CN116426473A - NKG2D positive exosome, preparation method and application - Google Patents
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Abstract
The invention relates to an NKG2D positive exosome, a preparation method and application, belonging to the field of exosomes. According to the invention, by utilizing the characteristic of innovative tumor source exosomes on NK cell culture, activated NK cell NKG2GD positive exosomes are extracted in a large scale, so that the aim of treating liver cancer is fulfilled.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an exosome, in particular to an NKG2D positive exosome, a preparation method and application.
Background
Liver cancer is a cancer that originates in the liver and is an invasive tumor, often occurring in the context of chronic liver disease and cirrhosis. Primary liver cancer or hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the seventh most common cancer in women, the third leading cause of cancer-related death worldwide. Liver cancer is a high-incidence malignant tumor, and the incidence rate of malignant tumors in the world is 6 th, and the death number of liver cancer patients is 3 rd in all cancers in 83 ten thousand deaths.Although in spite ofMethods of treating liver cancer have advanced, but it remains one of the most difficult cancers to treat. For early HCC patients, surgery, local destructive therapy, and liver transplantation offer therapeutic potential. However, recurrence of HCC after curative treatment remains a major problem, with morbidity exceeding 70% after 5 years. Even small liver cancer (3 cm) patients in early stages of surgery have unsatisfactory survival rates for 5 years. Often, diagnosis of HCC is often in an advanced stage, and many advanced patients are not eligible to receive treatment. At present, liver cancer is treated by interdisciplinary and multi-mode treatment schemes are generally selected individually according to complex interactions such as tumor stage, degree of liver basic diseases and overall health condition of patients. In the United states (national Integrated cancer network [ NCCN)]) European (European liver research institute-European cancer research and treatment organization [ EASLERTTC ]]) And asia (consensus statement of asian tumor peak society in 2009 [ AOS)]) There is also a difference in management of liver cancer among different guidelines. Current strategies for treating liver cancer include systemic therapy, standard first-line therapy, cytotoxic chemotherapy, immunotherapy, oncolytic virus therapy, new targeted therapies being developed, surgical resection, liver transplantation, local area therapy, adjuvant therapy after surgical resection, etc., while strategies for treatment with exosomes remain in the research stage.
The evolutionary dynamics of the immune system are mainly due to the stress exerted by environmental pathogens. However, both innate and adaptive immunity can utilize different complementary mechanisms to control and prevent tumor growth and spread. For example, cytolytic T lymphocytes recognize tumor-derived polypeptides in the context of MHC class I molecules, thus requiring MHC expression on tumor cells, whereas natural killer cells (NK) are perceived and activated in the absence of MHC molecules. These different properties provide important clues to the development of cancer treatment strategies. Thus, the identification of tumor antigens and immunogenic tumor peptides enables the development of tumor vaccines that better target tumor cells. On the other hand, the ability of NK cells to kill MHC class I deficient tumors has been exploited by different adoptive cell therapies, including infusion of lymphokine activated NK cells, hematopoietic Stem Cell Transplantation (HSCT), especially HLA-haplotype homoHSCT (hard-HSCT), to treat high-risk leukemia. T cells and more recently NK cells engineered to express Chimeric Antigen Receptors (CARs) also represent a new strategy of particular interest for the treatment of blood and solid tumors.
NK cells are key elements of innate immunity, capable of mediating strong anti-tumor and antiviral responses, directly killing cancer or infected cells, and indirectly improving antibody and T cell mediated responses. Indeed, NK cell mediated cytotoxicity and cytokine release can affect the activity of additional innate immune cells (dendritic cells, macrophages and neutrophils) and confer NK cell modulating functions that can affect subsequent antigen-specific, HLA-restricted T and B cell responses. NK cells express a single self iKIR offer advantages to the host because it allows the perception of loss of a single HLA class I allotype or peptide-induced changes, thereby killing damaged cells by a "lost self-recognition" mechanism. By the same mechanism, when cultured NK cells express only iKIR(s) which is not involved in HLA class I molecules, a heterologous reaction can occur. This provides a breakthrough therapeutic regimen for the treatment of heterologous diseases. NK is, however, after all self-replicating, susceptible to tumor microenvironment in vivo, and of uncertain variability.
