CN116333987A - Application of NK cell exosomes obtained by exosome extraction method in resisting tumor - Google Patents

Abstract

The invention relates to the technical field of exosomes, and particularly discloses an exosome extraction method, an NK (natural killer) exosome obtained by the extraction method and application thereof in resisting tumors, wherein the extraction method comprises various combinations of an ultracentrifugation method, an ultrafiltration method, a size exclusion chromatography, a precipitation method and an immunoaffinity separation method, in particular a combination method of the ultracentrifugation method and the size exclusion chromatography, namely, the exosome is firstly extracted by the ultracentrifugation method, and then the exosome is further extracted by the size exclusion chromatography to improve the purity of the exosome; the exosomes obtained by extraction through a combination method are high in purity, and when the exosomes of the NK cells are separated and purified by the combination method, the obtained NK cell exosomes are high in purity and can effectively inhibit the activity of tumor cells, so that the use of the NK cell exosomes is not limited.

Description

Application of NK cell exosomes obtained by exosome extraction method in resisting tumor

Technical Field

The invention relates to the technical field of exosomes, in particular to an application of NK cell exosomes obtained by an exosome extraction method in resisting tumors.

Background

In recent years, exosomes, which are "excreted waste" of cells, are in the field of view, and are extracellular vesicles secreted by cells, and have been found to have various functions, and they carry protein drugs, nucleic acids, etc., and thus have unique advantages in the in vivo transport of drugs such as genes and antitumor agents.

Natural killer cells, also called NK cells, belong to large-particle lymphocytes, are derived from bone marrow, are important immune cells of organisms, have broad-spectrum anti-tumor cell effects, and can directly kill tumor cells and virus-infected target cells in a nonspecific manner without depending on antigen stimulation; while the new NK cell action model focuses on the use of its derived exosomes (NK-EXOs), which are secretory vesicles of the phospholipid membrane that germinate through the cell membrane and endosome membrane and are rich in specific proteins, lipids, nucleic acids and glycoconjugates. The exosome of NK cells has various immunoregulatory functions, and is a promising antitumor nanometer biological preparation. However, how to significantly improve purity and yield of exosomes of NK cells to meet clinical treatment criteria is a necessary condition to achieve exosome transformation.

Since there is no standard method for producing exosomes of absolute purity at present, the purity of exosomes is greatly affected by contamination of lipoproteins, chylomicrons and the like, and low purity is the biggest obstacle for exosomes application, the exosomes NK-exosomes of NK cells are limited in use.

Disclosure of Invention

In order to overcome the defect of limitation of clinical application caused by low purity of exosomes extracted in the prior art, the invention provides an exosomes extraction method, an NK cell exosomes obtained by the extraction method and application of the exosomes in anti-tumor, so that the purity of the exosomes is obviously improved, and obvious progress is obtained in anti-tumor clinical research.

The invention adopts the following technical scheme:

the extraction method of the exosomes, provided by the invention, is used for separating the exosomes, and can improve the purity of the separated exosomes, and comprises various combinations of an ultracentrifugation method, an ultrafiltration method, a size exclusion chromatography method, a precipitation method and an immunoaffinity separation method.

The UC method is to separate and enrich exosomes by two different dimensions of particle size and density, and differential speed centrifugation or density gradient centrifugation is used singly or in combination, while differential speed ultracentrifugation is the most widely used exosome separation and purification method;

ultrafiltration is a relatively rapid and straightforward technique that is particularly useful for concentrating exosomes that have been isolated by other methods, and may also be used as the primary exosome separation/concentration technique. The membrane used in the ultrafiltration method is usually made of cellulose, polysulfone and other materials, the molecular weight cut-off is 10-1000kD, and the method is suitable for enriching exosomes from a large-volume sample;

size exclusion chromatography SEC, a chromatographic method that separates molecules in solution according to size or, in some cases, according to their molecular weight, alone or in combination with other separation methods;

the precipitation method is to separate exosomes from other compounds according to solubility, and the most common hydrophilic polymer precipitation method is a PEG precipitation method, so that the PEG has strong hydrophilicity and wide molecular weight;

the immune affinity separation method is to effectively separate exosomes by utilizing the antigen-antibody effect and the specific reaction between the receptor and the ligand in immunology, and mainly comprises an immune magnetic bead method and a microfluidic technology;

the six methods can be used for extracting exosomes, and combined extraction of the six methods can be performed for further improving the purity of exosomes.

