WO2017071380A1 - Tumor vaccine for use in treating liver cancer and preparation method for vaccine - Google Patents

Abstract

A tumor vaccine for use in treating liver cancer and a preparation method for the vaccine. The liver cancer vaccine of improved therapeutic effects comprises exosomes corpuscles secreted by liver cancer cells, where the liver cancer cells underwent a combined dosing treatment with Cephaloziellins J and SAHA, and the molar concentration ratio of Cephaloziellins J to SAHA is 0.8-1.2:1. By using Cephaloziellins J and SAHA for the dosing treatment of the liver cancer cells, the result shows that exosomes secreted by the treated liver cancer cells and soluble immune molecules of the exosomes can significantly improve the described inhibitory effect, significantly increase the therapeutic effects of the exosomes tumor vaccine, and provide important clinical application values.

Description

Tumor vaccine for treating liver cancer and preparation method thereof Technical field

The invention belongs to the field of tumor vaccines, and particularly relates to a liver cancer vaccine with improved therapeutic effect, and a preparation method of the liver cancer vaccine.

Background technique

More than 90% of China's primary liver cancer is hepatocellular carcinoma (HCC), followed by cholangiocarcinoma, and the incidence ratio between men and women is 2 to 5:1. Tumor cells are antigenic and can cause the body to produce an immune response, which is the theoretical basis of tumor immunotherapy. In recent years, due to the development of molecular immunology, cell biology and bioengineering technology, clinical applications such as tumor vaccines, monoclonal antibodies, cytokines, immunologically active cell infusion and gene transfer technologies have become possible, enabling immunotherapy of liver cancer. Research has grown tremendously. In particular, liver cancer vaccine has attracted much attention and has become one of the research hotspots of liver cancer immunotherapy. The liver cancer vaccine based on liver cancer cells, liver cancer antigens, dendritic cells and nucleic acids is used to stimulate the body's immune system to induce the body to produce an immune response against liver cancer-specific antigens, thereby killing tumor cells expressing liver cancer antigens. The purpose of anti-tumor effect. Through clinical trials, it has been found that liver cancer vaccine can reduce the recurrence and metastasis of liver cancer, improve the quality of life of patients and prolong the survival time, and has become an important part of comprehensive treatment of liver cancer.

Although there are many researches on tumor vaccines at home and abroad, but there have been few breakthroughs so far. The main problems are: the introduction of DNA into specific cells for expression is not yet mature, and the use of foreign sources The safety of DNA has not been solved; because tumor cells are highly heterogeneous, different antigens can be expressed on multiple tumor cells belonging to the same type of tumor, and T cells activated by one tumor antigen can only kill a part of tumor cells. Tumor cells that do not express the antigen cannot be killed; although tumor cell vaccines can contain almost all tumor antigens, current studies have shown that their role in activating T cells is limited, and the effect as a vaccine is not satisfactory. Therefore, how to provide a tumor vaccine, which does not have a safety problem of use and can be effective against almost all tumor antigens, has become a problem to be solved.

Exosomes are a class of bilayer membranous vesicles that originate in the endocytic system and are excreted outside the cell, between 40 and 100 nm in diameter. Exosomes can be secreted by a variety of cells including dendritic cells, tumor cells, etc., containing a large number of proteins and lipid components closely related to its source and function, as an important carrier of information transmission between cells, participating in a variety of pathophysiological processes. . Tumor cell-derived exosomes contain important immune molecules such as tumor common antigen and thermophilin, which can exhibit anti-tumor effects through various pathways, and as a new type of tumor vaccine, it has obvious advantages over DC vaccine.

However, in recent years, some related experiments have shown that some tumor-derived exosomes can inhibit or even destroy immune cells that play a role in tumors, such as down-regulating the expression of some NK receptors, affecting the activation of some innate immune cells in tumor immunity. Others can significantly inhibit IL-2 and inhibit the proliferation of human lymphocytes, thus playing a negative role in the immunotherapy of tumors. These tumor-derived exosomes may be the key factors for tumor tissue to escape the immune system clearance, which brings many difficulties and challenges to tumor immunotherapy. Therefore, how to improve the immunostimulatory ability of tumor cells derived from exosomes, and reduce its immunosuppressive ability has great practical significance in tumor immunotherapy.

