CN108753726B

CN108753726B - Exosome containing ECRG4mRNA and preparation method and application thereof

Info

Publication number: CN108753726B
Application number: CN201810594022.3A
Authority: CN
Inventors: 党喜同; 毛亮; 周锐
Original assignee: Southwest Medical University
Current assignee: Southwest Medical University
Priority date: 2018-06-11
Filing date: 2018-06-11
Publication date: 2020-10-09
Anticipated expiration: 2038-06-11
Also published as: CN108753726A

Abstract

The invention discloses an exosome containing ECRG4mRNA and a preparation method and application thereof; the exosome is loaded with ECRG4mRNA generated by cells over expressing ECRG4 gene and can be transported to receptor cells, so that the expression level of the ECRG4mRNA of the receptor cells is remarkably improved, and the expression of genes related to tumor cell proliferation/growth, angiogenesis and inflammatory reaction is further inhibited; meanwhile, the exosome has good compatibility with receptor cells, does not cause the immune reaction and inflammatory reaction of the receptor, and can obviously reduce the toxic and side effect of gene therapy; the exosome can obviously inhibit the proliferation and growth of tumor cells; has positive effect on treating tumor diseases.

Description

Exosome containing ECRG4mRNA and preparation method and application thereof

Technical Field

The invention relates to the field of cell biology, in particular to an exosome containing ECRG4mRNA, a preparation method and application thereof.

Background

Oral Squamous Cell Carcinoma (OSCC) is a malignant tumor that seriously threatens human health, and the search for safe and effective treatment regimens is a hotspot of medical research. Human esophageal cancer-associated Gene 4 (Esophageal cancer Related Gene 4, ECRG4) is a tumor suppressor Gene cloned from normal human esophageal epithelium and having a broad tumor suppression function by using mRNA differential display technology. ECRG4 is widely distributed in various tissues and organs of the human body, including the heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and the like. In the process of generating a plurality of tumors such as esophagus cancer, colon cancer, breast cancer, lung cancer, kidney cancer and the like, the ECRG4 expression level is obviously reduced, and the expression level is in positive correlation with the prognosis of a tumor patient. Experiments prove that: the recovery of ECRG4 expression can inhibit the proliferation and migration of tumor cells such as gastric cancer, colon cancer, esophageal cancer, liver cancer, brain glioma, etc. in vitro, and inhibit the growth and metastasis of various transplanted tumors in vivo. Therefore, the gene delivery technology can improve the expression level of ECRG4 in tumor cells, and has potential therapeutic value.

Exosomes (exosomes) are membrane-like bodies secreted by cells, with diameters of about 30-150 nm. Almost all cells in the human body secrete exosomes, which are also present in all body fluids. The exosomes are formed as follows: the inclusion bodies in the cells are invaginated, and simultaneously selectively wrap proteins, lipids, miRNA, mRNA, DNA, lncRNA and other nucleic acids in cytoplasm to form membrane bodies, when the inclusion bodies are fused with cell membranes, the membrane bodies are released to the outside of the cells and enter the systemic circulation, and the membrane bodies are exosomes. The exosome is combined with a receptor cell to release the content of the exosome to the receptor cell, so that the signal transmission and the genetic material exchange between cells are mediated, and the occurrence and development processes of the receptor cell diseases are further influenced. Research finds that exosome enters receptor cells through a high-efficiency mechanism similar to virus invasion into cells, has good biodistribution and biocompatibility, is natural, stable and nano-sized, can penetrate biological barriers, has low immunogenicity, can carry single or random combined therapeutic preparations, and is an ideal gene delivery vector.

The earlier stage research of the invention finds that the human serum exosome contains full-length ECRG4mRNA, and the expression level of ECRG4mRNA in the serum exosome of the oral squamous carcinoma patient is obviously lower than that of a healthy person, which suggests that the reduction of the expression level of ECRG4mRNA in the serum exosome is closely related to the occurrence of tumor, and the improvement of ECRG4mRNA in the serum exosome has potential tumor inhibition effect. However, since exogenous mRNA directly introduced into blood is easily degraded by nuclease in blood, it is necessary to use a gene delivery vector. At present, the traditional gene delivery vectors mainly comprise viral vectors or non-viral vector systems, and have the defects of vector toxicity, targetloss, low-efficiency gene delivery, short in vivo biological half-life, inflammation and immune response, and the like, so that the safety and the effectiveness of gene therapy are seriously affected, and a safer and more efficient gene delivery vector needs to be searched and developed. As the exosome has the excellent characteristics of natural stability, nano size, no immunogenicity, no cytotoxicity, capability of crossing biological barriers and the like, the exosome has great application value as a gene delivery vector.

Since exosomes are secreted by the cell itself and selectively encapsulate signal molecules in the cytoplasm during formation, the mechanism of their selectivity is unclear. Therefore, it is a technical difficulty how to load a therapeutically valuable gene into an exosome. Foreign genes are loaded into exosomes, and the following four methods are mainly used: (1) electroporation: introducing exogenous genes synthesized in vitro into exosomes in an electroporation mode; (2) repeated freeze thawing method: mixing exosomes and exogenous genes, repeatedly freezing and thawing, and packaging the exosomes into exosomes; (3) direct transfection: directly transfecting the exogenous gene synthesized in vitro into an exosome by using a transfection reagent such as Lipofectamine; (4) indirect transfection: the exogenous gene is cloned into an expression vector, an exosome 'generating cell' is transfected, the target gene is over-expressed in the 'generating cell', and the exogenous gene over-expressed in cytoplasm is packaged into the exosome in the generating process of the exosome.

The first three methods mainly change the physical structure of an exosome through a certain stimulation factor, so that the exosome is dynamically loaded with exogenous genes. In the 4 th method, because of the difference of expression modes, effects, limiting factors and the like of each gene in different cells, when different genes are loaded into exosomes, the exosomes with large number of loaded genes, good expression stability, good compatibility and good applicability can be obtained only by performing targeted selection according to the types of the genes, the types of 'producing cells' and culture methods, culture environments and the like.

Disclosure of Invention

The invention aims to provide an exosome loaded with a large amount of ECRG4mRNA, and provides a specific preparation method and related application of the exosome in gene therapy. The exosome is loaded with a large amount of ECRG4mRNA and can be conveyed to a receptor cell, so that the expression level of ECRG4 in the receptor cell can be obviously improved, and the expression of genes related to tumor cell proliferation/growth, angiogenesis and inflammatory reaction is further inhibited; meanwhile, the exosome has good compatibility with receptor cells, so that the exosome does not cause immune reaction and inflammatory reaction of the receptor, and the toxic and side effects of gene therapy can be obviously reduced; especially, the exosome can obviously inhibit the proliferation and growth of oral squamous cell carcinoma, and can also be used for inhibiting the growth of tumor cells such as esophageal cancer, colon cancer, breast cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, esophageal cancer, liver cancer, brain glioma and the like; has positive effect on tumor treatment.

