CN112011499B - Method for preparing extracellular vesicles and application thereof - Google Patents

Method for preparing extracellular vesicles and application thereof Download PDF

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CN112011499B
CN112011499B CN202010944380.XA CN202010944380A CN112011499B CN 112011499 B CN112011499 B CN 112011499B CN 202010944380 A CN202010944380 A CN 202010944380A CN 112011499 B CN112011499 B CN 112011499B
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张昊
秦西淳
周叶卿
贾才力
晁志祥
秦昊
刘修成
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Abstract

The invention discloses a method for preparing extracellular vesicles and application thereof, wherein the method comprises the steps of establishing a cell apoptosis model to induce cells to generate apoptosis; obtaining a pyrophorosome with a diameter of 1-5 μm and containing caspase-1. The preparation method of the small burnt body is convenient to operate and low in production cost, and the obtained small burnt body is high in purity, good in activity, convenient to store, low in toxic and side effects and beneficial to continuous production; meanwhile, the application approach is wide, the kit not only can be used as a specific marker for detecting the damaged part, but also can be used for immunotherapy and used as a tumor vaccine, so that accurate targeted therapy of cancer is realized, and development of the tumor vaccine or medicine is promoted.

Description

Method for preparing extracellular vesicles and application thereof
Technical Field
The invention belongs to the technical field of extracellular vesicle preparation, and particularly relates to a method for preparing extracellular vesicles and application thereof.
Background
Cell scorching is also called as cell inflammatory necrosis and is manifested by cell swelling and rounding, cell membrane rupture and inflammatory factor release. Recent research proves that a plurality of cells in a body can be burnt and die, and the burnt and die cells can cause nucleus shrinkage and exocytosis of vesicles to form burnt and die bodies.
Cell apoptosis is an important innate immune response in the body that plays an important role in combating infection, however, current research on the use and transformation of cell apoptosis is still lacking.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
As one aspect of the present invention, the present invention provides a method for preparing extracellular vesicles.
In order to solve the technical problems, the invention provides the following technical scheme: a method of preparing extracellular vesicles, comprising establishing a cell apoptosis model to induce apoptosis of cells; obtaining pyrophorosomes having a diameter of 2-5 μm and comprising caspase-1.
As a preferred embodiment of the method for preparing extracellular vesicles according to the present invention: the establishing of the cell apoptosis model to induce the cell to generate apoptosis adopts lipopolysaccharide and nigericin to co-culture the cell, wherein the concentration of the lipopolysaccharide is 0.5-10 mu g/ml, and the concentration of the nigericin is 0.5-20 mu mol/L.
As a preferred embodiment of the method for preparing extracellular vesicles according to the present invention: the cell scorching model is established to induce the cells to generate the scorchingThe temperature is 35-37 ℃ in the culture environment and 5% CO2，20％O2When the cell density is fused to more than eighty percent, adding lipopolysaccharide, culturing for 5 hours, adding nigericin, continuously culturing for 40-50 min, inducing the cells to generate scorching and generating the scorching corpuscles, and collecting cell supernatant.
As a preferred embodiment of the method for preparing extracellular vesicles according to the present invention: the method for obtaining the pyrophoric corpuscle with the diameter of 2-5 mu m and containing the cysteine aspartic acid specific protease-1 comprises the steps of collecting cell supernatant, centrifuging at the centrifugal force of 300-700 g for 5-30 min, discarding supernatant, and collecting precipitate; and (3) resuspending the precipitate with phosphate buffer solution, centrifuging at the centrifugal force of 300-700 g for 5-30 min, discarding the supernatant, and collecting the precipitate to obtain the small burnt body.
As a preferred embodiment of the method for preparing extracellular vesicles according to the present invention: the cells comprise umbilical vein endothelial cells, bronchial epithelium, vascular endothelial cells and lung adenocarcinoma A549 cells.
As another aspect of the invention, the invention provides the application of the extracellular vesicles in preparing a medicament for detecting the injured parts and the injured degree of a body.
The method is used for detecting the injury position and the injury degree of an organism, and comprises the step of detecting the injury degree of bronchial epithelial cells and vascular endothelial cells.
As another aspect of the invention, the invention provides the application of the extracellular vesicles in preparing tumor vaccines or antitumor drugs. The tumor, including lung adenocarcinoma.