Summary of the invention

It is an object of the present invention to provide a liver cancer vaccine which does not inhibit lymphocyte proliferation function.

The above object of the present invention is achieved by the following technical solutions:

A liver cancer vaccine with improved therapeutic effect, comprising an exosomes body secreted by liver cancer cells, wherein the liver cancer cells are treated by Cephaloziellins J and SAHA, and the molar concentration ratio of Cephalozielins J and SAHA is 0.8 to 1.2:1. .

Further, the molar ratio of Cephaloziellins J to SAHA is 1:1.

Further, the liver cancer vaccine having improved therapeutic effect further includes an adjuvant.

Further, the adjuvant is an aluminum adjuvant.

The preparation method of the liver cancer vaccine comprises the following steps: the liver cancer cells are cultured in a CO ₂ incubator with a DMEI culture medium containing fetal bovine serum, and the cells are grown in a single layer, and are subcultured once every 3 to 4 days. The cells were inoculated to log phase. After 24 hours of inoculation, the cells were treated with Cephaloziellins J and SAHA. After 24 hours of culture, the culture supernatant was collected and stored at low temperature. The collected liver cancer cell culture supernatant was centrifuged to remove cells. The supernatant is taken; the cell debris is removed by centrifugation, the supernatant is collected, concentrated by ultrafiltration, and centrifuged to obtain a concentrate. The separated and purified concentrate is transferred to a centrifuge tube, and ultracentrifuged at a low temperature to obtain an exosomes body. .

Further, the preparation method comprises the following steps: the liver cancer cells are cultured in a DMEI medium containing 100 ml/L fetal bovine serum, and cultured in a 50 ml/L CO ₂ incubator at 37 ° C, and the cells are grown in a single layer, each of which is 3 Passage once every ～4 days, inoculate 3×10 ⁶ /100ml when cells grow to log phase; use 1×10 ^-6 mol/L Cephaloziellins J and 1×10 ^-6 mol/L SAHA after 24 hours of inoculation The cells were treated, and the culture supernatant was collected for 24 hours, and the culture supernatant was collected and stored at 4 ° C. The collected liver cancer cell culture supernatant was centrifuged for 10 min to remove the cells, and the supernatant was taken; the cell debris was removed by centrifugation at 1500 g for 30 min, and the supernatant was collected. The solution was concentrated by ultrafiltration through a 100 kU MWCO Centriplus centrifugal ultrafiltration tube, and centrifuged at 1500 g for 30 min to obtain a concentrated solution. The separated and purified concentrated solution was transferred to a centrifuge tube, and centrifuged at 100 kg for 60 min at a horizontal angle of 4 ° C to obtain a precipitate. Contains exosomes body.

The liver cancer vaccine is used in the preparation of a medicament for preventing liver cancer.

Advantages of the invention: It is known that nano-sized small vesicle exosomes secreted by untreated liver cancer cells and soluble immune molecules thereof have significant inhibitory effects on lymphocyte proliferation function. The present invention creatively treats liver cancer cells with Cephaloziellins J and SAHA, and the results show that the exosomes secreted by the treated liver cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition. The invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.

detailed description

The substantial content of the present invention is further illustrated by the following examples, but is not intended to limit the scope of the present invention. While the present invention has been described in detail with reference to the preferred embodiments of the present invention, it is understood that the invention may be modified or equivalently modified without departing from the spirit and scope of the invention. Experimental methods in which no specific conditions are indicated in the examples are generally carried out according to conventional conditions, such as those described in the textbooks and experimental guides, or in accordance with the conditions recommended by the manufacturer. The chemical structure and preparation method of Cephaloziellins J can be found in the literature: Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri, J. Nat. Prod., 2013, 76, 1700-1708.

Example 1: Cell Culture

Experimental materials: H22-H8D8 cell line, 10% fetal bovine serum, and streptomycin mixture (Beijing Tiger Beauty Co., Ltd.).