To achieve the above object, the present invention provides an exosome loaded with ECRG4mRNA produced by a cell overexpressing ECRG4 gene; wherein the ECRG4mRNA comprises the complete ECRG4 encoding gene.

The invention provides an exosome, which is formed by secreting a cell expressing ECRG4 gene, is loaded with a large amount of ECRG4mRNA, can transport the loaded large amount of ECRG4mRNA to a receptor cell through excellent compatibility of the exosome and the receptor cell, and can remarkably improve the expression level of ECRG4 in the receptor cell, thereby remarkably inhibiting the growth and proliferation of tumor cells; and does not cause immune reaction and inflammatory reaction of the receptor; therefore, the appearance of exosomes loaded with large amounts of ECRG4mRNA has great promoting and guiding effects on exploring methods for gene therapy of tumors.

The exosome, wherein the cell overexpressing ECRG4 is a cell in which ECRG4 gene is transcribed and translated in a large amount, and therefore, an ECRG4 gene expression product exceeds a normal level.

The loading amount of ECRG4mRNA in the exosome is not less than 1000 times of that of the conventional exosome; the higher the content of ECRG4mRNA in the exosome is, the better the enhancement effect on the ECRG4 expression level in the receptor cell is, and the better the inhibition effect on the tumor cell is; the conventional exosome refers to an exosome secreted by a normal cell which does not over-express the ECRG4 gene.

The exosome, wherein, preferably, the cell over-expressing ECRG4 is ECRG4-HEK293 cell line; the cell line can stably over-express ECRG4, so that an exosome containing a large amount of ECRG4mRNA is secreted.

Preferably, the ECRG4-HEK293 cell line is prepared by the following preparation method:

A. construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: extracting total RNA, amplifying an ECRG4 gene by using reverse transcription PCR, loading the ECRG4 gene onto a pLVX-IRES-ZsGreen1 vector to obtain a pLVX-IRES-ZsGreen1-ECRG4 plasmid;

B. preparation of lentivirus: HEK293T cells were transfected with a first mixture containing three plasmids, pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ECRG 4; culturing the transfected HEK293T cells, collecting culture solution, and processing the culture solution to obtain lentivirus packaged with ECRG4 gene;

C. establishment of ECRG4-HEK293 cell line: infecting HEK293 cells with a second mixture comprising lentivirus, DMEM medium and polybrene; culturing the infected HEK293 cells, and sorting the cells to obtain an ECRG4-HEK293 cell line.

The ECRG4-HEK293 cell line prepared by the method can continuously and stably over-express ECRG4, the over-expression stability is better, the content of ECRG4mRNA in the obtained exosome is higher, the problem of source of the exosome containing a large amount of ECRG4mRNA is solved from the source, and the large-scale and standardized production of the exosome containing ECRG4mRNA is facilitated.

Preferably, in step a, the method for extracting total RNA comprises: total RNA was extracted from human cells by Trizol method.

Among them, preferably, in the step A, when the ECRG4 gene is amplified by reverse transcription PCR, the ECRG4 primer is designed by using the human ECRG4 gene sequence (GenBank: AF 325503.1) as a template, the upstream primer contains an XhoI cleavage site, and the downstream primer contains a BamHI cleavage site. The base sequence of the upstream primer is as follows: 5'-ATGACTCGAGTGCTCGCGCCCCGCCGCCATG-3', respectively; the base sequence of the downstream primer is as follows: 5'-TCATGGATCCTGTGGCAAGTCATGGTTAGT-3' are provided.

Wherein the lentiviral vector pLVX-IRES-ZsGreen1 used in step A is a bicistronic vector containing a gene encoding ZsGreen 1. ZsGreen1 is a third-generation green fluorescent protein, is a fluorescent protein with the highest brightness, and is convenient for monitoring the transfection efficiency of cells and sorting positive cells by flow. The pLVX-IRES-ZsGreen1-ECRG4 plasmid expresses ZsGreen1 fluorescent protein and ECRG4 gene simultaneously in a bicistronic form, the expression of the ZsGreen1 fluorescent protein can be used as an indirect 'indicator' of target gene expression, and the ZsGreen1 fluorescent protein is not fused with target protein, so that the normal expression of the target protein is not influenced, and the normal structure and function of the target protein are not influenced.

Preferably, in the step A, the ECRG4 gene is loaded on the pLVX-IRES-ZsGreen1 vector by the following method: both the ECRG4 gene and the pLVX-IRES-ZsGreen1 vector were digested simultaneously with XhoI and BamHI, and ligated using T4 DNA ligase.

Wherein, preferably, in the step A, the pLVX-IRES-ZsGreen1-ECRG4 plasmid is proliferated by a bacterial transformation method; the specific method comprises the following steps: the pLVX-IRES-ZsGreen1-ECRG4 plasmid is transformed into Escherichia coli, and positive monoclonal colonies are screened for amplification culture, so that a large amount of pLVX-IRES-ZsGreen1-ECRG4 plasmid is extracted.

Preferably, in the step B, the HEK293T cells are cultured in a DMEM medium (containing 10% FBS) without antibiotics 2-6 hours before the HEK293T cells are transfected, so that the cells can be in a better growth state, and the transfection effect is improved.

Preferably, in step B, lipofectamine2000 is used for transfection when HEK293T cells are transfected with the first mixture; this preference both ensures higher transfection efficiency and reduces the effect of the transfection reagent on cell viability.

Wherein, in step B, both pMD2.G and psPAX2 are lentivirus assembly plasmids; the pLVX-IRES-ZsGreen1-ECRG4 is an ECRG4 coding plasmid; preferably, the mass ratio of pMD2.G, psPAX2 to pLVX-IRES-ZsGreen1-ECRG4 in the first mixed solution is 2: 3: 4; under the condition of the mass ratio, the transfectability of the HEK293T cell is remarkably increased, the obtained virus titer is highest, the infection effect on the HEK293 cell is best, the expression amount is largest, and the quantity of ECRG4mRNA loaded in the obtained exosome is remarkably increased.

Wherein, in the step B, the transfection of the HEK293T cell refers to a process of enabling the HEK293T cell to be infected by ECRG4 gene lentivirus by using a transfection method; preferably, the transfection method comprises one of lipofection, calcium phosphate transfection or electroporation transfection; most preferably, the transfection method is lipofection; by optimizing the transfection method, the cytotoxicity of the transfection reagent can be reduced, the cells can be kept in a healthy growth state, and the virus packaging efficiency is improved.