The exosomes are subcellular vesicles with an average diameter of 100nm surrounded by a lipid bilayer membrane. They are found in almost all body fluids, including blood, sweat, tears, urine, saliva, breast milk, ascites fluid and cerebrospinal fluid. Exosomes are a novel intercellular communication system that carry and transmit signaling molecules that regulate the physiological state of cells, closely related to the occurrence and development of various diseases. Many studies have shown that exosomes have a close relationship with the occurrence and progression of cancer. In tumor microenvironments, exosomes can transfer bioactive molecules between tumor cells, immune cells and stromal cells, helping cancer cells to escape immune surveillance, inducing immune tolerance. In contrast, exosomes from immune cells can inhibit tumor growth, proliferation and metastasis. Thus, exosomes play a role in cancer immunity.
The primary goal of tumor immunotherapy is to stimulate immunosuppressive hosts to recognize and eradicate cancer cells. Exosomes can be used as delivery systems for immunotherapeutic drugs, antigens or genes by modifying the cells that secrete the exosomes. Both immature and mature dendritic cells can produce exosomes, and DC-Derived Exosomes (DEXs) can overcome tumor-induced immunosuppression by direct and indirect means. The level of MHC I, MHC II and costimulatory molecules in the exosomes released by mature DCs is higher than that of immature DCs, with stronger immunostimulation. The DEXs surface has MHC/peptide complex, can promote proliferation and activation of IL-15Ra and NKG2D dependent NK cells, and can enhance the T cell dependent antitumor activity. DeXs also activates T cells, enhances T cell mediated immune responses, and inhibits the growth of breast cancer cells. Anti-tumor vaccines based on DEXs have entered phase I and phase II clinical trials after animal model trials. Studies have shown that in 15 metastatic melanoma patients, exosomes are not secondarily toxic at the maximum tolerated dose, suggesting the safety of DEXs as a tumor vaccine. In treating non-small cell lung cancer patients, DEX may carry the melanoma antigen gene MAGE, which demonstrates the feasibility and potential effectiveness of DEX vaccines. In addition, exosomes from the mouse cell line expressing hMUC1 promote immune responses and anti-tumor activity to combat the growth of tumor cells expressing hMUC1 in vivo. CAR-T cell derived exosomes can replace CAR-T cells as a method of tumor immunotherapy. Exosomes secreted by antigen-presenting cells (APCs) are capable of stimulating proliferation of T cells in vitro and inducing anti-tumor immune responses in vivo. TEXs contain tumor-associated antigens, costimulatory molecules, MHC I and MHC II molecules, potentially inducing potent anti-tumor immune responses. Tex can cross-present to cytotoxic T lymphocytes via APC, thereby exerting tumor killing effects. This may provide a valuable new direction for immunotherapy for cancer treatment.
The application number is CN 202011196500.9 discloses an exosome sorting method in the NK cell activation stage, and the method mainly solves the problem that no technology for directionally sorting umbilical cord blood specific exosomes exists at present. The specific stimulation is adopted to separate the extracellular secretion of the activated umbilical cord blood NK cell period, the IL-2 is utilized to proliferate NK cells, and IL-12, IL-15, GM-SCF and Fit3-L can promote the activation of NK cells and induce CD34+ stem cells to differentiate into NK cells. And sorting and collecting NK cells in about 10d of in vitro culture to obtain more amount of exosomes secreted by the NK cells. The invention focuses on a sorting strategy and a culture method of NK cells of an umbilical cord blood source; sorting the exosomes by means of anion exchange chromatography columns. And (5) a heavy preparation process.
The application number is CN 202110021453.2 discloses a preparation method and application of NK cell exosomes, wherein the method comprises the following steps: NK cell culture preparation NK cell exosomes (NKE) were prepared from NKEM. The NKEM and NKE prepared by the invention not only have antiviral and anticancer effects, but also have the effect of beautifying the skin. NKEM production is higher than NKE; the same NK cells were cultured in half, NKE and NKEM were prepared separately, and the yield of the latter was about 15 times higher than that of the former. Not only overcomes the bottleneck that NKE cannot be produced in large quantities, but also has certain reference value for industrialization of other extracellular secretion products. The present invention lyses NK cells and uses the cell contents to expand the amount of exosomes. The cell contains a large amount of lysosomes, digestive enzymes and the like which easily cause inflammatory reaction after entering the human body, and the corresponding safety detection is not carried out.