Experimental method: H22-H8D8 cell line was cultured in DMEI medium containing 10% fetal bovine serum, penicillin 100 IU/mL, streptomycin 100×μg/mL, and cultured in a 37 ° C 5% CO ₂ incubator until growth. In the logarithmic phase, inoculate at 3 × 10 ⁶ /100 ml.

Example 2: Drug treatment

Experimental material: Cephaloziellins J was prepared by the same method (Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri, J. Nat. Prod., 2013, 76, 1700-1708), identified as Cephaloziellins J; SAHA was purchased from Sigma.

Experimental group: The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Cephaloziellins J), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Cephaloziellins J and SAHA combined dosing).

Experimental method: control group, no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was treated with Cephaloziellins J at a concentration of 1×10 ^-6 mol/L after 24 h of inoculation, after 24 h of dosing The culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1×10 ^-6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation. 1×10 ^-6 mol/L of Cephaloziellins J and 1×10 ^-6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.

Example 3: Drug treatment

Experimental material: Cephaloziellins J was prepared by the same method (Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri, J. Nat. Prod., 2013, 76, 1700-1708), identified as Cephaloziellins J; SAHA was purchased from Sigma.

Experimental group: The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Cephaloziellins J), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Cephaloziellins J and SAHA combined dosing).

Experimental method: control group, no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was treated with Cephaloziellins J at a concentration of 1×10 ^-6 mol/L after 24 h of inoculation, after 24 h of dosing The culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1×10 ^-6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation. 0.8×10 ^-6 mol/L of Cephaloziellins J and 1×10 ^-6 mol/L of SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.

Example 4: Drug treatment

Experimental material: Cephaloziellins J was prepared by the same method (Secondary Metabolites from the Chinese Liverwort Cephaloziella kiaeri, J. Nat. Prod., 2013, 76, 1700-1708), identified as Cephaloziellins J; SAHA was purchased from Sigma.

Experimental group: The volume of cell culture medium was strictly consistent in each group, blank control group, exosomes control group (no drug group), exosomes experimental group 1 (dosing Cephaloziellins J), exosomes experimental group 2 (dosing SAHA), exosomes experimental group 3 (Cephaloziellins J and SAHA combined dosing).

Experimental method: control group, no drug was added after inoculation, 150 ml of supernatant was collected as control after 24 h; drug group 1 was treated with Cephaloziellins J at a concentration of 1×10 ^-6 mol/L after 24 h of inoculation, after 24 h of dosing The culture supernatant was collected 150ml; the drug-added group 2 was treated with SAHA at a concentration of 1×10 ^-6 mol/L after 24 hours of inoculation, and 150 ml of the culture supernatant was collected after 24 hours of drug addition; the drug-added group 3 was used for 24 hours after inoculation. 1.2×10 ^-6 mol/L Cephaloziellins J and 1×10 ^-6 mol/L SAHA were treated. After 24 hours of dosing, 150 ml of the culture supernatant was collected and stored at 4 °C.

Example 5: Isolation and purification of exosomes

Experimental equipment: HMAC-CP7OG cryogenic ultracentrifuge, 100 KU MW COMilliporeAmicon high recovery high flow tangential flow ultrafiltration centrifuge tube (Millipore).

Experimental methods: The experimental and control cell culture supernatants collected in Examples 2 to 4 were centrifuged at 300 g for 10 min to remove the cells, and the supernatant was taken; the cell debris was removed by centrifugation at 1500 g for 30 min, and the supernatant was collected and passed through 100 kUMWCO Centriplus. Centrifugal ultrafiltration tube concentrated ultrafiltration, centrifuged at 1500g for 30min to obtain 6ml concentrate, the separated and purified concentrate was transferred to a 1.5ml centrifuge tube, and the precipitate obtained by ultracentrifugation at 100kg for 60min at a horizontal angle of 4°C contained exosomes .