Preferably, the preparation method of the first mixed solution in the step B includes: taking the volume ratio of 10-30: mixing the Opti-MEM culture medium of 1 with lipofectamine2000 to obtain a solution a; and adding plasmids pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4 into the same amount of Opti-MEM culture medium as the solution a, wherein the mass ratio of the three plasmids is 2: 3: 4, uniformly mixing to obtain a solution b; uniformly mixing the solution a and the solution b to obtain a first mixed solution; preferably, the transformability and stability of the first mixed solution can be improved.

The culture solution in step B is a solution remaining after removing cells and cell debris after the cells are cultured in the medium, and contains lentiviruses.

Preferably, the method for culturing the transfected HEK293T cell in the step B comprises the following steps: culturing the transfected HEK293T cells for 6-12h, then culturing by using a DMEM culture medium, and continuing culturing for 30-48h to complete the culture; by preference, the growth of transfected HEK293T cells was better.

Wherein, preferably, in the step B, the centrifugation temperature of the culture solution is 0-8 ℃, the centrifugation speed is 1500-; most preferably, the centrifugation temperature is 4 ℃, the centrifugation speed is 2000g, and the centrifugation time is 7 min; by optimizing the centrifugal conditions, residual cells and cell debris in the culture solution can be removed, so that the obtained virus solution has higher purity and better infection effect.

Wherein, in the step C, the concentration of polybrene in the second mixed solution is preferably 6-10 mug/ml; the most preferable concentration is 8 mug/ml, and the preferable polybrene concentration can reduce the cytotoxicity of polybrene and obviously improve the infection efficiency of viruses; the polybrene concentration is too high, the toxicity to cells is increased, the activity of the cells is influenced, and even a large amount of cells are killed; if the concentration of polybrene is too low, the effect of the polybrene on improving the virus infection efficiency is obviously reduced.

Wherein, preferably, in the step C, the HEK293 cell is infected by the second mixed solution for 2 times; the virus infection efficiency can be obviously improved through 2 times of infection; high infection efficiency, high proportion of positive cells, short sorting time, high efficiency and small influence on cell activity, and can ensure that the overexpression effect of ECRG4 in HEK293 cells is optimal and the content of ECRG4mRNA in secreted exosomes is maximum.

Preferably, in step C, before culturing the infected HEK293 cells, the HEK293 cells are centrifuged at 1800rpm, room temperature and 45 min; the centrifugal treatment helps the virus particles to be combined with the cell surface, and the infection efficiency of the virus can be further obviously improved.

Wherein, preferably, before the 2 nd infection of the HEK293 cell, the HEK293 cell after the first infection is cultured in a virus-free culture medium for 12-24 h; through the culture, the influence of the first infection on the HEK293 cell can be repaired, so that the HEK293 cell subjected to the first infection is recovered to a vigorous growth state, and the culture medium has a remarkable effect on improving the efficiency of the second infection.

Preferably, in the step (C), the sorting of the cells refers to sorting the GFP green fluorescence positive cells by flow cytometry, and the sum of the GFP green fluorescence positive cells is the ECRG4-HEK293 cell line.

Among them, the preferable culture conditions of the cells in step B, C are: temperature 37 ℃ CO₂The concentration is 5%, and preferably, the cells can maintain normal and stable growth rate and physiological state during the culture process.

Wherein, in the steps B and C, the culture medium for subculturing is a culture medium which can provide necessary nutrition for the growth and the maintenance of the HEK293 cells; preferably, the medium comprises one of DNEM or RPMI-1640, and most preferably, the medium is DNEM (containing 10% FBS, 1% streptomycin double antibody); through optimization, the cultured cells have high growth speed, normal shape and high transfection efficiency.

In order to achieve the above objects, the present invention further provides a method for preparing an exosome containing ECRG4mRNA, comprising the steps of:

1. preparing a cell overexpressing ECRG 4;

2. culturing the ECRG4 over-expressing cells in serum-free DMEM, and collecting a culture solution after the culture is finished;

3. and (3) separating and purifying the culture solution collected in the step (2) to obtain an exosome containing ECRG4 mRNA.

The preparation method of the exosome containing ECRG4mRNA utilizes the principle that a cell over-expressing ECRG4 can transcribe and generate a large amount of ECRG4mRNA in the cell and actively load ECRG4mRNA when the cell membrane is trapped to form the exosome; by culturing cells over expressing ECRG4, the cells secrete large amounts of ECRG4 mRNA-containing exosomes into the culture medium during growth and proliferation; then obtaining an exosome loaded with a large amount of ECRG4mRNA through separation and purification; the preparation method is simple to operate, stable and reliable, and suitable for large-scale and standardized production.

In the above method for producing an exosome containing ECRG4mRNA, preferably, the cell overexpressing ECRG4 in step 2 is cultured to a density of 80%, washed 2 times with PBS, and then replaced with serum-free DMEM medium; the preferred procedure removes the exosomes from the residual medium serum, increasing the purity of the exosomes secreted by the cells.

Wherein, preferably, the cell over expressing ECRG4 in step 2 is cultured in serum-free DMEM medium for 36-60 h; most preferably, the culture time is 48 h; the culture time is too short, the number of exosomes generated by cells is small, the separation yield is low, the culture time is too long, the cell viability can be obviously reduced, the cell death is increased, more cell fragments and small molecular structures related to the cell death are secreted into a culture medium, and the quality and the purity of the exosomes are reduced.

In the above method for producing an exosome containing ECRG4mRNA, preferably, the method for separating and purifying the culture fluid in step 3 comprises: a. centrifuging the culture solution at 4 deg.C and 200g for 10min, and collecting supernatant; b. centrifuging the supernatant obtained in step a for 10min at 4 ℃ under 3000g, and taking the supernatant for later use; c. centrifuging the supernatant obtained in step b for 30min at 4 ℃ under 10000g, and taking the supernatant for later use; d. centrifuging the supernatant obtained in step c for 70-120min at 4 ℃ under the condition of 100000-160000g, and taking the precipitate for later use; e. adding PBS into the precipitate obtained in the step d to resuspend the precipitate, and centrifuging for 70-120min under the conditions of 4 ℃ and 100000-160000g to precipitate the exosome containing ECRG4 mRNA; wherein, most preferably, the centrifugation speed in the steps d and e is 120000g, and the centrifugation time is 90 min; by preference, dead cells, cell debris and macromolecular protein particles in the cell culture medium can be removed, resulting in higher exosome purity.