Disclosure of Invention
In view of the above problems, the present invention employs exosomes derived from a liver cancer cell line to specifically treat umbilical cord blood NK cells/adult NK cells during the culture period, and collects exosomes containing specific NKG2G markers to inhibit proliferation of liver cancer cells. According to the invention, by utilizing the characteristic of innovative tumor source exosomes on NK cell culture, activated NK cell NKG2GD positive exosomes are extracted in a large scale, so that the aim of treating liver cancer is fulfilled.
The invention adopts the following specific scheme:
a method for preparing an NKG2D positive exosome comprising the steps of:
step one, removing the exosomes of the fetal bovine serum: removing exosomes in the fetal calf serum by adopting an ultracentrifugation combined immunoadsorption column method;
culturing HepG2 cells: the method comprises cell resuscitating and cell passaging, wherein the completely cultured cells are resuscitated by a complete culture medium, and then subculture is carried out; the complete medium is alpha-MEM+10% FBS without exosomes removed;
step three, collecting HepG2 cell exosomes: taking cell supernatant, adopting ultracentrifugation to separate exosomes, discarding the supernatant, and re-suspending the precipitate with PBS, so that the exosomes exist in the re-suspension;
step four, NK cell separation and culture:
(1) Centrifuging fresh blood at 2900r/min for 10min, sucking the upper layer pale yellow blood plasma and a 50ml centrifuge tube, carrying out water bath at 40 ℃ for 30min, centrifuging at 1500r/min for 10min to remove sediment, transferring the upper layer blood plasma to a new 50ml centrifuge tube, and placing the new 50ml centrifuge tube in a refrigerator at 4 ℃ for standby;
(2) On day 0, initial culture: initial culture volume was 20ml, cell seeding density 1×10 6 Per ml, the culture fluid is: GT-561;
(3) Supplementing liquid: the number of cells increases on day 1, the culture solution turns yellow, and 10ml GT-561 is added; if the cell density is not obviously increased, postponing until the 2 nd day fluid replacement;
(4) On day 3, the culture broth obtained in step (3) was centrifuged at 300 Xg for 8min, the supernatant was discarded, the pellet was suspended with fresh GT-561, and the cell density was adjusted to 1X 10 6 Culturing in incubator;
(5) Observing cells on the 4 th day, and if the cell state is good and the culture solution does not have obvious yellowing, not performing treatment; if the cell density is significantly increased, GT-561 is added to make the cell density 1×10 6 /ml；
(6) Day 5, if day 4Untreated, good cell state, high density, yellowing culture solution, and adding GT-561 to give cell density of 1×10 6 /ml；
(7) Transfer culture bag: counting on days 6-7, adding 5 feeder cells (1 ml) when the number of the cells is greater than 100M, and adding feeder cells when the number of the cells is 30-60 million, and adding the feeder cells until the 8 th day; GT-561 was added to give a total cell concentration of 1.0X10 6 /ml; transferring to T175 culture when the culture solution volume is larger than 100ml or the cell number is larger than 100M, gradually transferring to a cell culture bag for culture when the culture volume is increased;
(8) GT-561 was added to give a cell concentration of 1.0X10 when observed daily on days 8-11 6 /ml；
(9) GT-561 was added to give a cell concentration of 2.0X10 when observed daily on days 12-14 6 /ml；
(10) Harvesting the cells at 14-15 days;
step five, collecting and identifying NKG2D positive exosomes: coating an adsorption column with an anti-NKG2D biotin-labeled antibody to obtain an anti-NKG2D antibody-loaded adsorption column, collecting NKG2D positive exosomes by adopting the anti-NKG2D antibody-loaded adsorption column, and obtaining the NKG2D positive exosomes after identification is correct.
As a further optimization of the preparation method, in the first step, the immune adsorption column is filled with exosome adsorption material, the adsorption material is loaded with a polyclonal antibody with the specificity of CD9/CD963/CD81, and the antibody is attached to the surface of porous silica gel microspheres or high molecular porous microspheres. Further, the removal of the fetal bovine serum exosomes comprises the following steps:
(1) Subjecting FBS to high-speed centrifugation at 10000 Xg at 4deg.C for 30min, and collecting supernatant;
(2) Ultracentrifugation of the supernatant at 120000Xg, 4deg.C for 90min, and collecting the supernatant;
(3) The supernatant of the isolated FBS was filtered using a porous silica gel microsphere immunoadsorption column coated with a polyclonal antibody specific for CD9/CD963/CD 81.
