of the myocardial cells after starvation for 8 hours; c is control group myocardial cells added with PBS; d is myocardial inflammatory cells after being stimulated for 30 hours by adding LPS. FIG. 3 is a representative diagram of exosomes observed by transmission electron microscopy in example 1 of the present invention; wherein A is the form under the electron microscope of the exosome of the control group, and B is the form under the electron microscope of the exosome of the inflammation group.
FIG. 4 is a statistical chart of the exosome particle size distribution in example 1 of the present invention; wherein, A is a statistical chart of the grain size distribution of exosomes of a control group, and B is a statistical chart of the grain size distribution of exosomes of an inflammation group.
FIG. 5 is a representative diagram of Western detection of the exosome-specific proteins CD63 and TSG101, calnexin in example 1 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, suitable purification methods such as exosome extraction play an important role in subsequent content analysis and function research, and researchers have studied the collection and purification methods of cardiovascular exosomes such as endothelial cells and cardiac progenitor cells, but no report has been found on the extraction and purification methods of exosomes derived from human cardiomyocytes and myocardial inflammatory cells.
In view of the above, in an exemplary embodiment of the present invention, a method for extracting exosomes of cardiomyocytes and cardiomyocyte inflammatory cells is provided, the method comprising:
s1, culturing cardiac muscle cells and inducing the generation of cardiac muscle inflammatory cells; wherein the method for inducing and generating myocardial inflammatory cells comprises adding lipopolysaccharide of 10-20 μ g/ml and culturing for 24-48h to induce and generate myocardial inflammatory cells; and adding adiponectin at a concentration of 10-25ng/ml 0-8 hours after the addition of lipopolysaccharide;
s2, inducing the successful myocardial inflammatory cells to continue culturing, collecting a culture medium, and centrifuging to obtain a supernatant I;
s3, performing high-speed centrifugation on the supernatant I obtained in the step S2, and collecting a supernatant II;
and S4, filtering the supernatant II collected in the S3 into an ultracentrifuge tube through a filter, ultracentrifuging, and obtaining a precipitate which is an exosome after centrifuging.
In yet another embodiment of the present invention,
in the step S1, the cardiomyocyte is an AC16 cardiomyocyte, which is a cardiomyocyte line isolated from normal human ventricular tissue and immortalized;
the specific method of the step S1 comprises the following steps: the specific method for culturing the myocardial cells comprises the following steps: the complete culture medium containing 10% fetal calf serum is selected to culture the myocardial cells, and the culture conditions are as follows: 30-40 deg.C, 3-8% of CO 2 (preferably 37 ℃,5% 2 ) When the cell growth density reaches about 50%, the culture medium without serum is replaced for further 8-12 hours, 10-20 mu g/ml lipopolysaccharide is added for culture for 24-48 hours (preferably 20 mu g/ml lipopolysaccharide for culture for 30 hours) to induce the generation of myocardial inflammatory cells, and 10-25ng/ml adiponectin (preferably 15ng/ml adiponectin) is added 0-8 hours (preferably 4-6 hours) after the addition of lipopolysaccharide, so that the extracellular secretion yield is increased.
Meanwhile, in order to prevent the original exosomes in the fetal calf serum from interfering with the extraction method, fetal calf serum cells without exosomes need to be cultured. Preparation conditions of the secretion-removed fetal calf serum: serum and medium 1:4 after mixing, centrifugation at 100000g for 18 hours at 4 ℃.
The Lipopolysaccharide (LPS) selected in the invention is an endotoxin in the cell wall of gram-negative bacilli, the LPS can directly damage receptor cells after infection, or can be used as an important antigen molecule to be captured by an antigen presenting cell (apc) so as to cause the immune response of an organism, and the LPS is adopted to induce and generate myocardial inflammatory cells, so that the direct influence of the myocardial inflammatory cells on the myocardial cells can be evaluated, and signal molecules generated by the myocardial cells after infection can be obtained, and the information transmission and the signal exchange among cells in the inflammatory process can be researched. When lipopolysaccharide is added, the addition concentration and the addition time of the lipopolysaccharide are optimized, so that the apoptosis proportion of apoptotic cells is controlled, and experiments prove that the LPS concentration of 20 mu g/ml induces the stimulation condition for 30 hours, which is the optimal extraction condition of the exosomes of the myocardial inflammatory cells. Adiponectin, as a cardioprotective factor, plays a role in immune regulation and anti-inflammatory action by regulating signal pathways such as AMPK. Therefore, the inventors speculate that the addition of adiponectin during the culture of inflammatory cells promotes the production of extracellular exosomes, however, the present inventors found through research that the addition time of adiponectin had a significant effect on the amount of exosomes added, that the addition of adiponectin 2 hours after 20 μ g/ml LPS stimulates cardiomyocytes produced a significantly smaller total amount of exosomes than other periods, and that the addition of adiponectin 15ng/ml 4-6 hours after LPS stimulates cardiomyocytes produced the most exosomes.