In the above method for producing an exosome containing ECRG4mRNA, the culture conditions of the cells in steps 2 and 3 are preferably: temperature 37 ℃ CO₂The concentration is 5%, and preferably, the cells can maintain normal and stable growth rate and physiological state during the culture process.

The culture solution in steps 2 and 3 is a solution remaining after the cells are removed after the completion of the cell culture.

Furthermore, the invention also provides an application of an exosome containing ECRG4 mRNA; the exosome can obviously inhibit the proliferation and growth of tumor cells and has a positive effect on the treatment of tumors.

The exosome containing ECRG4mRNA can be used for treating oral squamous cell carcinoma, and can also be used for treating colon cancer, breast cancer, lung cancer, renal cancer, gastric cancer, colon cancer, esophageal cancer, liver cancer or brain glioma.

Compared with the prior art, the invention has the beneficial effects that:

1. the exosome is secreted by a cell over-expressing ECRG4 gene, and is loaded with ECRG4mRNA which exceeds the conventional exosome, and a large amount of loaded ECRG4mRNA can be transported to a receptor cell, so that the expression level of ECRG4 in the receptor cell can be obviously improved.

2. In the process of preparing an over-expression cell, the ECRG4mRNA amount loaded in the exosome can be obviously increased by optimizing the mass ratio of pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4, optimizing the concentration of polybrene and secondarily infecting the cell, so that the ECRG4mRNA loaded in the exosome reaches 1000 times or more of that of a conventional exosome, and the suppression effect on the growth and proliferation of tumor cells is obvious.

3. The ECRG4-HEK293 cell prepared by the invention can continuously and stably over-express ECRG4, the content of ECRG4mRNA in the obtained exosome is high, the problem of the source of the exosome is solved from the source, the large-scale and standardized production of the exosome is facilitated, and the exosome can be popularized to the loading, conveying and large-scale and standardized production of other genes with treatment potential.

4. The exosome can be used for treating tumor diseases, can obviously inhibit the proliferation and growth of tumor cells, and has positive effect on treating the tumor diseases.

Description of the drawings:

FIG. 1 is a photograph showing the ECRG4 gene detection scheme ((A) 100bp ladder indicates DNA molecular weight marker; ECRG4 Frag indicates a partial gene fragment of ECRG 4; ECRG4mRNA indicates the gene encoding ECRG 4; (B) Controls indicates the expression level of ECRG4mRNA in human serum exosomes of healthy humans, and Patients indicates the expression level of ECRG4mRNA in human serum exosomes of oral squamous cell carcinoma.

FIG. 2 is a graph showing the results of identification of HEK293 cell lines after infection in example 1 and comparative example 1 of the present invention ((A) is a statistical graph showing the results of real-time quantitative PCR, (B) is a graph showing the results of Western blot; ECRG4 HEK293 represents the ECRG4-HEK293 cell line in example 1, and Vector HEK293 represents the Vector-HEK293 cell line in comparative example 1).

Fig. 3 is a graph showing the morphology and particle size detection results of exosomes prepared in example 1 of the present invention ((a) is the structure diagram of exosome morphology observed under a transmission electron microscope and (B) is the particle size distribution diagram of exosomes detected by Nanoparticle Tracking Analysis (NTA)).

FIG. 4 is a graph showing the results of characteristic tests of exosomes prepared in example 1 of the present invention and comparative example 1; (A) a graph showing the result of detecting the western blot of an exosome-tagged protein, (B) shows the result of detecting the mRNA content of ECRG4 in exosomes, ECRG4-Exo shows the exosomes prepared in example 1, and Vector-Exo shows the exosomes prepared in comparative example 1).

FIG. 5 is a graph showing the results of uptake of the exosomes prepared in example 1 of the present invention into TCA8113 (DAPI represents the nucleus; ECRG4-Exo represents the exosomes extracted in example 1; α -Actin represents the cell Actin; and Merge represents the effect of fluorescence addition).

FIG. 6 is a graph showing the measurement of the intracellular ECRG4 gene expression level after TCA8113 cells take in the exosomes of example 1 and comparative example 1 of the present invention (ECRG 4-Exo means that TCA8113 cells are added to the exosomes of example 1, and Vector-Exo means that the exosomes of comparative example 1 are added to TCA8113 cells).

FIG. 7 is a graph showing the results of cell proliferation assay of TCA8113 cells inhibited by exosomes in example 1 and comparative example 1 of the present invention ((A) shows the results of cell cycle assay of TCA8113 cells added to exosomes in comparative example 1, (B) shows the results of cell cycle assay of TCA8113 cells added to exosomes in example 1, and (C) is a statistical graph showing the results of cell proliferation assay of TCA8113 cells with CCK8 after two exosomes are added).

FIG. 8 is a graph showing the results of the A549 cell proliferation assay using exosomes of example 1 and comparative example 1 of the present invention ((A) shows the results of the A549 cell cycle assay using exosomes of comparative example 1, (B) shows the results of the A549 cell cycle assay using exosomes of example 1, and (C) is a statistical graph showing the results of the A549 cell proliferation assay using CCK8 after two exosomes were added).

FIG. 9 is a graph showing the results of real-time quantitative PCR detection of the exosomes of example 1 of the present invention inhibiting the expression of the relevant genes in TCA8113 cells in comparative example 1 (ECRG 4-Exo indicates TCA8113 cells to which the exosomes of example 1 were added; Vector-Exo indicates TCA8113 cells to which the exosomes of comparative example 1 were added).

FIG. 10 is a graph showing a comparison of growth conditions of nude mice inoculated with TCA8113 cells treated with exosomes of example 1 of the present invention and comparative example 1 ((A) is a graph showing an average weight gain of nude mice during the experiment, (B) is a statistical graph showing an average food intake of nude mice during the experiment; ECRG4-Exo shows nude mice inoculated with TCA8113 cells treated with exosomes of example 1; Vector-Exo shows nude mice inoculated with TCA8113 cells treated with exosomes of comparative example 1).

FIG. 11 is a graph showing the results of the inhibition of the growth of transplanted tumor in nude mice by exosomes in example 1 and comparative example 1 of the present invention ((A) is a tumor-bearing nude mouse inoculated with TCA8113 cells treated with exosomes in example 1, (B) is a tumor-bearing nude mouse inoculated with TCA8113 cells treated with exosomes in comparative example 1, (C) is a statistical graph of tumor growth curve in nude mouse, (D) is a transplanted tumor in nude mouse exfoliated after 24 days, (E) is a statistical graph of transplanted tumor weight in nude mouse, (ECRG 4-Exo represents a nude mouse inoculated with TCA8113 cells treated with exosomes in example 1, and Vector-Exo represents a nude mouse inoculated with TCA8113 cells treated with exosomes in comparative example 1).

