CN115109750A - Macrophage exosome and extraction method and application thereof - Google Patents

Macrophage exosome and extraction method and application thereof Download PDF

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CN115109750A
CN115109750A CN202210957255.1A CN202210957255A CN115109750A CN 115109750 A CN115109750 A CN 115109750A CN 202210957255 A CN202210957255 A CN 202210957255A CN 115109750 A CN115109750 A CN 115109750A
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macrophage
exosome
precipitate
muscle
exosomes
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罗小毛
王海东
冀云燕
张慧玲
任华伟
于秀菊
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Shanxi Agricultural University
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Abstract

The invention discloses a macrophage exosome and an extraction method and application thereof, and relates to the technical field of preparation of medicinal extracts. The macrophage exosome is obtained by collecting supernatant of a starvation cell culture medium and performing gradient centrifugation. The invention discloses an extraction method of macrophage exosome, which comprises the following specific steps: culturing macrophage, starving, collecting supernatant, low-speed and ultrahigh-speed centrifuging, and collecting the precipitate as macrophage exosome. The invention takes macrophage as raw material, collects cell culture supernatant, adopts density gradient centrifugation method to extract, and has simple and convenient operation method. The macrophage exosome provides a new way for treating muscle development and muscle-related diseases while promoting muscle injury repair.