In another embodiment of the present invention, in the step S2, a specific centrifugation method includes: centrifuging the collected culture medium at 200-500g (preferably 300 g) at low temperature (such as 4 deg.C) for 5-20min (preferably 10 min) to remove suspended cells in the culture medium, centrifuging at 1500-2500g (preferably 2000 g) at low temperature (such as 4 deg.C) for 5-20 (preferably 10 min) to remove dead cells, and collecting supernatant I;
in another embodiment of the present invention, in the step S3, a specific high-speed centrifugation method includes: centrifuging at 8000-10000g at low temperature (such as 4 deg.C) for 20-50 min (preferably 30 min) to further remove cell debris;
in another embodiment of the present invention, the step S4 includes: filtering the collected supernatant II through a 0.22 mu m filter membrane into an ultracentrifuge tube, removing macromolecular substances with the diameter of more than 0.22 mu m in the supernatant, ultracentrifuging the filtrate at 90000-120000g (preferably 100000 g) at low temperature (such as 4 ℃) for 60-80min (preferably 70 min), discarding the supernatant, leaving 0.5-1mL of liquid, collecting the liquid in a plurality of centrifuge tubes after blowing and evenly mixing so as to enrich exosomes of the same source, and ultracentrifuging again at 90000-120000g (preferably 100000 g) at low temperature (such as 4 ℃) for 60-80min (preferably 70 min), wherein the obtained precipitate is the exosomes.
According to the invention, the exosome in the cell culture supernatant is extracted by adopting a mode of combining a differential centrifugation method and an ultrafiltration method, the obtained exosome has high purity and less impurities, and the subsequent exosome content and function research can be conveniently carried out.
In addition, although the extraction of exosome cells has been specifically described in the above extraction method using lipopolysaccharide-induced myocardial inflammatory cells as an example, the above extraction method is equally applicable to extraction of exosomes of normal myocardial cells, and is different only in that a step of using lipopolysaccharide (or lipopolysaccharide combined with adiponectin) -induced myocardial inflammatory cells is not used.
In another embodiment of the present invention, the extraction method further comprises the step of identifying the obtained cardiomyocytes and exosomes of the cardiomyocytes and the cardiomyocyte inflammatory cells, wherein the identifying step includes, but is not limited to, evaluating the morphology of the extracted exosomes by using a transmission electron microscope, verifying the exosome surface marker proteins CD63 and TSG101 by using Western blot, and evaluating the size and the number of the obtained exosomes by using a nanoparticle tracking analysis technology.
In another embodiment of the present invention, the exosome is extracted by the method for extracting an exosome from a cardiomyocyte and a cardiomyocyte.
In another embodiment of the present invention, the use of the above exosome in the basic research and clinical diagnosis and treatment of cardiovascular-related diseases is provided.
The present invention is further illustrated by the following specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental specific conditions not noted in the examples, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; the present invention is not particularly limited, and may be commercially available.