As a further optimization of the above preparation method, in step five, the collection of NKG2D positive exosomes comprises the following steps:
(1) Taking cell supernatant, adding the cell supernatant into a 250ml sterile centrifuge tube, and centrifuging at 300 Xg and 4 ℃ for 10min;
pouring the supernatant into a new 250ml centrifugal bottle, putting into a floor-type centrifugal machine, and centrifuging at 4 ℃ for 30min at 2000 Xg;
(3) Connecting a suction filter with a suction filter pump with a diameter of 0.45 μm, and suction-filtering the centrifuged supernatant to obtain a suction filtrate;
(4) Transferring the filtrate into a 50ml centrifuge tube, centrifuging at 10000 Xg and 4 ℃ for 30min;
(5) Coating an adsorption column with an anti-NKG2D biotin-labeled antibody;
(6) Washing the adsorption column with PBS buffer containing 5% ABS;
(7) Eluting exosomes from the adsorption column with eluent, and centrifuging at 110000Xg and 4deg.C for 90min; resuspended in PBS.
NKG2D positive exosomes prepared by the above preparation method.
A preparation comprising the above NKG2D positive exosomes. Preferably, the preparation is an NK cell culture nourishing activator prepared by taking exosomes or genetically engineered exosomes as carriers.
Use of the NKG2D positive exosomes described above for the manufacture of a medicament for the treatment of a disease, including cancer, diabetes, arthritis or hypertension. Further, the disease is liver cancer.
The beneficial effects are that: (1) Compared with the conventional method for extracting the NK cell exosomes at present, the NK cell exosomes have more pertinence in application effector aspect. The exosome stimulation of liver cancer cell line (exosome contains some special surface marks and plasma membrane contains several special nucleic acids and protein components) can make NK cell produce special correspondence to release exosome with specific function so as to implement inhibition effect.
(2) Research shows that liver cancer cells can express MICA and MICB molecules, which are important ligands of NKG 2D; increasing NKG2D screening performance of NK cell exosomes can improve targeting binding of exosomes to liver cancer cells, thereby improving therapeutic effect.
Drawings
FIG. 1 is a flow chart of exosome preparation according to the present invention;
FIG. 2 is a diagram of NKG 2D-positive exosome immunochromatography process;
FIG. 3 is a graph showing the results of electron microscopy of NK exosomes;
FIG. 4 is a graph showing the detection result of CD314 surface marker of NK exosomes;
FIG. 5 is a graph showing cytotoxicity of NK exosomes against hepatoma cells HepG2 cells.
Detailed Description
The invention provides a therapeutic or inhibitory exosome preparation of liver cancer cells, which can reduce or inhibit the progress of liver cancer. The invention adopts exosomes derived from liver cancer cell lines to carry out specific treatment on umbilical cord blood NK cells/adult NK cells in a culture period, and collects exosomes containing specific NKG2G markers to inhibit proliferation of liver cancer cells. According to the invention, by utilizing the characteristic of innovative tumor source exosomes on NK cell culture, activated NK cell NKG2GD positive exosomes are extracted in a large scale, so that the aim of treating liver cancer is fulfilled.
Nkg2d positive exosomes:
(1) The source is as follows: NK cells, NK92 cell lines and other cell lines capable of producing NKG2D or genetically engineered cell lines or cell lines capable of targeting NKG 2D.
(2) NKG2D expression positive exosomes: inhibit chimeric application of the following sequences or partial sequences of NKG2D in exosomes; chimeric applications of the following sequences or the following partial sequences of NKG2D in exosomes are prohibited and the application of the scope of the present patent is distinguished by the inclusion of other components.
mgwrgrrsrhswmshnyndkksdstrwkrcvvkskcrnasccavamgrmvawsavnsnvtsycgcknwcyknncydsknwysascmsnaskvyskddkvksyhwmgvhtngswwdgssntmkgdcayasskgyncstntycmrtv
(3) The application of the exosomes positive to the NKG2D expression in the treatment of diseases comprises the application of the exosomes which take the NKG2D as a main component or an auxiliary component, such as cancers, diabetes, arthritis, hypertension and the like in the diseases.
Sorting technique of nkg2d positive exosomes:
(1) An adsorption column loaded with an anit-NKG2D antibody is protected.
(2) Sorting technology step of protecting NKG2D positive exosome.