Preferably, the extraction method is a combination of ultracentrifugation and size exclusion chromatography, namely UC+SEC method, and the exosome is first extracted by ultracentrifugation, and then further extracted by size exclusion chromatography to improve the exosome purity.

Furthermore, the combined extraction method of the ultracentrifugation method and the size exclusion chromatography requires that the cell culture solution of the exosomes to be extracted is subjected to tangential flow concentration.

Furthermore, the packing of the chromatographic column used in the size exclusion chromatography is Sepharose CL-6FF, the gel filtration medium is formed by crosslinking Sepharose CL-6B and 2,3 dibromopropanol in multiple steps, and the Sepharose CL-6B resin can remarkably improve the purity of exosomes and has the best performance.

The invention also provides a technical scheme that:

the extraction method of the exosome is applied to NK cells and is used for separating and purifying the NK cells to obtain the exosome with high purity, and the extraction method of the exosome of the NK cells comprises the following steps:

s1: culturing and inducing NK cells in a culture medium, separating umbilical blood mononuclear cells from a healthy donor, and culturing and amplifying the umbilical blood mononuclear cells according to an IL-21 amplification system to obtain NK cells;

s21: filtering culture supernatant of NK cells with 0.45um membrane, concentrating by 100kDa tangential flow for 10 times, performing differential centrifugation, centrifuging at 2000g for 20 min, centrifuging at 10000g for 30 min to remove dead cells and cell fragments, centrifuging at 100000g at 4deg.C for 90 min with ultra-high speed centrifuge, collecting precipitate, washing the obtained precipitate in phosphate buffer solution, centrifuging again at 100000g for 70 min, and re-suspending exocrine in phosphate buffer solution;

s22: purifying the exosomes obtained by ultracentrifugation by size exclusion chromatography, connecting a chromatographic column with a clearFirst-3000 type protein purification system, monitoring the exosomes by using an ultraviolet detector of an instrument, and collecting the exosomes with peaks of the ultraviolet detector for subsequent detection and analysis;

s3: characterization and identification of NK cell exosomes by Transmission Electron Microscopy (TEM), nanoparticle Tracking Analysis (NTA), westernblotting (WB) and Flow Cytometry (FCM) for morphology, particle size and surface markers;

s4: proteomic analysis of NK cell exosomes was performed by protein mass spectrometry.

The invention also provides another technical scheme:

the application of the extraction method of the exosomes in resisting tumors, the NK cell exosomes after separation and purification are used for resisting tumors and treating cervical cancer, and the extraction method specifically comprises the following steps:

s5: recovering tumor cells, and culturing in complete culture medium;

s6: detecting the influence of the exosomes of NK cells on the survival rate and proliferation of tumor cells through a cell colony formation experiment and CCK-8;

s7: uptake of NK cell exosomes in tumor cells was observed under a confocal laser microscope and tumor cell apoptosis was detected by flow cytometry.

The cell colony formation experiment of step S6 specifically includes: tumor cells are added into an exosome of NK cells after being attached to the wall according to 1000 cells/Kong Chongban, liquid is changed after four days, giemsa staining is carried out after one week, and the cells are observed by a microscope and photographed;

the CCK-8 assay of step S6 is used to evaluate the activity of tumor cells, and specifically comprises: tumor cells and NK cell exosomes are placed in an orifice plate for co-culture, 10uL of CCK-8 enhanced solution is added into each orifice, the mixture is incubated at 37 ℃ in a dark place for 2 hours, an optical density reading is carried out at 450nm by using an enzyme-labeled instrument, and the proliferation condition of the tumor cells is calculated.