The invention has the beneficial effects that: the inventor finds that the exocytic vesicle generated in the cell apoptosis process can retain the membrane specific protein of the cell and can be released into an extracellular medium through repeated verification of the cell apoptosis morphology, and compared with the extracellular vesicle apoptotic bodies derived in the normal physiological state of the cell, the apoptosis body has stronger immunogenicity and good immune activation effect. According to the preparation method of the extracellular vesicle pyrophorosome, the obtained pyrophorosome with the diameter of 2-5 mu m reserves membrane specific protein of a primary cell and can be used as a basis for judging a specific damaged part and a specific damaged degree of an organism; the focal apoptotic body produced by the tumor cell has excellent anti-tumor effect, and can be used as a tumor vaccine for preventing or treating tumor and cancer.
The preparation method of the small burnt body is convenient to operate and low in production cost, and the obtained small burnt body is high in purity, good in activity, convenient to store, low in toxic and side effects and beneficial to continuous production; meanwhile, the application approach is wide, the kit not only can be used as a specific marker for detecting the damaged part, but also can be used for immunotherapy and used as a tumor vaccine, so that the accurate targeted therapy of cancer is realized, and the development of the tumor vaccine or medicament is promoted.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a transmission electron microscope scanning image of the focal death corpuscles of the present invention.
FIG. 2 is an immunoblot of apoptotic bodies of the invention.
FIG. 3 is a schematic diagram of small-volume flow analysis and detection of the present invention.
FIG. 4 is a statistical chart of the number of apoptotic bodies in bronchoalveolar lavage fluid of mice with acute lung injury at different time points.
FIG. 5 is a graph of HE staining of lung tissue of mice with acute lung injury at different time points.
FIG. 6 is a graph showing the results of nude mouse tumorigenesis.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
establishing a cell apoptosis model:
culturing Human Umbilical Vein Endothelial Cells (HUVEC) in a cell culture incubator at 37 deg.C and 5% CO2，20％O2When the cell density is fused to more than eighty percent, adding lipopolysaccharide (purchased from Sigma) for culture (1 mu g/ml), after 5 hours of culture, adding nigericin (purchased from Invitrogen) for continuous culture for 45 minutes (5 mu mol/L), inducing the cells to generate apoptosis, generating apoptosis corpuscles, collecting cell supernatant, and marking as cell supernatant A;
extraction of focal-death corpuscles:
(1) removal of cells and cell debris: centrifuging the cell supernatant A for 10min under the condition that the centrifugal force is 300g, discarding the precipitate, and taking the supernatant, and recording the supernatant as a supernatant B;
(2) precipitation of apoptotic bodies: centrifuging the supernatant B for 20min under the condition that the centrifugal force is 2000g, and discarding the supernatant to obtain a precipitate;
(3) purifying focal bodies: and (3) resuspending the precipitate obtained in the step (2) with sterile Phosphate Buffered Saline (PBS), and repeating the step (2) to obtain the precipitate, thereby obtaining the apoptotic body. The obtained precipitate is re-suspended with sterile normal saline to obtain suspension of focal corpuscle, and the suspension is stored in a refrigerator at the temperature of 80 ℃ below zero.
Example 2:
identification of coke-death corpuscles:
(1) morphological characteristics of coke-death corpuscles:
the morphology of the apoptotic bodies was observed by Transmission Electron Microscopy (TEM). And (3) taking the prepared pyrophoric corpuscle suspension, placing the pyrophoric corpuscle suspension into a copper net coated with amorphous carbon for soaking for 5min, placing the pyrophoric corpuscle suspension into a culture dish with filter paper, naturally drying the pyrophoric corpuscle suspension, then placing a sample into a sample tank of a transmission electron microscope, and observing, wherein the pyrophoric corpuscle is shown in figure 1 and has a uniform vesicle structure with the diameter of 1-5 mu m (standard bar is 2 mu m).
(2) Detection of markers of focal death bodies:
the total protein extraction kit (NO. C006225) for Shanghai biological engineering is adopted to extract the total protein from the apoptotic body according to the kit instruction. The specific method comprises the following steps: physiological extracellular vesicle pellets derived from normal cultured HUVEC cells and apoptotic body pellets derived from a cell apoptosis model were obtained according to the method of example 1, and a corresponding volume of pre-cooled lysate was added (1. mu.l protease inhibitor, 5. mu.l phosphatase inhibitor, 10. mu.l PMSF per 1ml of lysate). The sample is put on ice to be cracked for 30min, and is vibrated for 1min by an oscillator every 10 min; ultracentrifuge (4 ℃, 12000g, 15 min). Finally, the supernatant was aspirated and protein quantification was performed by BCA method. The immunoblotting method detects the expression of caspase-1 (caspase-1) in physiological extracellular vesicles and pyrophoromes. The results are shown in FIG. 2, where the pyrophoromes expressed caspase-1(β -tubulin is the loading control internal control protein) at a higher level than the physiological extracellular vesicles.
