CN108486060B

CN108486060B - Exosome for treating tumors and preparation method and application thereof

Info

Publication number: CN108486060B
Application number: CN201810200387.3A
Authority: CN
Inventors: 高博; 高翔; 李亚楠; 宣之胜; 张海明; 孙多灿
Original assignee: Shaoxing Youming Biotechnology Co Ltd
Current assignee: Shaoxing Youming Biotechnology Co., Ltd
Priority date: 2018-03-12
Filing date: 2018-03-12
Publication date: 2021-09-14
Anticipated expiration: 2038-03-12
Also published as: CN108486060A

Abstract

The invention discloses an exosome for treating tumors and a preparation method and application thereof. Specifically, the invention provides an exosome capable of remarkably inhibiting the growth and the tumorigenic capacity of a glioma cell strain and a preparation method thereof. The exosome can obviously inhibit the growth and the tumorigenic capacity of a glioma cell strain. Therefore, the compound can be used for treating brain glioma.

Description

Exosome for treating tumors and preparation method and application thereof

Technical Field

The invention belongs to the field of molecular biology and biomedicine, and particularly relates to an exosome for treating tumors and a preparation method and application thereof.

Background

The tumor tissue is composed of tumor cells and stroma, and the tumor cells are abnormal hyperplastic cells transformed from normal cells. Tumors can occur in many organs and tissues of the human body. The tumor is classified into benign tumor and malignant tumor according to the size of the harm of the tumor to human body and the growth characteristics of the tumor.

Benign tumors grow slowly and are in swelling growth, complete envelopes are usually arranged on the surface, less general symptoms except local symptoms, the benign tumors do not infiltrate into surrounding tissues and are not transferred to the whole body, the benign tumors are not easy to relapse after surgical excision, and the benign tumors have less harm to the body, such as lipoma, hemangioma, adenoma, cyst and the like.

Malignant tumor grows rapidly, often soaks to the surrounding tissue in growth, there is no capsule on the surface, often there is systemic metastasis, the pathological examination is seen atypical nuclear division, except local symptoms, the systemic symptoms are obvious, the late stage patient mostly presents cachexia, the recurrence rate after surgical resection is high, the harm to organism is large, such as bone cancer, esophageal cancer, liver cancer, lung cancer, leukemia, osteosarcoma, brain glioma, etc.

Malignant tumors are a group of diseases that currently seriously threaten human health and life, cells with division potential under the influence of carcinogenic factors, and neoplasms formed by malignant transformation and clonal proliferation. The occurrence of tumor is often caused by the damage and mutation of genetic material DNA caused by the sequential or synergistic manner of the genetic and environmental carcinogenic factors of the organism, and simultaneously, the activation of a plurality of oncogenes and the inactivation of tumor suppressor genes are accompanied, so that normal cells are continuously proliferated and transformed into final canceration.

Brain glioma is the most common nerve tumor in neurosurgery, and all current treatment means (such as comprehensive measures of operation, radiotherapy and chemotherapy, gene therapy, immunotherapy or combination thereof) cannot radically treat brain glioma and reduce the high recurrence rate of the disease due to the strong malignant proliferation capacity and invasiveness of the tumor, so that the brain glioma becomes a difficult problem and a hot research subject of neurosurgical clinical treatment. The biological characteristics of the brain glioma depend on the internal gene change, a reliable tumor marker gene is searched, and the characteristics of the brain glioma, such as proliferation, invasiveness, differentiation and the like, are judged, so that the method is an important prerequisite for diagnosing and prognosticating the brain glioma and determining effective treatment measures.

In view of the serious impact of tumors on human health, those skilled in the art are working to develop new, more effective tumor treatment techniques.

Disclosure of Invention

The invention aims to provide an exosome for treating tumors and a preparation method and application thereof.

In a first aspect of the invention, an exosome for treating a tumor is provided, wherein the exosome comprises a CD47 molecule on an exosome membrane.

In another preferred embodiment, the exosomes are encapsulated with siRNA molecules or precursors thereof.

In another preferred embodiment, the siRNA molecule is a tumor specific siRNA molecule or a precursor thereof.

In another preferred embodiment, the siRNA molecule targets the BPTF gene.