The method for extracting the exosomes by combining the ultracentrifugation method and the size exclusion chromatography can be applied to NK cells, can obtain the NK cell exosomes by separation and purification, and can treat cervical cancer by utilizing the NK cell exosomes for resisting tumors.

The invention has at least the following beneficial effects:

1. according to the extraction method of the exosomes, provided by the invention, the purity of the separated exosomes can be improved to a great extent by combining the ultracentrifugation method and the size exclusion chromatography;

2. the extraction method of the exosomes is applied to the NK cells, and the exosomes of the NK cells are extracted by ultracentrifugation and size exclusion chromatography, so that the purity and the yield of the obtained exosomes of the NK cells are improved;

3. the extraction method of the exosomes is applied to anti-tumor of the NK cell exosomes, so that the NK cell exosomes are favorable for anti-tumor treatment, the activity of tumor cells can be inhibited, and the treatment of cervical cancer is remarkably improved.

Drawings

FIG. 1 is a schematic flow chart of a method for extracting an exosome of NK cells;

FIG. 2 is a diagram showing the characterization of NK cell exosomes isolated by the UC method and the UC+SEC method;

FIG. 3 is a schematic diagram showing the particle number and protein content of NK cell exosomes isolated by the UC method and the UC+SEC method;

FIG. 4 is a schematic diagram of proteomic analysis of NK cell exosomes isolated by the UC method and the UC+SEC method;

FIG. 5 is a graph showing toxicity of HeLa cells to uptake NK cell exosomes;

FIG. 6 is a diagram showing NK cell exosome-induced tumor cell apoptosis.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The invention discloses an extraction method of exosomes, which is a combination of an ultracentrifugation method and a size exclusion chromatography method, and comprises the steps of carrying out tangential flow concentration treatment on a cell culture solution, then carrying out ultracentrifugation, then carrying out rough extraction on exosomes by the ultracentrifugation method, and then carrying out further extraction by the size exclusion chromatography method, wherein the exosomes obtained by the combination method have higher purity.

During practical application, the combination method can be used for extracting the exosomes of the NK cells, and specific experimental description is carried out on the fact that the combination method can improve the purity of the exosomes, and the specific experimental operation steps are as follows:

s1: culturing NK cells: firstly, NK cells are required to be obtained, 3 healthy donors (the donors are signed with written informed consent) are selected by a hospital and approved by an ethical committee, 3 healthy donor volunteers provide umbilical cord blood, then umbilical cord blood mononuclear cells CBMCs are separated by Ficoll (solabio, p 8900) respectively, the CBMCs are cultured and amplified according to an IL-21 amplification system to obtain NK cells, the CBMCs are added into a complete culture medium and feeder cells are cultured in a culture flask, after three days, centrifugal liquid exchange is carried out, after the seventh day, the feeder cells are added into a culture bag for culture, cell counting is carried out by using a cell counter (RWD-C100) every day, and the culture medium is supplemented, so that the cell concentration is maintained to be 0.8-1.0x10 ⁶ cell/ml, and obtaining NK cells induced by CBMCs after 15 days of culture;

s21: taking two groups of culture supernatants of 100ml NK cells, filtering the two groups by using a 0.45um membrane, concentrating 10 times by using a 100kDa tangential flow, performing differential centrifugation, centrifuging for 20 minutes at 2000g, centrifuging for 30 minutes at 10000g to remove dead cells and cell fragments, centrifuging for 90 minutes at 100000g at 4 ℃ by using a super-high speed centrifuge, collecting precipitates, washing the obtained precipitates in a phosphate buffer solution, centrifuging for 70 minutes again at 100000g, and re-suspending the exosomes in the phosphate buffer solution;