Sorting identification of NKG 2D-positive exosomes
(1) Protecting an immunomagnetic bead loaded with CD 63.
(2) And (3) an immunomagnetic bead separation technology step for protecting NKG2D positive exosomes.
Exosome stimulation of NK cells
(1) Protecting the application of HepG2 cell-derived exosomes in NK cell culture.
(2) All commercial applications of NK cells following stimulation of exosomes derived from HepG2 cells were protected.
(3) All commercial applications of NK cell culture supernatants following stimulation of HepG2 cell-derived exosomes were protected.
(4) All commercial applications of protecting all extracellular vesicles in NK cell culture supernatants after stimulation of HepG2 cell-derived exosomes.
(5) Protecting any NK cell culture nourishing activator prepared by taking exosomes or genetically engineered exosomes as carriers.
The technical scheme of the invention will be clearly and completely described below with reference to the embodiments of the invention and the accompanying drawings.
Example 1
A preparation for specifically inducing exosome for treating liver cancer is shown in figure 1.
1. Removal of bovine serum exosomes
The purpose of the process is as follows: reducing the content of exosomes in the treated serum so as to reduce the influence of exosomes in the animal serum on NK cells. At present, the method for removing the exosomes in the serum mainly adopts an ultracentrifugation method, an immunomagnetic bead method and the like, the ultracentrifugation method needs to be operated continuously for a plurality of times, and the ultracentrifugation method needs to be kept stand for a long time, but part of exosomes still remain in the obtained serum. The immunomagnetic beads method has high cost, can only treat a small amount of serum in one operation, and can only remove part of exosomes in the serum. When the serum is treated by the existing method, more exosomes still exist in the treated serum, and the research on downstream cell exosomes still can be influenced to a certain extent, so that there is room for improvement. The method adopts a method of combining ultracentrifugation with an immunoadsorption column to achieve the efficient removal effect.
1. High-speed centrifugation is carried out on FBS for 10000 Xg, 4 ℃ and 30min; the supernatant was collected under sterile conditions.
2. Ultracentrifugation of the supernatant at 120000 Xg, 4℃for 90min; the supernatant was collected under sterile conditions.
3. The supernatant of the isolated FBS was filtered using a porous silica gel microsphere adsorption column coated with a polyclonal antibody specific for CD9/CD963/CD 81.
The technology provides an exosome adsorption column, wherein exosome adsorption materials are filled in the exosome adsorption column, and the adsorption materials are loaded with CD9/CD963/CD81 specific polyclonal antibodies; a molecular marker capable of specifically recognizing transmembrane protein CD9/CD963/CD81 on the surface of exosomes; the antibodies in the three are attached to the surfaces of the full-porous silica gel microspheres or the high-molecular porous microspheres, and can be specifically adsorbed when the supernatant containing the exosomes passes through the packed column. The porous structure of the full porous silica gel microsphere has adsorption property, and the larger specific surface area enables the full porous silica gel microsphere to load more exosomes, so that serum and exosomes can be separated more efficiently. The three antibodies are adopted, so that the screening leakage phenomenon caused by the heterogeneity of exosomes can be prevented.
2. Culture of HepG2 cells
1. Cell resuscitation
1) Preparing a complete culture medium: alpha-mem+10% FBS (no exosomes removed);
2) Cell resuscitation: taking 9ml of complete culture based on a 15ml centrifuge tube, rapidly taking out frozen cells from liquid nitrogen, placing the frozen cells into a 37 ℃ metal bath, shaking to rapidly defrost the frozen cells, then transferring the thawed cells into an ultra-clean workbench, adding the thawed cells into the 15ml centrifuge tube, and centrifuging at 300 Xg for 5min;
3) The supernatant was discarded to give a cell pellet, and the cells were gently resuspended in 2ml of complete mediumAdding into T25 culture flask, marking, adding 37deg.C and 5% CO 2 Culturing in a saturated moderate incubator);
4) After 24h, the cell attachment was observed, the old medium was aspirated off, and fresh pre-warmed complete medium was added to continue the culture.
2. Cell passage
1) When the cells grow to 80-90% fusion degree, sucking old culture medium, washing once with PBS to remove residual culture medium and serum, adding 0.125% pancreatin, digesting in a 37 ℃ incubator, taking out the cells, and observing the cells under the mirror until the cells shrink and become round;
2) Adding a culture medium containing FBS, gently beating to enable cells to fall off to form single cell suspension, collecting cells in a 15ml sterile centrifuge tube, centrifuging at 1000rpm for 5min;
3) Cell pellet was collected, resuspended in complete medium, and finally inoculated into 5-10 fine layers of cell factory for continued culture using alpha-mem+10% FBS (no exosomes removed).