In another preferred embodiment, the siRNA molecule targets the sequence shown in SEQ ID No.3 in the BPTF gene.

In another preferred embodiment, the tumor is a brain glioma.

In a second aspect of the invention, there is provided a method of preparing exosomes, the method comprising the steps of:

(1) construction of Lentiviral expression vectors

Wherein the lentiviral expression vector comprises a polynucleotide sequence encoding CD 47;

(2) lentiviral packaging

Packaging the lentivirus expression vector constructed in the step (1) into virus particles;

(3) construction of cell lines

Infecting host cells with the virus particles obtained in step (2) to construct a cell line stably expressing CD 47;

(4) preparation of exosomes

And (4) culturing the cell strain constructed in the step (3), collecting cell supernatant, and extracting exosomes, thereby obtaining the exosomes.

In another preferred example, the method further comprises the steps of:

(5) preparing an exosome-siRNA complex.

In another preferred embodiment, the siRNA molecule is a tumor specific siRNA molecule.

In another preferred embodiment, the siRNA molecule targets the BPTF gene.

In another preferred embodiment, the tumor is a brain glioma.

In another preferred example, in the step (5), the siRNA molecule is encapsulated into the exosome by using an electrical transduction method, so as to prepare the exosome-siRNA complex.

In another preferred embodiment, in the step (5), the ratio of the amount of exosome particles to the amount of siRNA used during the electrical transduction is 10⁸～10¹⁰Individual exosome particles: 0.1-10 ug siRNA; preferably 10⁸～10¹⁰Individual exosome particles: 1ug siRNA; most preferably 10⁹Individual exosome particles: 1ug siRNA.

In another preferred example, in the step (1), the expression vector uses FUGW virus vector as a backbone.

In another preferred example, in said step (2), the helper plasmids used in the lentiviral packaging process are pCMV-dR8.2dvpr vector and pCMV-VSV-G vector.

In another preferred example, in the step (2), the packaging cells are 293T cells.

In another preferred example, in the step (3), the host cell is HEK293 cell.

In another preferred embodiment, the exosome for treating tumor according to the first aspect of the present invention is prepared by the method according to the second aspect of the present invention.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein said active ingredient is an exosome according to the first aspect of the invention.

In another preferred embodiment, the pharmaceutical composition is used for preventing or treating tumors.

In another preferred embodiment, the tumor is a brain glioma.

In a fourth aspect of the invention, there is provided the use of an exosome according to the first aspect of the invention for the prevention or treatment of a tumour.

In another preferred embodiment, the tumor is a brain glioma.

In a fifth aspect of the invention, there is provided a method of non-therapeutically inhibiting tumor cells in vitro, comprising the steps of: culturing the tumor cell in the presence of the exosome for treating a tumor according to the first aspect of the present invention, thereby inhibiting the tumor cell.

In another preferred embodiment, the tumor is a brain glioma; preferably, the tumor cell is a glioma cell line (e.g., U-87 cell line and U-251 cell line).

In another preferred embodiment, the tumor cell is a tumor cell growth inhibitor or a tumor cell tumor formation inhibitor.

In another preferred embodiment, the activity of the protein encoded by the BPTF gene is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, more preferably more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, most preferably completely free of the activity of the protein encoded by the BPTF gene in said tumor cell compared to a control tumor cell.

In another preferred embodiment, the expression of the BPTF gene in said tumor cell is reduced by more than 10%, preferably more than 20%, more preferably more than 30%, more preferably more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, more preferably more than 80%, more preferably more than 90%, most preferably completely free of BPTF gene expression, compared to a control tumor cell.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

FIG. 1A shows the knockdown efficiency of siRNA against different target sites of the ATM gene.

FIG. 1B shows the knockdown efficiency of siRNA against different target sites of the BPTF gene.

FIG. 1C shows the level of proliferation of cells knocked out for the BTPF and ATM genes, respectively.

FIG. 2A shows immunofluorescence identifying CD47-HEK293 cells.

FIG. 2B shows the electron microscopy of CD 47-siRNA-Exosome.

FIG. 3 shows the results of particle size measurement of CD 47-siRNA-Exosome.