s22: labeling one group of the two groups of NK cell exosomes obtained in the step S21 by an ultracentrifugation method as exosomes 1 (exosomes extracted by the ultracentrifugation method are control groups) and one group of the two groups of NK cell exosomes as exosomes 2 (exosomes extracted by the combination of the ultracentrifugation method and the size exclusion chromatography method are experimental groups);

s23: purifying exosomes 2 obtained by ultracentrifugation by size exclusion chromatography, wherein a chromatographic column in the step is purchased from SANJITECH (ND 4100) and filled with Sepharose CL-6FF, a gel filtration medium is formed by crosslinking Sepharose CL-6B and 2,3 dibromopropanol in multiple steps, the chromatographic column is connected with a clearFirst-3000 type protein purification system, an ultraviolet detector of an instrument is utilized for monitoring exosomes, and the exosomes in the step S21 are separated and purified and the exosomes with peaks detected by the ultraviolet detector are collected for subsequent detection analysis;

s3: in this step, characterization and identification of morphology, particle size and surface markers were performed for both exosomes 1 and exosomes 2, specifically as follows:

s31: observing the separated and purified NK cell exosomes by a Transmission Electron Microscope (TEM), taking out 10uL of the exosomes in the step S22 at room temperature, dripping the sucked sample on a copper mesh to precipitate for 1min, sucking floating liquid by filter paper, dripping 10uL of uranyl acetate on the copper mesh to precipitate for 1min, sucking the floating liquid by the filter paper, drying for a plurality of minutes at room temperature, and performing electron microscope (HitachihT-7700) detection imaging at 100kv to obtain a transmission electron microscope imaging result;

s32: the method comprises the steps of separating and purifying NK cell exosomes in a Nanoparticle Tracking Analysis (NTA) S22 step, taking out an appropriate amount of exosomes, diluting the exosomes to an appropriate multiple, firstly, performing zataview (20-602) instrument performance test on a standard substance to sample exosomes, taking care of gradient dilution to avoid sample blocking a sample injection needle, and obtaining particle size and concentration information of the exosomes after the sample is detected and processed by software of software Zetaview (version 8.05.14SP7);

s33: westernblotting (WB) A specific assay is carried out by adding a Loading Buffer (ZOMANBIO, ZS 306) into the exosome, boiling for 10min at 100deg.C, cooling to room temperature, separating by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (ZAMANBIO, ZD 304C-10), transferring the proteins in the gel onto polyvinylidene fluoride (PVDF) membrane (Millipore, billerica, MA, USA), sealing for 1h at room temperature with a rapid sealing solution (Biotech, p 30500) for 15min, incubating overnight at 4deg.C with a dilution of antibodies rabbit anti-human monoclonal antibody CD81 (1:1000) (definition, DF 2306) and Calnexin (1:1000 (Huabaio, ETT 1611-86)), washing the membrane 3 times with Tris-BufferedSalinewith Tween-20 (TBST) (bios, C5047), incubating the membrane with horseradish peroxidase (HRP) labeled anti-human monoclonal antibody CD81 (1:1000) (definition, DF 2306) for 5min, incubating the membrane with a final dilution of horseradish peroxidase (HRP) for 2 min, and washing with a light-sensitive antibody (ECMANEXI 2 min for 2 min each time;

s34: and (3) identifying the expression level of two groups of exosome positive markers by Flow Cytometry (FCM), diluting 30uL of exosome to 90uL in each group, respectively adding 20uL of fluorescent-labeled antibodies (CD 9, CD63 and CD 81) into the 30uL of diluted exosome, uniformly mixing, and incubating at the temperature of 37 ℃ in a dark place for 30 minutes. 1mL of pre-chilled phosphate buffer was added, the overspeed rotor was selected, ultracentrifuged for 70 minutes at 4 ℃,110000g, the supernatant carefully removed, 1mL of pre-chilled phosphate buffer was added, the overspeed rotor was selected, again ultracentrifuged for 70 minutes at 4 ℃,110000 g. The supernatant was carefully removed and resuspended with 50uL of pre-chilled 1-fold concentrated phosphate buffer. And (3) firstly, performing instrument performance test by using a standard substance, and then loading an exosome sample, wherein the sample is required to be subjected to gradient dilution to avoid the sample blocking a sample injection needle, and the protein index detection result of the NanoFCM instrument can be obtained after the sample is detected.