3. Collection of HepG2 cell exosomes
Centrifugal forces generated by ultracentrifugation, up to 1,000,000×g, are the best process for separating small particles including bacteria, viruses and organelles. Ultracentrifugation is therefore easily converted into isolation of exosomes and contributes to many open exosome searches.
1. Taking cell supernatant, adding into a 250ml sterile centrifuge tube, and centrifuging at 300 Xg and 4 ℃ for 10min;
2. pouring the supernatant into a new 250ml centrifugal bottle, putting into a floor type centrifugal machine, and centrifuging at 4 ℃ for 30min at 2000 Xg;
3. the centrifuged supernatant was filtered by a 0.45 μm filter pump connected to a filter pump.
4. The above filtrate was transferred to 10000 Xg in 50ml centrifuge tube and centrifuged at 4℃for 30min.
5. Transferring the liquid into a 38ml super-separation tube, balancing, and centrifuging at 110000 Xg and 4 ℃ for 90min;
6. discarding the supernatant, re-suspending the sediment with 200 μl PBS, repeatedly blowing the bottom of the super-release tube with a gun head, taking care to avoid too much foam, and allowing exosomes to exist in the re-suspension;
4. isolation and culture of NK cells
1. Fresh blood is centrifuged at 2900r/min for 10min, the upper pale yellow blood plasma and a 50ml centrifuge tube are sucked, water bath is carried out at 40 ℃ for 30min, sediment is removed after centrifugation at 1500r/min for 10min, the upper blood plasma is transferred to a new 50ml centrifuge tube, and the fresh blood plasma is placed in a refrigerator at 4 ℃ for storage (if sediment is separated out after the fresh blood plasma is placed in the refrigerator for two days, the fresh blood plasma is centrifuged again).
2. On day 0, initial culture (T75 square bottle): initial culture volume was 20ml, cell seeding density 1×10 6 /ml (initial procedure may be appropriate to increase cell seeding density). The culture solution is as follows: GT-561 (containing lymphocyte activating factor).
3. In general, the number of cells increases on day 1, the cell density increases significantly or the phenomenon of agglomeration occurs, and the culture solution turns yellow, and 10ml of GT-561 (containing lymphocyte activator) can be added. If the increase in cell density is not significant, the fluid replacement can be delayed until day 2.
4. On day 3, the culture broth obtained in the above step was centrifuged at 300g for 8min, the supernatant was discarded, and the supernatant was precipitated by suspension with fresh GT-561 (containing lymphocyte activator) and the cell density was adjusted to 1X 10 6 /ml (initial procedure may suitably increase cell density), in culture in an incubator.
5. On day 4, cells were observed, if the cell status was good, the culture medium was not significantly yellowing, and if the cell density was significantly increased, GT-561 (containing lymphocyte activator) was added as appropriate to give a cell density of 1×10 6 /ml (initial procedure may suitably increase cell density).
6. On day 5, if the cells are not treated on day 4 and the cell state is good and the density is high, GT-561 (containing lymphocyte activator) may be added as appropriate to give a cell density of 1×10 6 /ml (initial procedure may suitably increase cell density).
7. Transfer culture bag: counting on day 6-7, and adding 5 feeder cells (note centrifuging to remove lyophilized solution) with cell number greater than 100M, such as 3-6X10 6 Then the nourishment fine is added until the 8 th dayAnd (5) cells. GT-561 (containing lymphocyte activator) was added to give a total cell concentration of 1.0X10 6 /ml. The culture solution with the volume of more than 100ml or the cell number of more than 100M can be transferred to T175 for culture, and then gradually transferred to a cell culture bag for culture along with the increase of the culture volume.
8. On day 8-11, the GT-561 (containing lymphocyte activator) can be estimated empirically to give a cell concentration of 1.0X10 when the operator is skilled 6 About/ml. If the method is used for the first time, the culture medium is preferably added by counting so that the cell concentration is 1.0X10 6 /ml。
9. Day-to-day observations on days 12-14, GT-561 (containing lymphocyte activator) can be estimated empirically to give a cell concentration of 2.0X10 if the operator is skilled 6 About/ml. If the method is used for the first time, the culture medium is preferably added by counting so that the cell concentration is 2.0X10 6 /ml。
10. Cells are typically harvested at 14-15 days, when they are in the log phase, and the cell state is best, and slowly slides down further.