Description

Macrophage exosome and extraction method and application thereof
Technical Field
The invention belongs to the technical field of preparation of medicinal extracts, and particularly relates to a macrophage exosome, and an extraction method and application thereof.
Background
Skeletal muscle, an important component of animal products, has a profound impact on the development of the animal husbandry economy. However, nowadays, the incidence of diseases related to muscular dysplasia and muscular atrophy is higher and higher, and the development of livestock products is seriously hindered. Therefore, techniques that improve the repair of muscle damage and promote its healthy development are of great economic value.
Exosomes are small vesicles containing lipids, specific proteins and genetic material released by cells, the special membrane structure of which is often used as a transport vehicle for transmitting information content of the original cell, loading nucleic acids or drugs. The application of exosomes to improve muscle repair processes and promote efficient application thereof is an important direction in the drug research and development industry.
Therefore, a method for extracting macrophage exosomes with high concentration is needed to meet the requirement of drug development.
Disclosure of Invention
The invention aims to provide a macrophage exosome and an extraction method and application thereof, which can carry out highly concentrated extraction on the macrophage exosome, and the extracted macrophage exosome can promote muscle injury repair and provide a new way for treating muscle development and muscle-related diseases.
In order to achieve the above purpose, the invention provides a macrophage exosome and an extraction method and application thereof, comprising the following steps:
culturing macrophage, starving for 20-30h, collecting supernatant, performing gradient centrifugation, filtering, and collecting precipitate;
the starvation treatment comprises the following steps: culturing macrophage to 70-80%, washing cell with PBS buffer solution for 1-3 times, and replacing serum-free culture medium for continuous culture.
Preferably, the gradient centrifugation treatment specifically comprises the following steps:
(1) collecting supernatant, centrifuging with 300g and 2000g centrifugal force for 8-12min, and discarding precipitate;
(2) continuously centrifuging the supernatant prepared in the step (1) for 28-32min by using a centrifugal force of 10000g, and then discarding the precipitate;
(3) continuously centrifuging the supernatant prepared in the step (2) for 55-65min by using a centrifugal force of 100000g, and collecting precipitates;
(4) and (4) resuspending the precipitate collected in the step (3) by 25-30mL of PBS buffer solution, centrifuging for 55-65min by using a centrifugal force of 100000g, and taking the precipitate.
The macrophage exosome is extracted by the extraction method of the macrophage exosome.
The macrophage exosome extracted by the invention is applied to the preparation of the medicine for regulating muscle injury and repairing.
The application of the macrophage exosome extracted by the invention in preparing medicaments for promoting muscle development and treating muscle-related diseases.
In summary, the invention has the following advantages:
1. the invention collects supernatant of a cell culture medium subjected to starvation treatment based on macrophages, obtains concentrated macrophage exosomes after gradient centrifugation, finds that the macrophage exosomes have the effect of accelerating skeletal muscle injury repair, and widens the application prospect for the macrophage exosomes.
2. Compared with the prior art, the method takes the macrophages as the basis, adopts a gradient centrifugation method to extract the concentrated exosomes, has simple and convenient operation steps, is cheap and efficient compared with extraction methods such as a kit and the like, and has the function of remarkably promoting muscle injury repair.
3. The macrophage exosome extracted by the invention can be used for preparing a medicine for promoting gastrocnemius muscle injury repair.
Drawings
FIG. 1 shows the results of transmission electron microscopy and NTA identification of macrophage exosomes;
FIG. 2 shows the identification of PKH67 markers for macrophage exosomes;
FIG. 3 shows the results of in vivo enrichment of macrophage exosomes;
FIG. 4 is a graph showing the effect of macrophage exosome treatment on the cross-sectional area of gastrocnemius fibers;
FIG. 5 is the result of the action of macrophage exosome therapy on the gastrocnemius production key gene;
FIG. 6 shows the effect of macrophage exosome therapy on myosatellite myogenic factor MyoD;
FIG. 7 shows the effect of macrophage exosome therapy on myosatellite myogenic factor MyoG.
Detailed Description
The principles and features of this invention are described below in conjunction with embodiments, which are included to explain the invention and not to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The gradient centrifugation in the invention not only comprises single low-speed centrifugation and ultrahigh-speed centrifugation, but also can be low-speed centrifugation or ultrahigh-speed centrifugation with the same or different centrifugation speeds in two times.
The centrifugal speeds in the invention are all defined by centrifugal force, the types of the applied centrifuges are eppendorf (centrifu 5702 RH), eppendorf (centrifu 5804R) and Himac (CP1OOWX), and the centrifugal radii are respectively 13.5cm, 12.30cm and 6.20cm (Rav).
Example 1
The embodiment provides an extraction method of macrophage exosomes, which comprises the following steps:
macrophages were cultured (including the following culture criteria): and taking out mouse mononuclear macrophage RAW264.7 from a liquid nitrogen tank, incubating in a water bath at 37 ℃, re-warming, sucking out the cell suspension to a 15mL centrifuge tube, centrifuging to remove supernatant, adding 10% high-sugar DMEM (DMEM) to resuspend cells, and culturing in a 5% 37 ℃ incubator. After the culture density of the macrophages reaches 70%, carrying out starvation treatment, including washing the cells for 2 times by using PBS (phosphate buffer solution), starving for 24 hours, and collecting cell supernatants for gradient centrifugation;
and (3) standing the supernatant at 4 ℃ overnight after low-speed centrifugation, carrying out ultrahigh-speed centrifugation the next day to obtain a precipitate, namely a mixture of the macrophage exosomes and the contaminating proteins, carrying out heavy suspension on the precipitate by 27mL of PBS, and carrying out ultrahigh-speed centrifugation for 60min to obtain the precipitate, namely the macrophage exosomes.