S4: protein content of exosomes 1 and 2 was determined by BCA method, protein standard solutions were prepared according to the instructions of BCAproteinassay kit (meilunbio, MA 0082), and concentration determination was performed directly according to the instructions.

The experimental results are as follows:

data were analyzed using GraphpadPrism8 treatment, experimental data presented in the study were all shown with mean ± Standard Error (SEM), P < 0.05 was considered significant, the coefficient of variation of protein intensity was calculated by dividing the standard deviation by the duplicate mean, and Origin software (v.2021b) and Edrawmax were image processed.

In this experiment, two extraction methods were actually used to extract the exosomes of NK cells, the first method being exosomes 1 obtained by Ultracentrifugation (UC) extraction, the second method being exosomes 2 extracted by a combination of ultracentrifugation and size exclusion chromatography (uc+sec);

panel A in FIG. 2 shows Transmission Electron Microscope (TEM) images of NK cell exosomes obtained by both extraction methods, showing that exosomes isolated by both methods have a complete membrane structure and a typical cup-like morphology;

panel B of FIG. 2 is a graph showing the measurement of particle size of the separated components using Nanoparticle Tracking (NTA), mostly between 100-150 nm;

fig. 2C shows the size distribution (n=3) of exosomes separated by two separation and extraction methods using Nanoparticle Tracking (NTA), with UC on the left and uc+sec on the right;

fig. 2D is a graph of statistical analysis of the median of NK cell exosomes extracted by both methods, showing that the median diameter of exosomes 2 obtained by the combined extraction method is smaller than that of exosomes 1 obtained by the ultracentrifugation extraction method, and that there is a significant difference (p=0.0267);

the number of particles and the protein content per unit volume obtained by two separation methods from 100ml of culture supernatant of NK cells were evaluated for productivity, and the exosome purity was evaluated by calculating the ratio of the number of particles per unit volume to the protein content,

diagram a in fig. 3 shows: identifying the expression level of two groups of exosome positive markers including CD9, CD63 and CD81 according to a Flow Cytometry (FCM), wherein the positive markers of the exosome are reserved by separation and extraction of the two methods, but the expression level of the exosome CD9 separated by a UC+SEC method is reduced (p=0.0295), and the expression level of the CD81 is slightly increased, but no obvious difference exists;

panel B in FIG. 3 shows: based on NTA measurement results, the extract of UC+SEC method contains 7.9X10 of exosomes per unit volume ¹⁰ The particle number is 5.4X10 with UC method ¹¹ Compared with the method, the number is reduced, and the particles obtained by the UC+SEC method are obviously less than those obtained by the UC method, and the particles have obvious difference (p=0.0147);

panel C in fig. 3 shows: the total protein content of exosomes extracted by the two methods was evaluated according to BCA protein content assay, and the results showed that the total protein content per unit volume obtained by uc+sec method was significantly reduced by 0.3mg compared to uc3.4mg, with significant differences (p=0.0093), indicating that uc+sec method successfully cleared lipoprotein and other particle contamination;

fig. 3D shows: knot(s)The results show that the number of particles per unit volume of exosomes obtained by the UC+SEC method per protein content (10 ¹⁰ Per mg) was 26.6, significantly improved the purity of exosomes compared to 16 of UC method, with a significant difference p=0.0004.