Example 1 method for extracting exosomes from cardiac muscle cells and cardiac muscle inflammatory cells (first) preliminary experiment screening optimal stimulation conditions for extraction of exosomes from cardiac muscle inflammatory cells
Lipopolysaccharide (LPS) with the concentration of 10 mu g/ml and 20 mu g/ml can induce and generate a myocardial inflammatory cell model after being stimulated for 24-48 hours, so the experiment designs two concentration gradients of 10 mu g/ml and 20 mu g/ml and five time gradients of 24, 30, 36, 42 and 48, and 10 groups are respectively cultured and induced to generate the inflammatory cell model, and the markers are respectively as follows: 24-1 (24 hours of LPS concentration induction at 10. Mu.g/ml), 24-2 (24 hours of LPS concentration induction at 20. Mu.g/ml), 30-1 (30 hours of LPS concentration induction at 10. Mu.g/ml), 30-2 (30 hours of LPS concentration induction at 20. Mu.g/ml), 36-1 (36 hours of LPS concentration induction at 10. Mu.g/ml), 36-2 (36 hours of LPS concentration induction at 20. Mu.g/ml), 42-1 (42 hours of LPS concentration induction at 10. Mu.g/ml), 42-2 (42 hours of LPS concentration induction at 20. Mu.g/ml), 48-1 (48 hours of LPS concentration induction at 10. Mu.g/ml), and 48-2 (48 hours of LPS concentration induction at 20. Mu.g/ml). The percentage of apoptosis was determined by flow cytometry and the results were as follows: 6.3 percent of 24-1 apoptotic cells, 7.8 percent of 24-2 apoptotic cells, 8 percent of 30-1 apoptotic cells, 7.8 percent of 30-2 apoptotic cells, 8.3 percent of 36-1 apoptotic cells, 19.8 percent of 36-2 apoptotic cells, 15.5 percent of 42-1 apoptotic cells, 20 percent of 42-2 apoptotic cells, 17.6 percent of 48-1 apoptotic cells and 22 percent of 48-2 apoptotic cells. Screening culture conditions with the apoptosis percentage of less than 10 percent, selecting five culture conditions, namely 24-1, 24-2, 30-1, 30-2 and 36-1 respectively, inducing to generate myocardial inflammatory cells according to the five induction conditions respectively, collecting 100ml of culture supernatant respectively, extracting exosomes by a differential centrifugation method, detecting the concentration of exosome particles by NTA (nitrilotriacetic acid), and combining the proportion of the apoptotic cells in cell culture to obtain the stimulation condition of inducing the LPS concentration for 30 hours at 20 mu g/ml, wherein the stimulation condition is the optimal extraction condition of the myocardial inflammatory cell exosomes.
(II) culturing myocardial cells and inducing the production of myocardial inflammatory cells
Human cardiomyocytes (AC 16 cells, a cardiomyocyte line isolated from normal human ventricular tissue and immortalized, purchased from Qiao Xin Biotech, inc., shanghai) were cultured in T75 cell (Corning Inc) flasks, cultured in high-glucose medium containing 10% fetal bovine serum + DMEM, and cultured at 37 ℃ and 5% CO 2 In the incubator. When the cells grew to about 80% of the bottom of the flask, 0.25% pancreatin (GIBICO) digested cells, passaged at 1:3 for 24 hours, i.e., when the cell growth density reached about 50%, see FIG. 2A, the serum-free medium was replaced and cultured for 8 hours, see FIG. 2B, and Lipopolysaccharide (LPS) was added to the inflammation group at 20. Mu.g/ml to induceMyocardial inflammatory cells were generated and the control group was dosed with the same dose of PBS.
(III) exosome collection
After adding LPS to culture for 30 hours, the cell growth conditions are shown in figures 2C and 2D, wherein figure 2C is the cell growth condition of a control group, figure 2D is the cell growth condition of an LPS stimulation group, a culture medium is collected, 150mL of the culture medium is collected from an inflammation group and the control group respectively, centrifugation is carried out for 10min at the temperature of 4 ℃ and 300g, and suspended cells in the culture medium are removed; centrifuging at 4 deg.C for 10min at 2000g, removing dead cells, and collecting supernatant; centrifugation at 10000g for 30 minutes at 4 ℃ removed cell debris and the supernatant was carefully collected.
Filtering the collected supernatant into an ultracentrifuge tube through a 0.22 mu m filter membrane, removing macromolecular substances with the diameter larger than 0.22 mu m in the supernatant, ultracentrifuging the filtrate for 70min at 100000g at 4 ℃, carefully removing the supernatant, reserving 0.5-1mL of liquid, blowing and uniformly mixing, collecting the liquid in a plurality of centrifuge tubes into the same centrifuge tube so as to enrich the exosomes from the same source, and ultracentrifuging for 70min again at 100000g at 4 ℃, wherein the obtained precipitate is the exosomes.
The exosomes were resuspended in 200. Mu.l PBS and stored at 4 ℃ for a short period or-80 ℃ for a long period.
(IV) exosome identification
The morphology of the extracted exosomes is observed by a transmission electron microscope, and fig. 3 shows the size and morphology results of the exosomes observed by the transmission electron microscope. Wherein FIG. 3A is exosome derived from cardiomyocytes in a normal control group, and FIG. 3B is exosome derived from cardiomyocyte inflammatory cells. The transmission electron microscope result of the exosome shows that the size of the exosome is between 40 and 160nm, and the capsule-like ultramicrostructure is clear and visible and accords with the morphological characteristics of the exosome.