The centrifugal force of the low-speed centrifugation is 300g, the time of the low-speed centrifugation is 10min, the centrifugal force of the ultrahigh-speed centrifugation is 10000g, and the time of the ultrahigh-speed centrifugation is 60 min.
Example 2
The embodiment provides an extraction method of macrophage exosomes, which comprises the following steps:
culturing macrophages until the culture density of the macrophages reaches 80%, then carrying out starvation treatment, including washing the cells for 2 times by using PBS (phosphate buffer solution), carrying out starvation treatment for 24 hours, and further collecting cell supernatants for gradient centrifugation;
and (3) centrifuging the supernatant twice at different speeds at low speed, standing overnight at 4 ℃, centrifuging twice at different speeds twice at ultrahigh speeds the next day to obtain a precipitate, namely a mixture of the macrophage exosomes and the contaminating proteins, resuspending the precipitate with 27mL of PBS, and centrifuging at ultrahigh speed for 60min to obtain the precipitate, namely the macrophage exosomes.
The centrifugal force of the low-speed centrifugation is 300g and 2000g, the time of the low-speed centrifugation is 10min, the centrifugal force of the ultra-high-speed centrifugation is 10000g for the first time, and the centrifugation time is 30 min; the centrifugal force of the second ultra-high speed centrifugation is 100000g, and the centrifugation time is 60 min. The collected precipitate is a mixture of exosomes and contaminating proteins; and finally, resuspending the precipitate by 27mL of PBS, and centrifuging for 60min by using a centrifugal force of 100000g, wherein the precipitate is the macrophage exosome.
Test examples
Male 6-8 week-old C57BL/6 mice were selected and 0.1mL of sterile 1.2% BaCl was added 2 The solution was injected into the left gastrocnemius muscle of the mouse, or physiological saline was injected into the right gastrocnemius muscle of the mouse, and then exosomes of four macrophage sources (i.e., RAW-Ctrl-exo, RAW-KO-exo, RAW-OE-exo, and macrophage exosomes prepared in example 2) were injected into the ipsilateral gastrocnemius muscle of the injured mouse for treatment. The mouse was returned to the recovery cage until it was fully mobile.
At 12 h: in 12h light-dark cycle, the mice are raised at a constant temperature of 23 ℃, water and food can be freely obtained, the mice are anesthetized by ether after 7d and fixed on an operating table in a supine position, left gastrocnemius (or right gastrocnemius) of the mice is collected and fixed, and protein is extracted for application detection test.
Specifically, in the present test example, in order to verify the effect of the macrophage exosome of the present invention in promoting skeletal muscle injury repair, the mouse test was divided into a Control group (Control) and an injury group (BaCl) 2 ) An untreated exosome-lesion group (RAW-Ctrl-exo), a C/EBP- β knock-out exosome-lesion group (RAW-KO-exo), and a C/EBP- β overexpression exosome-lesion group (RAW-OE-exo).
Under the same feeding condition, BaCl with the volume fraction of 1.2 percent is used 2 Solution construction of a damaged mouse gastrocnemius muscle damage model, identification of macrophage exosomes extracted from various sources, and labeling of the exosomes by PKH 67. PBS and macrophage exosomes are respectively used as medicaments to carry out intramuscular injection treatment on mice with gastrocnemius muscle injury, and the treatment effect of different exosomes on the gastrocnemius muscle injury is observed.
As shown in figure 1, the detection of the particle size of the exosome by a transmission electron microscope shows that the exosome is in a double-layer membrane spherical vesicle structure, the size of the exosome meets the size of the exosome small vesicle, and the exosome has high purity.
As shown in fig. 2, confocal laser microscopy showed successful labeling of macrophage exosomes from various sources as green.
As shown in fig. 3, almost all of the exosomes were found to be enriched in the site of the damaged gastrocnemius muscle by the small animal imaging experiment, and the enrichment degree of the three exosome-treated groups differed significantly.
As shown in figure 4, after macrophage exosome treatment for 3d, 5d and 7d, HE staining is carried out on each group, and it is found that fibers in the 3d damaged area are polygonal, interstitial spaces of fibers are enlarged, the 5d muscle fiber damage condition is particularly obvious, and the 7d damaged muscle inflammation level is reduced, which indicates that BaCl 2 Approximately 80% of gastrocnemius injuries were severe after treatment. With BaCl 2 In comparison, the recovery of the distribution of the sizes of the gastrocnemius muscle fibers was substantially similar in the RAW-KO-exo-treated group, with 3d and 5d regenerated fibers present in the damaged muscle and co-localized with the distribution of inflammatory cells, and 7d still showed no significant re-growthA phenomenon occurs. While the 7d group treated with RAW-Ctrl-exo and RAW-OE-exo showed a large number of central nuclear fibers, and the diameter of regenerated muscle fibers in the RAW-OE-exo treated group was closer to the normal level, indicating that the RAW-OE-exo treated group had the best effect of promoting repair of gastrocnemius muscle injury (black arrows indicate inflammatory cells, gray indicates regenerated muscle fibers).
As shown in FIG. 5, it was found by expression analysis of myogenic key genes that MyoD and MYH3 were significantly up-regulated at 5d in the RAW-KO-exo treatment group, and MyoD and MYH3 were significantly increased at 3d in the RAW-Ctrl-exo and RAW-OE-exo treatment groups, and the expression levels were higher than those in the RAW-KO-exo treatment group, indicating that C/EBP-beta carried on macrophage exosomes could accelerate myogenic regeneration.
As shown in FIG. 6, it was further found by immunofluorescence assay of a section of gastrocnemius tissue that, together with BaCl 2 Compared with the group, the muscle satellite cells of the RAW-Ctrl-exo and RAW-KO-exo treatment groups are activated in a small amount, while the muscle satellite cells of the RAW-OE-exo treatment groups are activated and proliferated in a large amount, and the differentiation of myoblasts is also obviously enhanced as shown in figure 7, which indicates that the RAW-OE-exo treatment group accelerates the repair process of gastrocnemius,
in conclusion, the macrophage exosome extracted by the invention can be used for preparing a medicine for promoting gastrocnemius muscle injury repair.
While the present invention has been described in detail with reference to the specific embodiments thereof, it should not be construed as limited by the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims (5)