It can be seen that the combined extraction method (uc+sec) successfully separated NK cell exosomes of high purity from the cell culture supernatant.

Proteomics of exosomes extracted by two methods were analyzed by mass spectrometry MS as in panel a of fig. 4: the UC+SEC combination method can separate more protein types from NK cell supernatant, and 1817 protein can be separated from cell supernatant by the UC+SEC combination method, which is far higher than 1567 protein separated by the UC method; FIG. 4B is a heat map showing the types of proteins in exosomes extracted by different separation methods;

by non-labeled quantification technique LFQ, both sets of all detected proteins were ranked according to the level of quantitative data, LFQ values reflecting the abundance values of each protein assessed by MS. Lipoproteins (Apo-A1, apo-A2, APOC3 and HDLBP) are the most common contaminants in NK cell exosomes proteomic analysis, whereas lipoprotein contaminants are the highest protein-intensity contaminants Apo-A1 and Apo-A2 and the lowest intensity contaminants HDLBP in NK cell exosomes extracted in both methods. In NK cell exosomes isolated by uc+sec combination, the exosome related hybrid proteins showed lower protein intensity (shown in figure 4, panel C), furthermore we also quantitatively analyzed the lipoprotein contaminant content, finding that they all decreased by more than 50%, with the Apo-A2 content decreasing most significantly, then Apo-A1, APOC3 and HDLBP in sequence, another more important finding that the NKP30/NCR3 content increased by more than 2-fold (shown in figure 4, panel D);

further, the UC+SEC combination method can greatly reduce protein impurities such as lipoprotein and the like in exosomes and improve enrichment of toxic proteins.

Further, in the analysis of protein composition differences of NK cell exosomes extracted by the two methods, compared with the UC method, the up-regulation of B2M, MBL, CYTH2 was most pronounced in the uc+sec combination method, and the down-regulation of STK24, RPL17, FN1 was most pronounced, as shown in the E-graph in fig. 4, the upper diagonal represents the first three of significantly up-regulated proteins in the uc+sec method: B2M, MBL and CYTH2, the oblique lower panels represent the first three of the significant downregulated proteins in the uc+sec method: STK24, RPL17 and FN1.

The comprehensive experimental result shows that the combined extraction method (UC+SEC) successfully separates the NK cell exosomes with high purity from the cell culture supernatant, greatly reduces protein impurities such as lipoprotein and the like in the exosomes, improves the enrichment of toxic proteins, and enables the NK cell exosomes to play a role.

Example two

The invention also discloses application of the NK cell exosomes separated by the UC+SEC combined extraction method in resisting tumor and treating cervical cancer, wherein the NK cell exosomes used in the embodiment are exosomes extracted by the combined method of the first embodiment, and the NK cell exosomes obtained by extraction and purification by the combined method of ultracentrifugation and size exclusion chromatography have killing effect on tumor cells, and the specific experimental steps are as follows:

s1: heLa cells were recovered and cultured in complete medium: culturing in complete culture medium after resuscitating, wherein the culture medium adopts DMEM+10% foetal calf serum+1% double antibody. The cells are passaged, heLa cells with the confluence degree of 70-80% are selected, the HeLa cells are digested by trypsin of 0.25% trypsin and EDTA, the cells are washed once by phosphate buffer solution PBS, the trypsin is inactivated by complete culture medium of 10% fetal bovine serum, the HeLa cells are centrifugated for 5 minutes at 1000 revolutions per minute, and then HeLa cell suspension is inoculated to a new culture dish according to 1/3;

s2: incubating NK cell exosomes with HeLa cells;

s3: detecting the influence of the exosomes of NK cells on the survival rate and proliferation of HeLa cells through a cell colony formation experiment and CCK-8;

s31: cell colony formation experiments specifically included: digestion of HeLa cells in the logarithmic phase, counting after centrifugation, attaching HeLa cells to the wall according to 1000 cells/Kong Chongban, adding exosomes of NK cells, changing liquid after four days, and staining with Giemsa after one week;