FIG. 12 shows the fluorescence patterns of HEK293 cells after lentivirus infection in examples of the present invention ((A) shows the fluorescence pattern of HEK293 cells after lentivirus infection in example 1, (B) shows the fluorescence pattern of HEK293 cells after lentivirus infection 1 time in example 2, (C) shows the fluorescence pattern of HEK293 cells in example 3, (D) shows the fluorescence pattern of HEK293 cells in example 4, DAPI shows the nucleus, Ecrg4 shows positive cells with ECRG4 overexpression, and Merge shows the effect of fluorescence superposition.

FIG. 13 is a flow cytometric image of HEK293 cells after lentivirus infection in examples 1 and 2 of the present invention (A) is a flow cytometric image of HEK293 cells after lentivirus infection in example 1, (B) is a flow cytometric image of HEK293 cells after lentivirus infection 1 time in example 2, and P2 represents positive cells with over-expression of ECRG 4).

Detailed Description

The present invention will be described in further detail with reference to specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.

Example 1

1. Construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: extracting total RNA of human umbilical vein endothelial cells by a Trizol method, performing double enzyme digestion on an ECRG4 gene, an ECRG4 gene and a pLVX-IRES-ZsGreen1 vector by using a reverse transcription PCR (polymerase chain reaction), connecting the vectors by using T4 DNA ligase, transforming the vectors into escherichia coli, screening positive clones, extracting plasmids, and identifying by using double enzyme digestion and DNA sequencing. Wherein the ECRG4 primer is designed by taking a human ECRG4 gene sequence (GenBank: AF 325503.1) as a template, the upstream primer comprises an XhoI restriction site, and the downstream primer comprises a BamHI restriction site. The base sequence of the upstream primer is as follows: 5'-ATGACTCGAGTGCTCGCGCCCCGCCGCCATG-3', respectively; the base sequence of the downstream primer is as follows: 5'-TCATGGATTCTGTGGCAAGTCATGGTTAGT-3' are provided.

2. Cell preparation: recovering HEK293T cells, and culturing with DMEM medium containing 10% fetal calf serum and 1% streptomycin at 37 deg.C and 5% CO₂Culturing in constant temperature incubator, subculturing for 1-2 times, inoculating 0.8 × 10⁶Transferring the cells to a 6cm culture dish, transfecting by lipofectamine2000 the next day, packaging the lentivirus, and changing to a DMEM culture medium without antibiotics 2h before transfection;

3. and (3) preparing lentivirus: preparing a solution a comprising 450. mu.l of Opti-MEM and 18. mu.l of lipofectamine 2000; preparing a solution b comprising 450. mu.l of Opti-MEM, 2. mu.g of plasmid pMD2.G, 3. mu.g of plasmid psPAX2 and 4. mu.g of plasmid pLVX-IRES-ZsGreen1-ECRG 4; adding the solution b into the solution a, gently mixing to obtain a first mixed solution, standing at room temperature for 5min, adding the first mixed solution into HEK293T cells obtained in the step 1, standing at 37 ℃ with 5% CO₂Washing with PBS for 2 times after 6h in the constant temperature incubator, changing into DMEM culture medium, collecting culture solution after 36h, centrifuging for 7min at 4 deg.C and 2000g, and collecting supernatant as virus solution;

4. establishment of ECRG4-HEK293 cell line: recovering HEK293 cells by DMEM medium (DMEM medium for short) containing 10% fetal calf serum and 1% streptomycin at 37 deg.C and 5% CO₂Culturing in constant temperature incubator, subculturing for 1-2 times, inoculating 0.8 × 10⁶Cells to 6cm petri dish; the next day of the day,and (3) mixing the virus solution obtained in the step (2) with a DMEM culture medium according to the ratio of 3: 1, adding polybrene to make the concentration of polybrene 8 mug/ml to obtain a second mixed solution; adding the second mixed solution into HEK293 cells, centrifuging for 45min at 1800rpm and 37 ℃, changing to a DMEM culture medium after culturing for 12h, continuing to culture for 12h, adding the second mixed solution into the HEK293 cells again, centrifuging for 45min at 1800rpm and 37 ℃, changing to the DMEM culture medium after culturing for 12h, continuing to culture for 48h, and then performing flow cytometry sorting to obtain an ECRG4-HEK293 cell line;

5. production of exosomes ECRG4-HEK293 cell line was inoculated in 10cm dishes in an amount of 2.2 × 10⁶Culturing until the cell density is about 80%, washing the cells for 2 times by using PBS, changing the cells into a serum-free DMEM culture medium, and collecting a culture solution after culturing for 48 hours;

6. extraction of exosomes: centrifuging the culture solution collected in the step 4 for 10min at the temperature of 4 ℃ under the condition of 200g, and taking the supernatant for later use; centrifuging the obtained supernatant at 4 deg.C and 3000g for 10min, and collecting supernatant; centrifuging the obtained supernatant at 4 deg.C and 10000g for 30min, and collecting supernatant; centrifuging the obtained supernatant at 4 deg.C and 120000g for 90min, and collecting precipitate; the obtained precipitate was resuspended in PBS and centrifuged at 120000g at 4 ℃ for 90min to obtain an exosome containing ECRG4mRNA, which was named ECRG 4-Exo.

7. Preservation of exosomes: adding a proper amount of PBS into the exosome for resuspension, quantifying by using a BCA protein concentration detection kit, wherein the volume of the PBS is 1 percent of the volume of the original culture medium, and storing at-80 ℃.

Example 2

1. Construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: the specific operation was the same as in example 1.

2. Cell preparation: the specific operation was the same as in example 1.

3. And (3) preparing lentivirus: the specific operation was the same as in example 1.

4. Establishment of ECRG4-HEK293 cell line: recovering HEK293 cells, culturing in DMEM medium (DMEM medium for short) containing 10% fetal calf serum and 1% streptomycin at 37 deg.C,5% CO₂culturing in constant temperature incubator, subculturing for 1-2 times, inoculating 0.8 × 10⁶Cells to 6cm petri dish; the next day, the virus solution obtained in step 2 was mixed with DMEM medium in a ratio of 3: 1, adding polybrene to make the concentration of polybrene 8 mug/ml to obtain a second mixed solution; adding the second mixed solution into HEK293 cells, centrifuging at 1800rpm and 37 ℃ for 45min, culturing for 12h, changing into DMEM culture medium, continuously culturing for 48h, and performing fluorescence observation and flow cytometry sorting;

5. production of exosomes: the specific operation was the same as in example 1.