Example 6: Electron microscopic identification of exosomes

Drop 20-30μl exosomes suspension on the loaded copper mesh, let stand for 1min at room temperature, use the filter paper to suck the liquid from the side, add 20ml/L phosphotungstic acid solution (pH6.8) about 30μl on the copper network, and negatively dye at room temperature for lmin. The negative staining solution was blotted through a filter paper, and air-dried at room temperature for about 10 minutes to observe the photograph under a transmission electron microscope. The results showed that the exosomes secreted by H22-H8D8 cells under electron microscope showed a membrane-like microcapsule structure with a diameter of 30-80 nm, which was round or elliptical, and the cavity was low in electron density.

Example 7: Detection of proliferation of PBMC cells by H3-TdR incorporation assay

Experimental materials: H3-TdR (Shanghai Institute of Nuclear Research), Streptomycin Mixture (Beijing Taigemei), fetal bovine serum

Experimental instrument: β-liquid scintillation counter measurement (FJ-2107G type)

Experimental group: blank control group (plant lectin and PBMC cells only), exosomes control group (no drug group), exosomes experimental group 1 (dosing Cephaloziellins J), exosomes experimental group 2 (dosing SAHA), exosomes experiment Group 3 (Cephaloziellins J and SAHA combined dosing), exosomes extracted after supernatant control group (no drug group), exosomes extracted after supernatant group 1 (dosing Cephaloziellins J), exosomes extracted after supernatant group 2 ( Addition of SAHA), exosomes extraction supernatant group 3 (Cephaloziellins J and SAHA combined dosing), each group of three holes.

Experimental method: Separated PBMC cells were added to 5 ml of fresh RPMI 1640 medium containing 10% fetal bovine serum. After cell counting, the dilution was 5000/50 μl. According to the previous grouping, 50 μl/well was added to the 96-well plate. At the same time, a mixture of penicillin 100 IU/mL and streptomycin 100×μg/mL was added in a ratio of 1:1000, 50 μl of the sample was further added, and finally 100 μl of IPHA was added. Placed in a 50 ° CO ₂ incubator at 37 ° C overnight, the next day, PBMC cells were observed to grow in a pellet under a 10 × inverted microscope, and H3-TdR was added to the culture plate at 3.7 × 10 ⁴ Bq per well. After 6 hours of culture, the medium was removed, washed 3 times with 1×PBS, cell membrane was broken with 1 mol/L NaOH, scintillation fluid and anti-quenching agent were added, and the cpm value (minute count) was recorded with a β-liquid scintillation counter. The results are shown in Table 1.

Table 1 Effect of exosomes before and after combined drug treatment and supernatant after extraction on proliferation of PBMC cells

Group	Number of groups	Cpm value (x±S)
Blank control	3	24096±2207.62
Exosomes control group	3	5023±25.34

Exosomes experimental group 1	3	13472±774.28
Exosomes experimental group 2	3	13689±767.74
Exosomes experimental group 3	3	23998±2041.31
Supervised control group after exosomes extraction	3	4966±415.45
Supervised experimental group 1 after exosomes extraction	3	14926±5504.52
Exosomes extracted after supernatant experiment group 2	3	14714±5721.52
Supervised experimental group 3 after exosomes extraction	3	23839±5202.46

The results showed that compared with the blank control group, the untreated drug treated exosomes and PBMC cells had a significant effect on lymphocyte proliferation, which was significantly decreased (P<0.05). After drug treatment, the exosomes could be significantly improved. Inhibition of proliferative function, in which exosomes (exosomes experimental group 3) obtained after treatment with the pharmaceutical composition had the least inhibition of lymphocyte proliferation function, which was significantly less than that obtained by exosomes (exosomes experimental group 1 and exosomes experimental group 2). The inhibition of lymphocyte proliferation, PBMC proliferation value and the exosomes control group were statistically significant (P<0.05), and there was no significant difference compared with the blank control group (P>0.05). In the experimental group, the drug-treated supernatant extracted by exosomes and the PBMC cells co-cultured had little effect on cell proliferation, and there was no statistically significant difference compared with the blank control group (P>0.05), without drug treatment. The supernatant had a significant effect on lymphocyte proliferation, and there was a statistically significant difference compared with the blank control group or the supernatant group (P<0.05). The medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.