Example 3:
detecting the source and the quantity of the burnt corpuscles to judge the injured parts and the injury degree of the organism:
experimental animals: healthy adult C57BL/6 mice (purchased from the animal center of Xuzhou university of medical science) weighing 22-28g were modeled for acute lung injury using tracheal instillation of an aqueous solution of Lipopolysaccharide (LPS) (2 mg/kg). Equal amount of physiological saline was instilled into trachea of the blank group, and the group was recorded as 0 h. After 4h, 12h and 24h of animal model establishment, mice were sacrificed by cervical dislocation, neck skin was incised, trachea was exposed, and lung bronchi were lavaged with sterile physiological saline (0.5 ml each time, repeated 3 times) to obtain bronchoalveolar lavage fluid.
Pyrophorosomes were obtained using the method of example 1, and then stained on ice with 1 μ g EpCAM-FITC antibody (bronchial epithelial cell marker), 1 μ g CD31-APC (vascular endothelial cell marker) for 30 minutes, centrifuged at 2000g for 20min, the pellet was left, the supernatant removed for further analysis, the pelleted pyrophorosomes resuspended in 500 μ L PBS, 10 μ L of calibration counting beads of 1 μm size and 10 μ L of 5 μm size were added, 2-5 μm size pyrophorosomes were gated by calibration counting beads and the number of pyrophorosomes was calculated (as shown in figure 3). Apoptotic bodies from bronchial epithelial cells were defined as EpCAM positive events and apoptotic bodies from vascular endothelial cells as CD31 positive events. The statistical results are shown in FIG. 4.
And (3) pathological examination: and (3) taking the right lung middle lobe of the mouse, placing the right lung middle lobe of the mouse in a 4% paraformaldehyde solution for fixation for 24 hours, then carrying out conventional paraffin-embedded section and HE staining, and observing the pathological condition of lung tissues of the mice of each group. The results are shown in FIG. 5.
As shown in fig. 4 and 5, with the progression of acute lung injury, the lung tissue morphology is gradually disturbed, the edema is gradually deepened, the damage to bronchial epithelium and vascular endothelium is continuously increased, and the quantity of apoptotic bodies from epithelial cells and endothelial cells is proportional to the degree of histomorphological damage. The results show that the detection of the focal corpuscle has high specificity and high sensitivity, and can effectively reflect the damage degree of bronchial epithelium and vascular endothelium. FIG. 4 is a statistical graph of the number of pyrophoric bodies released from bronchial epithelium and vascular endothelial cells from mice with acute lung injury at various time points; FIG. 5 is the HE staining observation result of lung tissue of mice with acute lung injury, wherein column 5A represents the blank group, column 5B represents the group of mice with acute lung injury of 4h, column 5C represents the group of mice with acute lung injury of 12h, and column 5D represents the group of mice with acute lung injury of 24 h. Magnification times 200.
In vitro anti-tumor effects of pyrophoric bodies:
establishing a tumor cell apoptosis model: human lung adenocarcinoma A549 cells were taken and cultured and a cell apoptosis model was established according to the method of example 1 to obtain apoptotic bodies produced by tumor cells.
Nude mouse tumorigenesis experiment: in nude mice (purchased fromNanmo organism) axillary subcutaneous injection of lung adenocarcinoma A549 cells 2X 106Establishing a tumor-bearing mouse model. Divided into a control group, a physiological extracellular vesicle group and a focal corpuscle group. 7 days after molding, the focal corpuscle suspension is injected one time (1X 10) beside the tumor in the focal corpuscle group6Only). The physiological extracellular vesicle group was injected with the same amount of physiological extracellular vesicles, and the blank group was not treated. Dissections were performed 28 days later.
The results are shown in FIG. 6: compared with the blank group and the physiological extracellular vesicle group, the focal-death corpuscles can obviously inhibit the growth of the lung cancer subcutaneous tumor. Column 6A represents the blank group, column 6B represents the physiological extracellular vesicle group, and column 6C represents the focal apoptotic body group.
The method establishes a cell apoptosis model to induce the cells to generate apoptosis, and prepares apoptosis bodies with the diameter of 2-5 mu m and containing caspase-1 (caspase-1). The research of the invention finds that the source and the quantity of the burnt corpuscles prepared by the invention can help to judge the damaged parts and the damage degree of the organism; the pyrophorome with the diameter of 2-5 mu m generated by the tumor cells can activate an immune system and serve as a tumor vaccine or a medicament.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims (5)