FIG. 4 shows the detection of CD47-siRNA-Exosome marker protein by Western blot.

FIG. 5 shows the result of fluorescent microscope examination of exosomes and glioma cells incubated together.

FIG. 6 shows the MTT assay results of exosomes incubated with glioma cells.

FIG. 7 shows the results of a clonogenic assay with exosomes incubated with glioma cells.

FIG. 8 shows the results of testing the invasiveness of exosomes incubated with glioma cells.

FIG. 9 shows the experimental results of exosome injection tumorigenic nude mice.

Detailed Description

The inventor of the invention has conducted extensive and intensive research to find for the first time that the BPTF gene inhibitor can significantly inhibit the growth and the tumorigenic capacity of brain glioma cell strains. Therefore, the BPTF gene plays an important role in the brain glioma and is a novel brain glioma treatment target. The present invention has been completed based on this finding.

Term(s) for

Exosomes

Exosomes (exosomes) are membrane vesicles which widely exist and are distributed in various body fluids and can be secreted by various cells, have a lipid bilayer membrane structure, have a diameter generally between 30-120 nm, contain cell-specific proteins, lipids and nucleic acids, can carry and transmit important signal molecules, form a brand-new intercellular information transfer system so as to change the functions of other cells, and play an important role in many physiological and pathological aspects.

BPTF gene and protein encoded by the same

The BPTF Gene (NCBI serial number Gene ID:2186) is located on 17q24 chromosome, is the subunit with the largest molecular weight in chromosome remodeling complex NURF, can recruit other subunits of NURF complex to promoter or enhancer region of Gene, and promotes Gene transcription by regulating nucleosome sliding. The existing research finds that BPTF plays a very important role in the process of neural metaplasia of zebra fish. BPTF functionally and structurally interacts with Smad 2. BPTF and Smad2 synergistically regulate nucleosome slippage of the bound Wnt8a promoter region to regulate target gene expression, thereby exerting a role in nervous system development.

The invention carries out gene chip detection on a non-small cell lung cancer sample related to the brain metastasis tumor, and through large-scale screening, the abnormal expression of BPTF in the brain metastasis tumor is discovered unexpectedly, which shows that the gene is related to the morbidity of the brain metastasis tumor; after silencing BPTF gene in lung embryo fiber cell by RNAi technology, the clone forming ability of the cell is obviously inhibited; finding the BPTF gene with mutation in liver cancer sample; BPTF has a mutant type also found in bladder cancer, and the BPTF is knocked out by aiming at 3 bladder cancer cell lines, so that the clonality of the cells can be remarkably reduced.

In a preferred embodiment of the invention, the sequence of the BPTF gene is as shown in SEQ ID No. 1.

In a preferred embodiment of the invention, the sequence of the protein encoded by the BPTF gene is shown in SEQ ID No. 2.

The BPTF gene encoding protein of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide.

As used herein, the term "RNAi" (RNA interference) refers to a highly conserved, double-stranded RNA (dsrna) -induced phenomenon of highly efficient and specific degradation of RNA with complementary pairing sequences during evolution. Since the expression of a specific gene can be specifically turned off by using the RNAi technology, the technology has been widely used in the fields of gene therapy for exploring gene functions and infectious diseases and tumors. The phenomenon of dsRNA mediated RNAi is found in various eukaryotes such as fungi, Drosophila, Arabidopsis thaliana, trypanosomes, hydroids, vortexes, zebra fish and the like, and the phenomena of posttranscriptional gene silencing (PTGS), cosuppression (cosuppression) and RNA-mediated virus resistance in plants and inhibition (quelling) of fungi also belong to the expression forms of RNAi in different species.

As used herein, the term "siRNA" refers to a Small RNA molecule (about 21-25 nucleotides) that can be processed by Dicer (an enzyme of rnase iii family that is specific for double-stranded RNA) from its precursor (e.g., dsRNA, shRNA, etc.), or can be synthesized chemically or produced by other protein processing. siRNA is a main member of siRISC, and stimulates target RNA with a complementary sequence to be rapidly cut and degraded, so that a target gene is silenced, and the siRNA becomes a key functional molecule in RNAi. Given a particular gene target sequence, one skilled in the art can design and obtain an siRNA directed against that target sequence by routine methods.