6. Extraction of exosomes: the specific operation was the same as in example 1.

7. Preservation of exosomes: the specific operation was the same as in example 1.

The test shows that: example 2, step 4, only 1 virus infection treatment was performed on HEK293 cells, and the proportion of HEK293 cells positive after infection was about 54.8%, as shown in fig. 12 (B) and 13 (B), and the proportion of HEK293 cells positive in example 1 was about 97.2%, as shown in fig. 12 (a) and 13 (a). Example 2 with 1 virus infection treatment, the positive rate of the cells is obviously reduced, and the expression level of ECRG4 is lower than that of example 1, which is not beneficial to flow sorting of the cells and extraction of exosomes. Therefore, the adoption of the 2-time infection method can greatly improve the infection efficiency, accelerate the establishment of stable cell strains and improve the expression quantity of ECRG4mRNA and protein in HEK293 cells, thereby greatly increasing the content of ECRG4mRNA in exosomes.

Example 3

2. Cell preparation: the specific operation was the same as in example 1.

3. And (3) preparing lentivirus: preparing a solution a comprising 450. mu.l of Opti-MEM and 18. mu.l of lipofectamine 2000; preparing a solution b comprising 450. mu.l of Opti-MEM, 2. mu.g of plasmid pMD2.G, 2. mu.g of plasmid psPAX2 and 2. mu.g of plasmid pLVX-IRES-ZsGreen1-ECRG 4; adding the solution b into the solution a, gently mixing uniformly to obtain a first mixed solution, standing at room temperature for 5min, and mixing the first mixed solutionAdding the mixture into HEK293T cells obtained in step 1, standing at 37 deg.C and 5% CO₂Washing with PBS for 2 times after 6h in the constant temperature incubator, changing into DMEM culture medium, collecting culture solution after 36h, centrifuging for 7min at 4 deg.C and 2000g, and collecting supernatant as virus solution;

4. establishment of ECRG4-HEK293 cell line: the specific operation was the same as in example 1.

5. Production of exosomes: the specific operation was the same as in example 1.

6. Extraction of exosomes: the specific operation was the same as in example 1.

The test shows that: in example 3, the mass ratio of pmd2.g, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4 in the first mixture of step 3 was not the preferred ratio, the prepared virus fluid contained less lentivirus carrying ECRG4 gene, the infectivity of the virus fluid was reduced, the positive cell rate (the positive cell ratio was less than 30%) and the expression level of ECRG4 in the HEK293 cells were significantly lower than those of example 1 (the positive cell ratio was about 97.2%) after infection of the HEK293 cells, see fig. 12 (a), (C), which affected the loading level of ECRG4 gene in exosomes. Therefore, the optimal mass ratio of pMD2.G, psPAX2 to pLVX-IRES-ZsGreen1-ECRG4 is adopted, the titer of ECRG4 virus can be greatly improved, the infection efficiency of HEK293 cells and the over-expression effect of ECRG4 are increased, and the content of ECRG4mRNA in secreted exosomes is maximum.

Example 4

2. Cell preparation: the specific operation was the same as in example 1.

4. Establishment of ECRG4-HEK293 cell line: recovering HEK293 cells by DMEM medium (DMEM medium for short) containing 10% fetal calf serum and 1% streptomycin at 37 deg.C and 5% CO₂Culturing in constant temperature incubator, subculturing for 1-2 times, inoculating 0.8 × 10⁶Cells to 6cm petri dish; the next dayAnd (3) mixing the virus solution obtained in the step (2) with a DMEM culture medium according to the ratio of 3: 1, adding polybrene to ensure that the concentration of polybrene is 12 mug/ml, and obtaining a second mixed solution; adding the second mixed solution into HEK293 cells, centrifuging for 45min at 1800rpm and 37 ℃, changing to a DMEM culture medium after culturing for 12h, continuing culturing for 12h, adding the second mixed solution into the HEK293 cells again, centrifuging for 45min at 1800rpm and 37 ℃, changing to the DMEM culture medium after culturing for 12h, continuing culturing for 48h, and then performing fluorescence detection;

5. production of exosomes: the specific operation was the same as in example 1.

6. Extraction of exosomes: the specific operation was the same as in example 1.

The test shows that: in example 4, the concentration of polybrene in the second mixed solution in step 4 was not the optimal concentration, and compared to example 1, the second mixed solution increased the toxicity of the cells, more HEK293 cells died, and the infectivity of the virus solution decreased. Therefore, the proportion of positive cells (less than 30% of positive cells) in the ECRG4-HEK293 cell line obtained is significantly less than that of example 1 (the proportion of positive cells is about 97.2%), which is not beneficial to flow sorting and exosome extraction of positive cells. Therefore, the infection efficiency of the second mixed solution can be greatly improved by adopting the optimal polybrene concentration, so that the overexpression effect of ECRG4 in HEK293 cells is optimal, and the content of ECRG4mRNA in secreted exosomes is larger.

Comparative example 1

1. Cell preparation: recovering HEK293T cells, and culturing with DMEM medium containing 10% fetal calf serum and 1% streptomycin at 37 deg.C and 5% CO₂Culturing in constant temperature incubator, subculturing for 1-2 times, inoculating 0.8 × 10⁶Cells were plated on 6cm dishes and transfected the next day with lipofectamine 2000; changing to a DMEM medium without antibiotics 2h before transfection;

2. and (3) preparing lentivirus: preparing a solution a comprising 450. mu.l of Opti-MEM and 18. mu.l of lipofectamine 2000; a plasmid containing 450. mu.l of Opti-MEM, 2. mu.g of plasmid pMD2.G, 3. mu.g of plasmid psPAX2 and 4. mu.g of plasmid pLVX-IRE was preparedSolution b of S-ZsGreen 1; adding the solution b into the solution a, gently mixing to obtain a first mixed solution, standing at room temperature for 5min, adding the first mixed solution into HEK293T cells obtained in the step 1, standing at 37 ℃ with 5% CO₂Washing with PBS for 2 times after 6h in the constant temperature incubator, changing into DMEM culture medium, collecting culture solution after 36h, centrifuging for 7min at 4 deg.C and 2000g, and collecting supernatant as virus solution;