1. The application of the extracellular vesicles in preparing antitumor drugs is characterized in that: the tumor is lung adenocarcinoma, and the extracellular vesicle is a focal apoptotic body produced by lung adenocarcinoma cells; the extracellular vesicle is prepared by establishing a cell apoptosis model to induce cells to generate apoptosis; obtaining the pyrophores with a diameter of 1-5 μm and containing caspase-1.
2. Use according to claim 1, characterized in that: the cell apoptosis model is established to induce the cells to generate apoptosis, and the cells are co-cultured by lipopolysaccharide and nigericin, wherein the concentration of the lipopolysaccharide is 0.5-10 mu g/ml, and the concentration of the nigericin is 0.5-20 mu mol/L.
3. Use according to claim 2, characterized in that: the establishment of the cell apoptosis model to induce the cells to generate apoptosis is carried out in a culture environment at 35-37 ℃ and 5% CO2，20％O2When the cell density is fused to more than eighty percent, adding lipopolysaccharide, culturing for 5 hours, adding nigericin, continuously culturing for 40-50 min, inducing the cells to generate scorching and generating the scorching corpuscles, and collecting cell supernatant.
4. Use according to claim 2, characterized in that: the method for obtaining the pyrophoric corpuscle with the diameter of 1-5 mu m and containing the cysteine aspartic acid specific protease-1 comprises the steps of collecting cell supernatant, centrifuging at the centrifugal force of 300-700 g for 5-30 min, discarding supernatant, and collecting precipitate; and (3) resuspending the precipitate with phosphate buffer solution, centrifuging at the centrifugal force of 300-700 g for 5-30 min, discarding the supernatant, and collecting the precipitate to obtain the small burnt body.
5. Use according to claim 1, characterized in that: the cells, including lung adenocarcinoma a549 cells.
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CN108904504A (en) * 2018-06-20 2018-11-30 暨南大学 Application of the rutaecarpin in the drug of preparation enhancing NLRP3 inflammation corpusculum activation
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CN109010811A (en) * 2018-09-04 2018-12-18 庆云堂生物科技（北京）有限公司 Apoptotic body vaccine immunity prepares broad-spectrum anti-tumor mouse model

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Pyroptotic cells externalize eat-me and release find-me signals and are efficiently engulfed by macrophages;Qiang Wang等;《International Immunology》;20130226;第25卷(第6期);第363-372页 *
李敬贤.细胞焦亡和细胞坏死的形态学差异及其机理的探究.《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》.2019,A006-8. *
细胞焦亡和细胞坏死的形态学差异及其机理的探究;李敬贤;《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》;20190615;第37-51页 *

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