s32: CCK-8 assays were used to assess HeLa cell activity, and specifically included: placing exosomes of HeLa cells and NK cells in an orifice plate for co-culturing for 12 hours and 24 hours, then evaluating the cell survival rate, adding 10uL of CCK-8 enhanced solution into each orifice, incubating for 2 hours at 37 ℃ in a dark place, and then performing optical density reading at 450nm by using an enzyme-labeled instrument;

s4: uptake of NK cell exosomes in tumor cells was observed under a confocal laser microscope and HeLa apoptosis was detected by flow cytometry.

To further explore the internalization process of NK cell exosomes in HeLa cells: heLa cells were treated with DAPI labeling and the left panel of panel A in FIG. 5 shows nuclei; treatment of NK cell exosomes with PKH26 markers, the middle panel of panel a in fig. 5 representing exosomes; after 10h incubation, the images were imaged with a confocal laser microscope, and the overlap was seen in the right panel of panel A in FIG. 5, showing that PKH 26-labeled NK cell exosomes could be taken up by HeLa cells, making the experiment interesting.

To further explore the relationship between NK cell exosomes cytotoxicity to HeLa cells, inhibition of cell proliferation and exosome dose and time, three groups of exosome concentrations of 50ug/ml, 100ug/ml and 200ug/ml were selected as experimental groups in step 2, and a group of control groups: no NK cell exosomes were added; four groups are simultaneously tested, each group is cultured for 12 hours and 24 hours to record the activity of HeLa cells, as shown in a B diagram and a C diagram in fig. 5, the final result is that the HeLa cells treated by 100ug/ml exosomes obviously inhibit the activity rate of the HeLa cells compared with a control group, the effect of 200ug/ml exosomes is stronger, the effect of 24 hours is better, and the NK cell exosomes are verified to have killing effect on the HeLa tumor cells.

In order to further explore the killing effect of NK cell exosomes on HeLa cells, detecting apoptosis of tumor cells by using a flow cytometer, detecting two groups of exosomes extracted by a UC method and a UC+SEC method in the first embodiment, and performing a group of control groups without exosomes, adding the HeLa cells into a prepared medicament in advance in a liquid change mode according to a kit operation method, and performing apoptosis induction treatment for 24 hours; centrifuging at 300g for 5min, discarding the supernatant, collecting cells, washing once with PBS, and counting; subsequently, 1-5×10 is taken ⁵ Centrifuging 300g of the cell suspension for 5min, discarding PBS, collecting cells, and adding 500uL of 1 XBing Buffer; adding 5uL of annexin v-FITC, adding 10uL of PI-PerCP/Cy5.5, gently vortex mixing, incubating at room temperature in dark place, detecting by commercial machine,

as shown in fig. 6, the final results show that, compared with the control group, the apoptosis ratio of NK cell exosomes extracted by the UC method and the uc+sec method is significantly increased, whereas the apoptosis-inducing activity of the uc+sec combination method is stronger than that of the UC method, i.e. the exosomes separated by the uc+sec method have stronger apoptosis-inducing activity on tumor cells.

According to the first and second embodiments, the purity of the NK cell exosomes extracted by the combination method of the ultracentrifugation method and the size exclusion chromatography is higher, the NK cell exosomes extracted by the combination method can effectively inhibit the proliferation of tumor cells, has a killing effect on the tumor cells, has the characteristic of remarkable anti-tumor activity, and can play a great role in the subsequent research of treating cervical cancer.

Although the embodiment of the invention only illustrates that the purity of the exosome separated and extracted by the UC+SEC combination method is obviously improved, the method for extracting the exosome by each combination method of the ultracentrifugation method, the ultrafiltration method, the size exclusion chromatography, the precipitation method and the immunoaffinity separation method which are performed by the experimental steps of the embodiment of the invention is within the protection scope of the invention and can improve the purity of the exosome.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (10)

1. An extraction method of exosomes for separating exosomes, capable of increasing the purity of the separated exosomes, characterized in that the extraction method comprises various combinations of ultracentrifugation, ultrafiltration, size exclusion chromatography, precipitation, immunoaffinity separation.