5. Collection of NKG2D positive exosomes.
1. Taking cell supernatant, adding into a 250ml sterile centrifuge tube, and centrifuging at 300 Xg and 4 ℃ for 10min;
2. pouring the supernatant into a new 250ml centrifugal bottle, putting into a floor type centrifugal machine, and centrifuging at 4 ℃ for 30min at 2000 Xg;
3. the centrifuged supernatant was filtered by a 0.45 μm filter pump connected to a filter pump.
4. The above filtrate was transferred to 10000 Xg in 50ml centrifuge tube and centrifuged at 4℃for 30min.
5. The adsorption column was coated with anti-NKG2D biotin-labeled antibody.
6. The column was washed once with 5% abs in PBS buffer.
7. Eluting exosomes from the adsorption column with eluent, and centrifuging at 110000×g, 4deg.C for 90min; resuspended in PBS.
A diagram of the NKG 2D-positive exosome immunochromatography procedure is shown in FIG. 2.
6. Morphological identification of exosomes
(1) Morphological identification of exosomes
Electron microscope detection results: taking out 10 mu L of exosomes; 10 mu L of the sample is sucked and dripped on a copper net to be deposited for 1 min, and floating liquid is sucked by filter paper; dripping 10 mu L of uranyl acetate on a copper net to precipitate for 1 min, and sucking floating liquid by filter paper; drying for several minutes at normal temperature; 100 kv was subjected to electron microscopy imaging. The results are shown in FIG. 3.
From the figure, it can be seen that Nk exosomes present a disk-like or cup-like structure, with a diameter around 100nm, with typical resulting features and dimensions of the exosomes.
Detection of surface markers: the exosomes were labeled with Beads-anti-CD63 and APC-anti-CD314 (NKG 2D) and were subjected to flow detection. The results are shown in FIG. 4. From the detection results, the selected exosomes are enriched with the specific marker NKG2D surface molecules.
7. Functional identification of exosomes
Cytotoxic effects of exosomes on hepatoma cells HepG2 cells: the mitochondrial membrane potential of tumor cells (HepG 2) was detected with JC-1 probe. Cells were seeded in 6-well plates (1X 10 per well) 5 Cells), with NK-Exo (40 g/ml), control (40 g/ml), incubation for 24h, respectively. Cells were then loaded with JC-1 (1 mg/L) at 37℃for 20min. After washing with cold PBS, the fluorescence intensities of each group were analyzed with a fluorescence confocal laser microscope. The results are shown in FIG. 5.
JC-1 staining results show that the mitochondrial membrane potential of tumor cells is obviously increased after NK-Exo treatment, which indicates that the sorted NK exosomes have obvious killing effect on liver cancer cells.
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (8)
1. A method for preparing an NKG2D positive exosome, characterized by: the method comprises the following steps:
step one, removing the exosomes of the fetal bovine serum: removing exosomes in the fetal calf serum by adopting an ultracentrifugation combined immunoadsorption column method;
culturing HepG2 cells: the method comprises cell resuscitating and cell passaging, wherein the completely cultured cells are resuscitated by a complete culture medium, and then subculture is carried out; the complete medium is alpha-MEM+10% FBS without exosomes removed;
step three, collecting HepG2 cell exosomes: taking cell supernatant, adopting ultracentrifugation to separate exosomes, discarding the supernatant, and re-suspending the precipitate with PBS, so that the exosomes exist in the re-suspension;
step four, NK cell separation and culture:
(1) Centrifuging fresh blood at 2900r/min for 10min, sucking the upper layer pale yellow blood plasma and a 50ml centrifuge tube, carrying out water bath at 40 ℃ for 30min, centrifuging at 1500r/min for 10min to remove sediment, transferring the upper layer blood plasma to a new 50ml centrifuge tube, and placing the new 50ml centrifuge tube in a refrigerator at 4 ℃ for standby;
(2) On day 0, initial culture: initial culture volume was 20ml, cell seeding density 1×10 6 Per ml, the culture fluid is: GT-561;
(3) Supplementing liquid: the number of cells increases on day 1, the culture solution turns yellow, and 10ml GT-561 is added; if the cell density is not obviously increased, postponing until the 2 nd day fluid replacement;