As used herein, the term "siRNA precursor" refers to an RNA molecule that can be processed in mammalian cells to produce siRNA, and in particular, selectively processed by Dicer or other similar proteins to produce mature siRNA for RNAi.

As used herein, the term "construct" is a construct comprising an shRNA of the invention.

As used herein, the term "expression cassette" refers to an expression cassette comprising a coding sequence for an shRNA of the invention, and a promoter and termination signal operably linked to the coding sequence, which expression cassette, upon transcription, produces the shRNA of the invention.

As used herein, the term "miRNA" (microRNA) is a class of non-coding single-stranded RNA molecules of about 20-24 nucleotides in length encoded by endogenous genes, involved in the regulation of expression of a large number of genes in animals and plants. To date, over four thousand miRNA molecules have been found in animals and plants as well as viruses. Most miRNA genes exist in the genome in single copy, multiple copies or gene clusters (cluster). Each miRNA can regulate and control a plurality of target genes, and several miRNAs can also be jointly involved in regulating the same gene to form a complex regulation network. It is speculated that mirnas regulate the expression of more than half of the human genes. mirnas exist in a variety of forms, the most primitive being pri-mirnas; pri-miRNA becomes pre-miRNA, namely miRNA precursor, with the length of about 50-90 nucleotides after being processed by Drosha; the pre-miRNA is cut by Dicer enzyme to become mature miRNA with length of about 20-24 nucleotides. miRNA inhibits target gene expression mainly by inhibiting translation and accelerating mRNA polyadenylation, a mechanism different from siRNA-mediated mRNA degradation.

One way to generate "small interfering RNA" (siRNA) in vivo is to clone the siRNA sequence into a plasmid vector as part of a "short hairpin". When delivered to an animal, the hairpin sequence is expressed to form a "double-stranded RNA" (shRNA) with an apical loop structure, which is recognized and processed by intracellular Dicer proteins to produce functional siRNAs.

As used herein, the terms "shRNA", "shRNA" are used interchangeably and refer to a particular shRNA constructed with the human miR-26b precursor as the backbone. The shRNA comprises the following components from 5 'end to 3' end in sequence: (a) 5' end flanking sequence region; (b) 5' end pairing siRNA region; (c) a top ring region; (d) a 3 ' paired siRNA region, and the 5 ' paired siRNA region and the 3 ' paired siRNA region form a double-stranded region; (e) 3' end flanking sequence region; the shRNA generates siRNA, and the nucleotide sequence of the siRNA corresponds to the 3 'paired siRNA region or the 5' paired siRNA region.

Generalized shRNA is an abbreviation for short hairpin RNA, i.e., "short hairpin RNA". The shRNA comprises two short reverse complementary sequences, the middle of the two short reverse complementary sequences is separated by a top ring (loop) sequence, a hairpin structure is formed, the transcription of the shRNA is usually controlled by an RNA polymerase III (RNAPLyMERaseIII) promoter endogenous to a cell, and the tail end of the shRNA sequence is connected with 5-6T which are used as a transcription terminator of the RNA polymerase III. shRNA can also be produced by transcription from promoters of other RNA polymerases.

Pharmaceutical composition

The invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and effective amounts of the following active ingredients: an exosome according to the first aspect of the invention.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the active ingredient of the invention is administered at a daily dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight). For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

Applications of

The invention provides an in vitro non-therapeutic method for inhibiting tumor cells, comprising the following steps: culturing a tumor cell in the presence of the exosome of the first aspect of the invention, thereby inhibiting the tumor cell, the tumor cell being a glioma cell.

In a preferred embodiment of the present invention, the tumor cell inhibition is tumor cell growth inhibition or tumor formation inhibition.

Preferably, the activity of the protein encoded by the BPTF gene in said tumor cell is reduced by more than 10%, preferably by more than 20%, more preferably by more than 30%, more preferably by more than 40%, more preferably by more than 50%, more preferably by more than 60%, more preferably by more than 70%, more preferably by more than 80%, more preferably by more than 90%, most preferably completely free of the activity of the protein encoded by the BPTF gene, compared to a control tumor cell.