1. A method for extracting macrophage exosomes is characterized by comprising the following steps:
culturing macrophage, starving for 20-30h, collecting supernatant, performing gradient centrifugation, filtering, and collecting precipitate;
the starvation treatment comprises the following steps: culturing macrophage to 70-80%, washing cell with PBS buffer solution for 1-3 times, and replacing serum-free culture medium for continuous culture.
2. The method for extracting macrophage exosomes according to claim 1, wherein the gradient centrifugation treatment specifically comprises the following steps:
(1) collecting supernatant, centrifuging with 300g and 2000g centrifugal force for 8-12min, and discarding precipitate;
(2) continuously centrifuging the supernatant prepared in the step (1) for 28-32min by using a centrifugal force of 10000g, and then discarding the precipitate;
(3) continuously centrifuging the supernatant prepared in the step (2) for 55-65min by using a centrifugal force of 100000g, and collecting precipitates;
(4) and (4) resuspending the precipitate collected in the step (3) by 25-30mL of PBS buffer solution, centrifuging for 55-65min by using a centrifugal force of 100000g, and taking the precipitate.
3. The macrophage exosome extracted by the method for extracting macrophage exosome according to claim 1 or 2.
4. Use of macrophage exosomes according to claim 3 for the preparation of a medicament for modulating muscle damage repair.
5. Use of macrophage exosomes according to claim 3 for the preparation of a medicament for promoting muscle development and treating muscle-related disorders.
CN202210957255.1A 2022-08-10 2022-08-10 Macrophage exosome and extraction method and application thereof Pending CN115109750A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102215237B1 (en) * 2020-06-29 2021-02-15 (주)지에프씨생명과학 Method of isolating high yield exosomes from cell culture supernatants
CN113046316A (en) * 2021-04-07 2021-06-29 中南大学湘雅医院 M2 type bone marrow macrophage exosome, application thereof and spinal cord injury treatment preparation
WO2021147127A1 (en) * 2020-01-21 2021-07-29 武汉生之源生物科技股份有限公司 Kit for separating exosomes from cell supernatant and use method of kit
CN113797229A (en) * 2021-10-14 2021-12-17 天津市天津医院 Macrophage-derived exosome preparation for resisting inflammation and promoting tissue regeneration and preparation method and application thereof
CN114107205A (en) * 2021-11-29 2022-03-01 厦门大学附属翔安医院 Method for stimulating cells to quickly secrete exosomes and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021147127A1 (en) * 2020-01-21 2021-07-29 武汉生之源生物科技股份有限公司 Kit for separating exosomes from cell supernatant and use method of kit
KR102215237B1 (en) * 2020-06-29 2021-02-15 (주)지에프씨생명과학 Method of isolating high yield exosomes from cell culture supernatants
CN113046316A (en) * 2021-04-07 2021-06-29 中南大学湘雅医院 M2 type bone marrow macrophage exosome, application thereof and spinal cord injury treatment preparation
CN113797229A (en) * 2021-10-14 2021-12-17 天津市天津医院 Macrophage-derived exosome preparation for resisting inflammation and promoting tissue regeneration and preparation method and application thereof
CN114107205A (en) * 2021-11-29 2022-03-01 厦门大学附属翔安医院 Method for stimulating cells to quickly secrete exosomes and application thereof

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