(4) On day 3, the culture broth obtained in step (3) was centrifuged at 300 Xg for 8min, the supernatant was discarded, the pellet was suspended with fresh GT-561, and the cell density was adjusted to 1X 10 6 Culturing in incubator;
(5) Observing cells on the 4 th day, and if the cell state is good and the culture solution does not have obvious yellowing, not performing treatment; if the cell density is significantly increased, GT-561 is added to make the cell density 1×10 6 /ml；
(6) On day 5, if the cells are not treated on day 4 and the cell state is good, the density is high, the culture solution turns yellow, and GT-561 is added to make the cell density 1×10 6 /ml；
(7) Transfer culture bag: count on day 6-7, cell numberIf the amount is more than 100M, adding 5 1ml of trophoblasts, if the number of the trophoblasts is 30-60 million, adding the trophoblasts until the 8 th day; GT-561 was added to give a total cell concentration of 1.0X10 6 /ml; transferring to T175 culture when the culture solution volume is larger than 100ml or the cell number is larger than 100M, gradually transferring to a cell culture bag for culture when the culture volume is increased;
(8) GT-561 was added to give a cell concentration of 1.0X10 when observed daily on days 8-11 6 /ml；
(9) GT-561 was added to give a cell concentration of 2.0X10 when observed daily on days 12-14 6 /ml；
(10) Harvesting the cells at 14-15 days;
step five, collecting and identifying NKG2D positive exosomes: coating an adsorption column with an anti-NKG2D biotin-labeled antibody to obtain an anti-NKG2D antibody-loaded adsorption column, collecting NKG2D positive exosomes by adopting the anti-NKG2D antibody-loaded adsorption column, and obtaining the NKG2D positive exosomes after identification is correct.
2. The method of manufacturing according to claim 1, characterized in that: in the first step, the immune adsorption column is filled with exosome adsorption material, the adsorption material is loaded with CD9/CD963/CD81 specific polyclonal antibody, and the antibody is attached to the surface of porous silica gel microsphere or high molecular porous microsphere.
3. The preparation method according to claim 2, characterized in that: the removal of the exosomes of the fetal bovine serum comprises the following steps:
(1) Subjecting FBS to high-speed centrifugation at 10000 Xg at 4deg.C for 30min, and collecting supernatant;
(2) Ultracentrifugation of the supernatant at 120000Xg, 4deg.C for 90min, and collecting the supernatant;
(3) The supernatant of the isolated FBS was filtered using a porous silica gel microsphere immunoadsorption column coated with a polyclonal antibody specific for CD9/CD963/CD 81.
4. The method of manufacturing according to claim 1, characterized in that: in the fifth step, the collection of NKG2D positive exosomes comprises the following steps:
(1) Taking cell supernatant, adding the cell supernatant into a 250ml sterile centrifuge tube, and centrifuging at 300 Xg and 4 ℃ for 10min;
(2) Pouring the supernatant into a new 250ml centrifugal bottle, putting into a floor-type centrifugal machine, and centrifuging at 4 ℃ for 30min at 2000 Xg;
(3) Connecting a suction filter with a suction filter pump with a diameter of 0.45 μm, and suction-filtering the centrifuged supernatant to obtain a suction filtrate;
(4) Transferring the filtrate into a 50ml centrifuge tube, centrifuging at 10000 Xg and 4 ℃ for 30min;
(5) Coating an adsorption column with an anti-NKG2D biotin-labeled antibody;
(6) Washing the adsorption column with PBS buffer containing 5% ABS;
eluting exosomes from the adsorption column with eluent, and centrifuging at 110000Xg and 4deg.C for 90min; resuspended in PBS.
5. NKG2D positive exosomes prepared according to the method of any one of claims 1-4.
6. A formulation comprising the NKG2D positive exosomes of claim 5.
7. Use of the NKG2D positive exosome according to claim 5 or the formulation according to claim 6 for the manufacture of a medicament for the treatment of a disease comprising cancer, diabetes, arthritis or hypertension.
8. The use according to claim 7, characterized in that: the disease is liver cancer.
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| CN116904397B (en) * | 2023-09-04 | 2023-12-26 | 山东德升细胞治疗工程技术有限公司 | Preparation and application of extracellular vesicles of NK cells |
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