The number and the size of exosomes are measured by adopting a nanoparticle tracking analysis technology (NTA), and the particle concentration and the particle size distribution of exosomes are analyzed by using ZataView8.04.02 software. FIG. 4A shows that the average particle size of the control group is 144.2 + -1.82 nm by the statistical analysis of particle sizes, and FIG. 4B shows that the average particle size of the exosome of the LPS inflammation stimulation group is 137.8 + -0.57 nm by the statistical analysis of particle sizes.
Western blotting is adopted to detect the expression conditions of the exosome surface specific proteins CD63 and TSG 101. Total protein was first extracted with RIPA, separated by 10% sdds-polyacrylamide gel electrophoresis, electrotransferred to PVDF membrane, 5% skim milk powder was blocked at room temperature, and after shaking overnight at 4 ℃, primary anti-1.
Table 1 exosome particle concentration (particles/ml) and apoptotic cell ratio (%)' under each stimulation condition in example 1
| Group of | Particle concentration (particles/ml) | Proportion of apoptotic cells (%) |
| 24-1 | 6.9×10 10 | 6.3 |
| 24-2 | 2.4×10 11 | 7.8 |
| 30-1 | 9.5×10 10 | 8.0 |
| 30-2 | 2.6×10 11 | 7.8 |
| 36-1 | 1.2×10 11 | 8.3 |
Example 2
Culturing myocardial cells and inducing the production of myocardial inflammatory cells
Human cardiomyocytes (AC 16 cells, a cardiomyocyte line isolated from normal human ventricular tissue and immortalized, purchased from Qiao Xin Biotech, inc., shanghai) were cultured in T75 cell (Corning Inc) flasks, cultured in high-glucose medium containing 10% fetal bovine serum + DMEM, and cultured at 37 ℃ and 5% CO 2 In the incubator. When the cells grow to about 80% of the bottom of the culture bottle, 0.25% pancreatin (GIBICO) digests the cells, the cells are passaged according to 1:3 for 24 hours, after the passage, namely when the cell growth density reaches about 50%, the serum-free culture medium is replaced for 8 hours, 20 mu g/ml Lipopolysaccharide (LPS) is added into an inflammation group, myocardial inflammation cells are induced to generate, and adiponectin with the concentration of 10ng/ml is added for 30 hours.
Example 3
The difference from example 2 is that the adiponectin concentration is different, and the adiponectin concentration added simultaneously in this example is 15ng/ml.
Example 4
The difference from example 2 is that the adiponectin concentration is different, and the adiponectin concentration added simultaneously in this example is 20ng/ml.
Example 5
The difference from example 2 is that the adiponectin concentration is different, and the adiponectin concentration added simultaneously in this example is 25ng/ml.
TABLE 2 exosome particle concentration (particles/ml) and main peak particle size (nm) for the inflammation group of each example
| Group of | Particle concentration (particles/ml) | Major peak particle size (nm) |
| Example 1 | 2.4×10 11 | 142.0 |
| Example 2 | 3.1×10 11 | 144.0 |
| Example 3 | 4.2×10 11 | 140.7 |
| Example 4 | 4.0×10 11 | 141.5 |
| Example 5 | 3.9×10 11 | 140.0 |
As can be seen from examples 1-5, the addition of adiponectin was found to increase the exosome yield of myocarditis cells in the inflammatory group, and the particle concentration was increased, with the highest exosome yield with adiponectin added at 15ng/ml (i.e., example 3).
Example 6
To further investigate whether adiponectin addition time affects secretion of exosomes by inflammatory cardiomyocytes, adiponectin was added at 0 hr (i.e., example 3), 2 hr, 4 hr, 6 hr, and 8 hr after LPS stimulation with 20 μ g/ml, respectively, and the results are shown in table 3, and addition of adiponectin at different time periods increases the amount of exosomes produced by inflammatory cardiomyocytes, however, we found that adiponectin was added 2 hr after LPS stimulation, and the amount of exosomes produced by inflammatory cells was significantly reduced compared to the group. It has been shown that adiponectin, as a cardioprotective factor, can play a role in immune regulation and anti-inflammatory action by regulating signal pathways such as AMPK. Therefore, we speculate that the addition of adiponectin during the culture of inflammatory cells promotes the production of extracellular exosomes and simultaneously reduces the level of cellular inflammation, adiponectin is added 2 hours after the inflammatory cells are stimulated by LPS to produce the strongest anti-inflammatory effect, the cellular inflammation is reduced, and the total amount of exosomes produced is reduced compared with other groups because the number of exosomes produced by normal cells is obviously reduced compared with the number of exosomes produced by inflammatory cells, and the adiponectin is added 2 hours after the myocardial cells are stimulated by LPS.