Or preferably, the expression level of the BPTF gene in said tumor cell is reduced by more than 10%, preferably by more than 20%, more preferably by more than 30%, more preferably by more than 40%, more preferably by more than 50%, more preferably by more than 60%, more preferably by more than 70%, more preferably by more than 80%, more preferably by more than 90%, most preferably completely free of BPTF gene expression, compared to a control tumor cell.

The main advantages of the invention are:

(1) provides an exosome capable of obviously inhibiting the growth and the tumorigenic capacity of a glioma cell strain for the first time and a preparation method thereof.

(2) Provides a method for efficiently preparing exosome.

(3) The exosome can obviously inhibit the growth and the tumorigenic capacity of a glioma cell strain, so that the exosome can be used for treating glioma.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Parts and percentages are by weight unless otherwise indicated.

Example 1 Gene chip screening assay

Total RNA samples from brain gliomas and control tissues were analyzed by agilent 2100, and aRNA (amplified RNA) was prepared using GeneChip 3' IVT Express Kit. That is, cDNA is obtained through one-strand synthesis, a double-stranded DNA template is obtained through further two-strand synthesis, and then aRNA (amplified RNA) with biotin labeling is obtained through in vitro inversion. aRNA was purified and then fragmented before hybridization to chip probes. After hybridization, washing and dyeing the chip, finally scanning to obtain pictures and original data, carrying out deep analysis on the original data to obtain a final report, wherein specific results are shown in table 1.

TABLE 1

The gene expression difference between a pathological group sample and a normal group sample is detected through a chip, and gene information with obvious expression difference is selected and processed, for example, ATM closely related to a tumor is also highly expressed in the detection, and in addition, the expression of BPTF in a glioma sample is similar to the expression of ATM, is also abnormally highly expressed and is a potential target gene.

The present inventors designed a plurality of siRNAs for the ATM gene (Gene No.: NM-000051) and the BPTF gene, respectively, and examined them.

Typical target information for sirnas against ATM genes is as follows:

ATM siRNA 1#：GCGATTGGCTTATACGCGC(SEQ ID NO.:5)

ATM siRNA 2#：GCGCCTGATTCGAGATCCT(SEQ ID NO.:6)

ATM siRNA 3#：TTACAGTAATTGGAGCATT(SEQ ID NO.:7)

siRNA aiming at different target positions is chemically synthesized and then transfected into a U-87 glioma cell line, and the expression abundance of a target gene ATM is detected through qPCR, so that the most effective target position is screened out.

The screening results are shown in fig. 1A, and the results show that the siRNA specifically targeting target 2(ATM siRNA # 2, KD2) sequence in ATM gene is most effective, and the knockdown efficiency can reach about 60%.

Typical target information for sirnas against the BPTF gene is as follows:

BPTF siRNA 1#：CAGGAGAGTTCTCAAGTAGAT(SEQ ID NO.:3)

BPTF siRNA 2#：CAGCACAGAGAAGACCATGAT(SEQ ID NO.:8)

BPTF siRNA 3#：GAGACTGAGAATGACTCTAAA(SEQ ID NO.:9)

different siRNAs aiming at target genes are chemically synthesized and then transfected into a U-87 glioma cell line, and the expression abundance of the target gene BTPF is detected through qPCR, so that the most effective target spot is screened out.

The screening results are shown in fig. 1B, and the results show that the siRNA specifically targeting target 1(BPTF siRNA # 1, KD1) sequence in BTPF gene is most effective, and the knockdown efficiency can reach about 80%.

Further, applicants compared proliferation levels of U87 glioma cells after both gene knockouts using preferably siRNA to knock out the BTPF and ATM genes, respectively. Specifically, siRNA aiming at different genes is transferred into a glioma cell line, and then the influence of different siRNA target points on the proliferation level of cells is detected through an MTT experiment.

The experimental result is shown in fig. 1C, and through comparative analysis of the MTT experiment, it can be seen from the experimental result that, after the BPTF is knocked out, the proliferation level of the cell is significantly reduced, and the effect of knocking off the ATM gene is also significantly better.

Example 2 construction of CD47 lentiviral vectors:

(1) using CD47cDNA clone as a template, carrying out PCR reaction by an amplification primer, and amplifying to obtain a PCR product with the size of about 1 Kb.