2. The method for extracting exosomes according to claim 1, wherein the extraction method is a combination of ultracentrifugation and size exclusion chromatography, the exosomes are first coarsely extracted by ultracentrifugation, and then further extracted by size exclusion chromatography to increase exosome purity.

3. The method of claim 2, wherein the method comprises subjecting the cell culture fluid to tangential flow concentration followed by ultracentrifugation.

4. A method of extracting exosomes according to claim 3, wherein the packing material of the chromatographic column used in the size exclusion chromatography is sepharose cl-6FF, and the gel filtration medium is formed by multi-step cross-linking sepharose cl-6B with 2,3 dibromopropanol.

5. An application of an extraction method of exosomes in NK cells for separating and purifying NK cells to obtain high-purity exosomes, comprising the extraction method of any one of claims 2-4, specifically comprising the following steps:

s1: culturing induced NK cells in a culture medium;

s2: separating NK cell exosomes from the NK cell culture supernatant by ultracentrifugation and size exclusion chromatography;

s3: carrying out characterization and identification on morphology, particle size and surface markers of the NK cell exosomes by a transmission electron microscope, nanoparticle tracking analysis, western blotting and flow cytometry;

s4: proteomic analysis of NK cell exosomes was performed by protein mass spectrometry.

6. The method for extracting exosomes according to claim 5 for use in NK cells, wherein step S2 specifically comprises:

s21: filtering culture supernatant of NK cells with 0.45um membrane, concentrating by 100kDa tangential flow for 10 times, performing differential centrifugation, centrifuging at 2000g for 20 min, centrifuging at 10000g for 30 min to remove dead cells and cell fragments, centrifuging at 100000g at 4deg.C for 90 min with ultra-high speed centrifuge, collecting precipitate, washing the obtained precipitate in phosphate buffer solution, centrifuging again at 100000g for 70 min, and re-suspending exocrine in phosphate buffer solution;

s22: purifying the exosomes obtained by ultracentrifugation by size exclusion chromatography, connecting a chromatographic column with a clearFirst-3000 type protein purification system, monitoring the exosomes by using an ultraviolet detector of an instrument, and collecting the exosomes with peaks detected by the ultraviolet detector for subsequent detection and analysis.

7. An application of the extraction method of exosomes in resisting tumors, which uses the separated and purified NK cell exosomes in resisting tumors, characterized by comprising the following steps:

s5: recovering tumor cells, and culturing in complete culture medium;

s6: the effect of NK cell exosomes on tumor cell viability and proliferation was examined by cell colony formation assay and CCK-8.

8. The use of the exosome extraction method according to claim 7 for anti-tumor, wherein the cell colony formation assay of step S6 specifically comprises: tumor cells were stained with 1000 cells/Kong Chongban, the exosomes of NK cells were added after tumor cells had been attached, the solution was changed after four days, giemsa staining was performed after one week, and the cells were observed with a microscope.

9. The use of an exosome extraction method according to claim 7 for anti-tumor, wherein the CCK-8 assay of step S6 is used to evaluate the activity of tumor cells, comprising: the exosomes of tumor cells and NK cells are placed in an orifice plate for co-culture, CCK-8 enhanced solution is added into each orifice, after light-shielding incubation is carried out for 2 hours at 37 ℃, an enzyme-labeled instrument is used for optical density reading, and proliferation conditions of the tumor cells are calculated.

10. The use of the method for extracting exosomes according to claim 7 for anti-tumor, further comprising: s7: uptake of NK cell exosomes in tumor cells was observed under a confocal laser microscope and tumor cell apoptosis was detected by flow cytometry.

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