TABLE 3 addition of adiponectin at 15ng/ml for different time periods, exosome particle concentration (particles/ml) and main peak particle size (nm) for the resulting inflammatory group
| Addition time (hours) | Particle concentration (particles/ml) | Major peak particle size (nm) |
| 0 | 4.2×10 11 | 140.7 |
| 2 | 3.5×10 11 | 135.8 |
| 4 | 6.0×10 11 | 137.7 |
| 6 | 5.9×10 11 | 146.7 |
| 8 | 4.7×10 11 | 139.0 |
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A method for extracting exosomes of myocardial cells and myocardial inflammatory cells, which is characterized by comprising the following steps:
s1, culturing cardiac muscle cells and inducing the generation of cardiac muscle inflammatory cells; wherein the method for inducing and generating myocardial inflammatory cells comprises adding lipopolysaccharide of 10-20 μ g/ml and culturing for 24-48h to induce and generate myocardial inflammatory cells; and adding adiponectin at a concentration of 10-25ng/ml 0-8 hours after the addition of lipopolysaccharide;
s2, inducing the successful myocardial inflammatory cells to continue culturing, collecting a culture medium, and centrifuging to obtain a supernatant I;
s3, performing high-speed centrifugation on the supernatant I obtained in the step S2, and collecting a supernatant II;
and S4, filtering the supernatant II collected in the S3 into an ultracentrifuge tube through a filter, ultracentrifuging, and obtaining a precipitate which is an exosome after centrifuging.
2. The extraction method according to claim 1, wherein in the step S1, the cardiomyocytes are AC16 cardiomyocytes.
3. The extraction method according to claim 1, wherein the step S1 specifically comprises: the specific method for culturing the myocardial cells comprises the following steps: the complete culture medium containing 10% fetal calf serum is selected to culture the myocardial cells, and the culture conditions are as follows: 30-40 deg.C, 3-8% of CO 2 When the cell growth density reaches 50%, replacing serum-free culture medium to continuously culture for 8-12 hours, and adding 10-20 microgram/ml lipopolysaccharide to culture for 24-48 hours to induce and generate myocardial inflammatory cells; and adding adiponectin at a concentration of 10-25ng/ml 4-6 hours after lipopolysaccharide addition.
4. The extraction method according to claim 3, wherein the fetal bovine serum is exosome-removed fetal bovine serum.
5. The extraction method according to claim 1, wherein in the step S2, the specific centrifugation method includes: centrifuging the collected culture medium at 200-500g for 5-20min at low temperature to remove suspended cells in the culture medium, centrifuging at 1500-2500g for 5-20min at low temperature to remove dead cells, and collecting supernatant I.
6. The extraction method according to claim 1, wherein in the step S3, the specific high-speed centrifugation method comprises: centrifuging at 8000-10000g for 20-50 min.
7. The extraction method according to claim 1, wherein the step S4 specifically includes: filtering the collected supernatant II into an ultracentrifuge tube through a 0.22 mu m filter membrane, removing macromolecular substances with the diameter larger than 0.22 mu m in the supernatant, ultracentrifuging the filtrate at low temperature of 90000-120000g for 60-80min, discarding the supernatant, reserving 0.5-1mL of liquid, blowing and uniformly mixing, collecting the liquid in a plurality of centrifuge tubes into the same centrifuge tube so as to enrich exosomes from the same source, and ultracentrifuging at low temperature of 90000-120000g for 60-80min again, wherein the obtained precipitate is the exosomes.
8. The extraction method according to claim 1, wherein the extraction method further comprises the step of identifying the obtained cardiomyocytes and cardiomyocyte exosomes, wherein the identification step includes but is not limited to the steps of assessing the morphology of the extracted exosomes by transmission electron microscopy, verifying the exosome surface marker proteins CD63, TSG101 by Western blot, and assessing the size and number of the obtained exosomes by nanoparticle tracking analysis.
9. The exosome extracted by the method for extracting exosomes of myocardial cells and myocardial inflammatory cells according to any one of claims 1 to 8.
10. The use of the exosome of claim 9 in the basic research and clinical diagnosis and treatment of cardiovascular-related diseases.
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