(2) The PCR product and the FUGW viral vector (purchased from Addgene) were subjected to double digestion with BamHI and AgeI.

(3) The transformation reaction was carried out after ligation with T4DNA ligase (purchased from Takara).

(4) Through the identification of the transformant, a positive clone is selected and tested, and a correct clone with a sequence consistent with an expected sequence is selected.

The gene sequence of the CD47cDNA is shown in SEQ ID NO. 4.

Example 3 packaging of CD47 lentiviral vectors:

(1) DNA solutions of 3 plasmids (20. mu.g of CD47 lentiviral vector, 15. mu.g of pCMV-dR8.2dvpr vector (available from Addgene) and 10. mu.g of pCMV-VSV-G vector (available from Addgene) in a lentiviral packaging system were prepared, mixed with a corresponding volume of Opti-MEM and diluted uniformly to adjust the total volume to 2.5ml, and incubated at room temperature for 5 minutes.

(2) Mu.l Lipofectamine2000 (ex invitrogen) reagent was mixed and diluted in another tube with 2.4ml Opti-MEM (ex invitrogen) and incubated for 5 min at room temperature.

(3) The diluted DNA described in (1) and the diluted Lipofectamine2000 described in (2) were mixed and mixed by gentle inversion within 5 minutes. Incubate at room temperature for 20 min.

(4) The DNA and Lipofectamine2000 mixture was transferred to 293T cell (purchased from ATCC) culture medium, mixed, and incubated at 37 ℃ with 5% CO₂Culturing in a cell culture box. After 8 hours of incubation, the medium containing the transfection mixture was poured off, 20ml of PBS was added to each flask of cells, the flask was gently shaken side to wash the residual transfection mixture, and then poured off.

(5) Adding 25ml of cell culture medium containing 10% serum into each flask of cells, and culturing at 37 deg.C with 5% CO₂The incubator was allowed to incubate for 48 hours.

(6) The supernatant of 293T cells 48 hours after transfection was collected. Cell debris was removed by centrifugation at 4000g for 10min at 4 ℃. The supernatant was filtered through a 0.45 μm filter into a 40ml ultracentrifuge tube. The viral crude extract sample was added to the filter cup and the lid was closed, and the filter cup was inserted into the filtrate collection tube. After the combination, the balance is well made and placed on the rotating head. Centrifuge at 4000g for about 10-15 minutes. After centrifugation is completed, the centrifuge is removed and the filter cup is separated from the lower filtrate collection cup. The sample in the sample collecting cup is virus concentrated solution.

(7) Removing the virus concentrated solution, subpackaging and storing in a virus tube, and storing at-80 ℃ for a long time. Is named as LV-CD 47.

Example 4 construction of Stable Strain

The stable cell line was constructed by the following experimental steps:

(1) HEK293 cells (purchased from ATCC) were seeded in 6-well plates 12-18 hours prior to infection with approximately 30% -50% confluency.

(2) The virus was removed from the freezer at-80 ℃ and thawed on ice.

(3) According to the MOI value of 100, the amount of CD47-GFP-LV lentivirus was added.

(4) After mixing evenly, the mixture is put back to the incubator with 5 percent CO2 at 37 ℃ for culturing for 72 h.

(5) The monoclonal cell lines were selected 72 hours after infection.

(6) After the GFP selection, the cell line was frozen and named CD47-HEK293 stable cell line.

(7) Expression of CD47 was detected by immunofluorescence.

FIG. 2A shows the identification result of CD47-HEK293 cell by immunofluorescence, and the result shows that the cell strain constructed in the embodiment can stably express the target protein.