3. establishment of Vector-HEK293 cell line: recovering HEK293 cells by DMEM medium (DMEM medium for short) containing 10% fetal calf serum and 1% streptomycin at 37 deg.C and 5% CO₂Culturing in constant temperature incubator, subculturing for 1-2 times, inoculating 0.8 × 10⁶Cells to 6cm petri dish; the next day, the virus solution obtained in step 2 was mixed with DMEM medium in a ratio of 3: 1, adding polybrene to make the concentration of polybrene 8 mug/ml to obtain a second mixed solution; adding the second mixed solution into HEK293 cells, centrifuging for 45min at 1800rpm and 37 ℃, changing to a DMEM culture medium after culturing for 12h, continuing culturing for 12h, adding the second mixed solution into the HEK293 cells again, centrifuging for 45min at 1800rpm and 37 ℃, changing to the DMEM culture medium after culturing for 12h, continuing culturing for 48h, and then performing flow cytometry sorting to obtain a Vector-HEK293 cell line;

4. production of exosomes Vector-HEK293 cells were seeded in 10cm dishes in a number of 2.2 × 10⁶Culturing until the cell density is about 80%, washing the cells for 2 times by using PBS, changing the cells into serum-free DMEM culture medium, and collecting the culture medium after culturing for 48 hours;

5. extraction of exosomes: centrifuging the culture medium collected in the step 4 for 10min at the temperature of 4 ℃ and under the condition of 200g, and taking the supernatant for later use; centrifuging the obtained supernatant at 4 deg.C and 3000g for 10min, and collecting supernatant; centrifuging the obtained supernatant at 4 deg.C and 10000g for 30min, and collecting supernatant; centrifuging the obtained supernatant at 4 deg.C and 120000g for 90min, and collecting precipitate; PBS was added to the obtained pellet to resuspend the pellet, and the pellet was centrifuged at 120000g at 4 ℃ for 90min to obtain exosomes (Vector-Exo).

6. Preservation of exosomes: adding a proper amount of PBS into the exosome for resuspension, quantifying by using a BCA protein concentration detection kit, wherein the volume of the PBS is 1 percent of the volume of the original culture medium, and storing at-80 ℃.

Experimental example:

(1) ECRG4-HEK293 cell line and ECRG4-Exo identification

The ECRG4-HEK293 and Vector-HEK293 cells established in example 1 were tested for ECRG4mRNA and protein expression levels by real-time quantitative PCR and western blot, respectively, and the results show that ECRG4-HEK293 cells ECRG4mRNA expression level is 7.8 × 10 of Vector-HEK293 cells⁵See fig. 2A. And a clear and heavy ECRG4 protein band with the molecular weight of about 17kDa is detected in the ECRG4-HEK293 cell lysate, while an ECRG4 protein band is not detected in the Vector-HEK293 cell lysate with the same mass, as shown in FIG. 2B. Therefore, the ECRG4-HEK293 cell line established in example 1 has strong over-expression of ECRG4mRNA and protein, and provides good precondition for loading ECRG4 gene for exosome.

Equal amounts of ECRG4-HEK293 and Vector-HEK293 cells were cultured in serum-free DMEM medium for 48h, the medium was collected to extract exosomes, and the exosomes were shown to have a typical "cup-shaped" vesicle structure by transmission electron microscopy, with the particle size mainly concentrated at 93nm, as shown in FIG. 3. Western blot results show that the expression levels of positive marker CD9 and Alix of exosomes are obviously higher than that of cell lysates, the expression levels of negative marker GRP94 and Calnexin of exosomes are obviously lower than that of cell lysates, see figure 3, and the expression level of ECRG4mRNA in ECRG4-Exo is 1162 times that of Vector-Exo, see figure 4. This indicates that the ECRG4-Exo exosomes extracted in example 1 have a morphological structure typical of exosomes and are loaded with a high level of ECRG4 gene.

(2) Experimental detection of exosome transport ECRG4 mRNA:

ECRG4-Exo prepared in example 1 was added to a Cell Tracker^TMCM-DiI (Thermo, C7000), final concentration 1. mu.g/ml, incubation at 37 ℃ for 30min, standing at 4 ℃ for 15min, transferring to an ultracentrifuge tube, centrifuging at 4 ℃ for 90min at 120000g, discarding supernatant, dissolving precipitate in PBS solutionStoring at-80 deg.C; the TCA8113 cell culture medium is RPMI-1640 containing 10% FBS, the TCA8113 cells are inoculated on a cell climbing sheet, the density is about 60%, the cells are cultured in a 6-pore plate overnight, 50 mu g of ECRG4-Exo is added, and the cells are cultured for 12 h; taking out the cell slide, fixing with 4% paraformaldehyde at room temperature for 15min, washing with PBS for 5min, adding precooled anhydrous methanol, standing at-20 ℃ for 20min, washing with PBS for 5min, sealing with 5% skimmed milk at room temperature for 1h, adding anti-actin primary antibody (Sigma) for incubation at 4 ℃ overnight, washing with PBS for 5min, repeating for 3 times, adding green fluorescent secondary antibody (Abcam), incubating at room temperature for 1h, washing with PBS for 5min, repeating for 3 times, adding 1% nuclear dye Hoechst, incubating at room temperature for 10min, washing with PBS for 5min, repeating for 3 times, sealing with a sealing agent, and detecting with a fluorescence microscope. The results showed that red fluorescently labeled ECRG4-Exo was able to pass through the TCA8113 cell membrane, enter the recipient cells, distribute mainly in the cytoplasm, and surround the nucleus, as shown in FIG. 5.

In order to further confirm whether the ECRG4-Exo exosomes can transport the carried ECRG4 gene to TCA8113 cells, ECRG4-Exo and Vector-Exo are respectively added to the TCA8113 cells, the cells are collected after 12 hours, and real-time quantitative PCR detection is carried out. The results showed that ECRG4mRNA expression levels in TCA8113 cells in the ECRG4-Exo (example 1) treated group were 29.48 times higher than in the Vector-Exo (comparative example 1) treated group, as shown in FIG. 6, and it was fully demonstrated that ECRG4mRNA was transported to recipient cells via exosomes.

(3) ECRG4-Exo inhibits tongue cancer cell TCA8113 (oral squamous cell carcinoma) cell proliferation experiment detection:

TCA8113 cells were inoculated in 96-well plates at 2000 cells/well for 24h of culture, 2. mu.g ECRG4-Exo was added to the experimental group (example 1), 2. mu.g Vector-Exo was added to the control group (comparative example 1), culture was continued for 48h, 10. mu.l CCK-8 solution was added to each well, culture was carried out at 37 ℃ for 1h, absorbance at 450nm was measured, SPSS was performed for statistical analysis of data, TCA8113 cells were inoculated in 6-well plates at 0.3 × 10⁶And/well, after overnight culture, the culture was synchronized with serum-free DMEM medium for 12h, and then changed to DMEM medium, and 20ug ECRG4-Exo was added to the experimental group (3 wells), 20ug Vector-Exo was added to the control group (3 wells), and the culture was continued for 24 h. Cells were trypsinized and washed with PBSOnce, the cells were fixed with 70% ethanol overnight at 4 ℃ and washed 2 times with PBS, 500. mu.l of PI/RNase stain (BD 550825) was added and detected by flow cytometry.