Example 8: In vivo anti-tumor experiment

Experimental materials: healthy Kunming mice, H22-H8D8 liver cancer mice, mouse liver cancer cells (H22-H8D8), DMEI medium (purchased from GIBCO), fetal bovine serum.

experimental method:

(1) Establish a tumor-bearing mouse model: aseptically take ascites from H22-H8D8 liver cancer mice, trypan blue staining, tumor cell count under light microscope, live tumor cells 9000, adjust the cell concentration to 1×10 ⁷ /ml, 0.2 ml of each healthy Kunming mouse was inoculated into the right sac, and a solid tumor-bearing mouse model was prepared.

(2) Preparation of tumor vaccine: The mouse hepatoma cells (H22-H8D8) were cultured according to the above Example 1; the cultured cells were divided into 4 groups, the first group was not administered, and the second group was administered with Cephaloziellins J, Three groups of drug-added SAHA, a fourth group of Cephaloziellins J and SAHA were combined, and the specific drug treatment was referred to Example 2; the collected cell culture supernatants were prepared according to Example 5 to prepare exosomes tumor vaccine.

(3) Grouping and treatment: 30 healthy Kunming mice were randomly divided into 5 groups, 6 in each group. Blank control group: On the day of tumor cell inoculation, physiological saline was injected subcutaneously into the root of the mouse; exosomes control group: the day of tumor cell inoculation, the first group of prepared tumor vaccine (10 μg/mouse) was injected subcutaneously into the leg root of the mouse; exosomes experiment Group 1: On the day of tumor cell inoculation, a second group of prepared tumor vaccines (10 μg/mouse) was injected subcutaneously into the roots of the mice; exosomes experimental group 2: on the day of tumor cell inoculation, the third group was prepared by subcutaneous injection into the roots of the mice. Tumor vaccine (10 μg/mouse); exosomes experimental group 3: On the day of tumor cell inoculation, a fourth group of prepared tumor vaccine (10 μg/head) was subcutaneously injected into the root of the mouse leg. Repeat 1 time every 1 day for 3 times. All mice were sacrificed on the 15th day after tumor inoculation, and the tumor was taken. Weigh, measure tumor volume, calculate tumor weight inhibition rate, tumor volume inhibition rate.

(4) Weigh the tumor weight and calculate the tumor inhibition rate

After the mice were sacrificed, the tumor tumors were taken out separately, and the blood was stained with filter paper. The tumor weight was weighed and the tumor inhibition rate was calculated. The tumor inhibition rate (%) = (control group tumor size - treatment group tumor size) / Control group tumor size × 100%.

Table 2 Inhibition of tumor vaccine on liver cancer xenografts

Experimental grouping	Tumor average
Blank control group	3.34±0.32
Exosomes control group	2.72±0.18
Exosomes experimental group 1	1.74±0.25
Exosomes experimental group 2	1.65±0.27
Exosomes experimental group 3	0.85±0.19

The growth of liver cancer xenografts was inhibited after treatment with exosomes tumor vaccine. The tumor masses of exosomes group 1, exosomes group 2 and exosomes group 3 were (1.74±0.25) g, (1.65±0.27) g, (0.85±0.19), respectively. g, compared with the blank control group (3.34±0.32) g, exosomes control group (2.72±0.18) g ratio was significant (P<0.05), Cephaloziellins J and SAHA combined drug vaccine prepared by the tumor vaccine had the strongest inhibition, the tumor quality was (0.85±0.19) g, the tumor inhibition rate reached 74.55%. The medicated group 3 of Examples 3 and 4 and Example 2 was able to prepare a vaccine having similar therapeutic activity.