The results showed that the proliferation rate of TCA8113 cells was decreased by 58% in the ECRG4-Exo treated group compared to the control group, and the number of cells in the G0/G1 phase was significantly higher than that in the control group (ECRG 4-Exo group 63.97%, control group 54.34%), and the number of cells in the S phase was lower than that in the control group (ECRG 4-Exo group 24.16%, control group 29.23%), as shown in FIG. 7, indicating that ECRG4-Exo blocked the G0/G1 → S phase process of TCA8113 cells. In order to more fully confirm the effect of ECRG4-Exo, the invention further confirms that ECRG4-Exo delays the process of the S → G2 phase of A549 cells (ECRG 4-Exo group 11.83%, and control group 16.04%) in human lung cancer cells A549 by using the same test method, and finally leads to that the proliferation speed of the A549 cells is reduced by 42%, as shown in figure 8. In vitro experiments at the cellular level well demonstrated that ECRG4-Exo effectively inhibited tumor cell proliferation.

(3) Inhibition experiment of inflammation and angiogenesis-related gene expression:

inoculating TCA8113 cells into 12-well plates at a density of 70%, culturing overnight, adding 20ug ECRG4-Exo into experimental group (6 wells), adding 20 μ g Vector-Exo into control group (6 wells), culturing for 24 hr, collecting cells, extracting total RNA, and performing real-time quantitative PCR 2^-CTThe method detects the expression of NF-kappa B p50, IL-1b, IL-6, MCP1, CDK4 and VEFGA related genes of cell proliferation, angiogenesis and inflammation, and is shown in figure 9. The results show that ECRG4-Exo remarkably inhibits the expression level of NF-kappa Bp50, IL-1b, IL-6, MCP1, CDK4 and VEFGA of TCA8113 cells.

(4) Nude mouse transplanted tumor growth inhibition experiment:

in order to further verify the effect of ECRG4-Exo in vivo, the invention uses ECRG4-Exo and Vector-Exo to pretreat TCA8113 cells respectively, then an equal amount of cells are taken to inoculate nude mice subcutaneously, and a transplantation tumor model is replicated, and the specific operation is as follows.

Culturing TCA8113 cell in 10cm culture dish to reach density of 80-90%, adding ECRG4-Exo 400 μ g in experimental group, adding Vector-Exo 400 μ g in control group, culturing for 24 hr, digesting with trypsin, diluting with PBS to 10⁷One per ml. 6-month-old male BALB/C immunodeficient nude mice were randomly divided into 2 groups of 6 mice each. Two groups of cells were inoculated subcutaneously on the right anterior limb back of nude mice in a number of 10⁶On days 3, 6 and 9 after cell inoculation, 100 μ g ECRG4-Exo was injected into the tail vein of the nude mice in the experimental group, 100 μ g Vector-Exo was injected into the tail vein of the nude mice in the control group, three times in total, the longest diameter (L) and the shortest diameter (W) of the tumor were measured with a vernier caliper from day 2 after the nude mice were inoculated with the cells, and the tumor volume (V) was calculated, wherein V = L × W²× 0.5.5, the amount of feed consumed and the weight of the nude mice were recorded 1 time every 2 days until day 24, the nude mice were sacrificed, the tumors were detached, and weighed, the data were expressed as "mean ± standard deviation", and independent sample T-test analysis was performed using SPSS19.0 software.

Statistical results showed no significant difference in mean body weight and food intake during the trial period in the two groups of nude mice, see figure 10, but significant difference in tumor size. ECRG4-Exo group had significantly lower tumor volumes than Vector-Exo group on

days

8, 14, 16, 20, 22, 24 (seep<0.05), see fig. 11. After the nude mice die, the tumor is stripped, the size of the tumor in the experimental group is obviously smaller than that of the control group, and the weight of the tumor in the experimental group is obviously lower than that of the control group through statistical analysis (p<0.05), see fig. 11. The ECRG4-Exo is fully proved to obviously inhibit the growth of oral squamous cell carcinoma cells and tumors from molecular level, cell level and overall level respectively through in vitro and in vivo tests, and the effect is realized mainly by inhibiting the proliferation of tumor cells and inhibiting the angiogenesis of tumors and the expression of genes related to inflammatory reaction.

Claims

1. An exosome loaded with ECRG4mRNA produced by a cell overexpressing ECRG4 gene;

the cell over expressing ECRG4 is ECRG4-HEK293 cell line;

the ECRG4-HEK293 cell line is prepared by the following preparation method:

A. construction of pLVX-IRES-ZsGreen1-ECRG4 plasmid: extracting total RNA, amplifying an ECRG4 gene by using reverse transcription PCR, cloning the ECRG4 gene to a pLVX-IRES-ZsGreen1 vector to obtain a pLVX-IRES-ZsGreen1-ECRG4 plasmid;

B. preparation of lentivirus: the weight ratio of the components is 2: 3: 4 pmd2.g, psPAX2 and pLVX-IRES-ZsGreen1-ECRG4 a first mixture of three plasmids to transfect HEK293T cells; culturing the transfected HEK293T cells, collecting culture solution, and processing the culture solution to obtain lentivirus packaged with ECRG4 gene;

C. establishment of ECRG4-HEK293 cell line: 2 times of infection of HEK293 cells is carried out by using a second mixed solution containing ECRG4 gene lentivirus, DMEM medium and polybrene with the concentration of 6-10 mug/ml; culturing the infected HEK293 cells, and sorting the cells to obtain an ECRG4-HEK293 cell line.

2. Exosome according to claim 1, characterized in that the amount of ECRG4mRNA loaded is not less than 1000 times that of a conventional exosome.

3. Exosome according to claim 1, characterized in that in step C, the first infected HEK293 cells are cultured in DMEM medium containing 10% FBS for 12-24h before infection of HEK293 cells for the 2 nd time.

4. A method for the preparation of exosomes according to any one of claims 1-3, comprising the steps of:

(1) preparing a cell overexpressing ECRG 4;

(2) culturing the cell over expressing ECRG4 in serum-free DMEM, and collecting the culture solution after the culture is finished;

(3) and (3) separating and purifying the culture solution collected in the step (2) to obtain an exosome containing ECRG4 mRNA.

5. Use of an exosome according to any one of claims 1-3 in the preparation of a medicament for treating a neoplastic disease.