Example 9: Selection of liver cancer vaccine adjuvant

Adjuvants generally refer to a class of substances that specifically bind to an antigen by physical or chemical means to enhance their specific immunity. The most widely used adjuvant in the world is the aluminum adjuvant. There are two main preparation methods for the aluminum adjuvant vaccine: one is to add an aluminum adjuvant to the antigen solution to form an aluminate protein precipitate; the other is to add the antigen solution to the previously prepared aluminum hydroxide, aluminum phosphate, hydrogen. An adsorptive vaccine is formed in a mixture of alumina and aluminum phosphate or in alumina. The aluminum phosphate adjuvant and the aluminum hydroxide adjuvant are positively charged at physiological pH, and the negatively charged protein is adsorbed in the lattice structure of the aluminum salt, and can be used for the treatment of the liver cancer vaccine in the preparation of the liver cancer vaccine of the present invention. effect.

Liposomes can also be used as an adjuvant for the liver cancer vaccine of the present invention to improve the therapeutic effect of the liver cancer vaccine.

The present invention creatively treats liver cancer cells with Cephaloziellins J and SAHA, and the results show that the exosomes secreted by the treated liver cancer cells and their soluble immune molecules can significantly improve the aforementioned inhibition. The invention significantly improves the therapeutic effect of the exosomes tumor vaccine and has important clinical application value.

The above embodiments are intended to illustrate the substantial content of the present invention, but do not limit the scope of the present invention. A person skilled in the art should understand that the technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. The invention relates to a liver cancer vaccine with improved therapeutic effect, which comprises: exosomes small body secreted by liver cancer cells, wherein the liver cancer cells are treated by Cephaloziellins J and SAHA, and the molar concentration ratio of Cephalozielins J and SAHA is 0.8. ~1.2:1.
2. The liver cancer vaccine having improved therapeutic effect according to claim 1, wherein the ratio of the molar concentration of Cephalozielins J and SAHA is 1:1.
3. The liver cancer vaccine improved in therapeutic effect according to claim 1 or 2, which further comprises an adjuvant.
4. The liver cancer vaccine improved in therapeutic effect according to claim 3, wherein the adjuvant is an aluminum adjuvant.
5. The method for preparing a liver cancer vaccine according to claim 1 or 2, comprising the steps of: culturing the liver cancer cells with a DMEI medium containing fetal bovine serum in a CO ₂ incubator, and the cells are grown in a single layer, each of which is 3 After 4 days of passage, the cells were inoculated until the logarithmic phase. After 24 hours of inoculation, the cells were treated with Cephaloziellins J and SAHA. After 24 hours of culture, the culture supernatant was collected and stored at low temperature; the collected liver cancer cells were collected. The culture supernatant is centrifuged to remove the cells, and the supernatant is taken; the cell debris is removed by centrifugation, the supernatant is collected, the ultrafiltration is concentrated, and the concentrate is centrifuged, and the separated and purified concentrate is transferred to a centrifuge tube, and ultracentrifuged at a low temperature. The resulting precipitate contains exosomes bodies.
6. The preparation method according to claim 5, comprising the following steps: the liver cancer cells are cultured in a DMEI medium containing 100 ml/L fetal bovine serum, and cultured in a 50 ml/L CO ₂ incubator at 37 ° C, and the cells are single-layered. Wall growth, passage once every 3 to 4 days, inoculate 3×10 ⁶ /100 ml when the cells grow to log phase; use 1×10 ^-6 mol/L Cephaloziellins J and 1×10 ^-6 after 24 h of inoculation The cells were treated with mol/L SAHA. After 24 hours of culture, the culture supernatant was collected and stored at 4 ° C. The collected liver cancer cell culture supernatant was centrifuged for 10 min to remove the cells, and the supernatant was taken. The cells were removed by centrifugation at 1500 g for 30 min. Fragment, collect the supernatant, concentrate the ultrafiltration through a 100kU MWCO Centriplus centrifugal ultrafiltration tube, centrifuge at 1500g for 30min to obtain a concentrate, transfer the separated and purified concentrate to a centrifuge tube, and centrifuge at 100kg with a horizontal corner at 4°C. At 60 min, the resulting precipitate contained exosomes bodies.
7. The liver cancer vaccine according to claim 1 or 2 is used for the preparation of a medicament for preventing liver cancer.