Example 5 exosome preparation and extraction

Replacing serum-free culture medium, continuously culturing CD47-HEK293 stable cell strain for more than 2 days, collecting cell supernatant, and extracting exosome, wherein the experimental steps are as follows: (refer to Shanghai Yubo Biotechnology Ltd., exosome extraction kit instruction manual operation)

(1) Removing cells: the culture supernatant of the stable cell strain is centrifuged for 10 minutes at 4 ℃/300g, and the supernatant is taken;

(2) removal of dead cells: removing cell supernatant, centrifuging at 4 deg.C/2000 g for 10min, and collecting supernatant;

(3) removing cell debris: removing the supernatant of dead cells, centrifuging for 30 minutes at 4 ℃/10000g, and taking the supernatant;

(4) pretreatment of supernatant fluid: adding Exosomoe Concentration Solution (ECS reagent) to the centrifuged supernatant with impurities removed, and adding 5mL of ECS reagent to every 20mL of supernatant;

(5) precipitating exosomes: centrifuging the resuspension solution at 4 deg.C/100000 g for 70 min to obtain precipitate as exosome, resuspending with appropriate amount of PBS, packaging, and storing in-80 deg.C refrigerator.

(6) Solution mixing: adding ECS reagent, tightly covering the centrifugal tube, uniformly mixing for 1min by using a vortex oscillator, and standing for 2h at the temperature of 2-8 ℃;

(7) precipitating exosomes: taking out the centrifuge tube filled with the mixed solution, centrifuging at 4 ℃ for 60min at 10000g, discarding the supernatant, and allowing the precipitate to be rich in exosome particles; (Note: supernatant was removed as far as possible)

(8) Suspension of the exosome: uniformly blowing 100 mu L of 1 XPBS into the centrifugal precipitate, and transferring the heavy suspension into a new 1.5mL centrifuge tube after the centrifugal precipitate is uniformly suspended in the PBS;

(9) harvesting exosome particles: a1.5 mL centrifuge tube containing the resuspension was centrifuged at 12000g for 2min at 4 ℃ and the supernatant, which was enriched with exosome particles, was retained.

(10) Purifying the exosomes: transferring the obtained crude product of the exosome particles into an upper chamber of an exosome purification Filter (EPF column), centrifuging at 4 ℃ for 10min at 3000g, and collecting liquid at the bottom of the EPF column after centrifugation, wherein the liquid is the purified exosome particles;

(11) preservation of exosomes: the purified exosomes were stored in a-80 ℃ low-temperature refrigerator for later use in experiments.

Example 6 exosome identification

(1) Transmission electron microscopy inspection

The screened exosome samples were processed as follows:

fixing: 2.5% glutaraldehyde fixation for 2 hours; rinsing with 0.1M phosphoric acid rinsing solution for three times;

1% osmate fixation for 2 hours; rinsing with 0.1M phosphoric acid rinsing solution for three times;

and (3) dehydrating: 50% ethanol, 20 minutes; 70% ethanol for 20 minutes; 90% ethanol, 20 minutes; 90% ethanol: 90% acetone (1: 1), 20 minutes; 90% acetone for 20 minutes; the above is carried out in a refrigerator with 4 ℃,100 percent acetone at room temperature for 20 minutes;

embedding: pure acetone + embedding solution (2: 1) at room temperature for 3 hours; pure acetone + embedding solution (1: 2) at room temperature overnight; pure embedding liquid, 2 hours at 37 ℃;

and (3) curing: oven at 37 deg.C, overnight; in a 45-degree oven, 12 hours; in a 60-degree oven, 24 hours;

slicing (microtome slicing 50-60 nm): installing a sample, and selecting a knife edge to enable the knife edge to be parallel to the sample; checking each clamp locking device, opening a locking switch, and manually contacting the sample with the cutter face through the adjustment of fine and rough adjustment; adding distilled water to refract the water to control the water surface to be parallel to the knife surface; switching automatic slicing operation, collecting slices on water surface, and fumigating with chloroform; then, fishing out the slices by using a copper net;

dyeing: 3% uranium acetate-lead citrate double dyeing.

And (3) detection: and (5) observing and taking a picture by using a transmission electron microscope and shooting the picture.

(2) Nano laser particle size meter detection

The screened exosome samples were processed as follows: (taking Zetaview particle sizer in USA as an example)

Turning on a computer and an instrument power supply, preheating the instrument for 10 minutes, starting operation software, setting a particle size mode and parameters, putting the screened exosome sample into a sample pool, putting the sample pool on an embedded sample detection device of the instrument, closing a sliding cover, setting detection time and times, starting to detect the sample, and observing a front value of a panel, wherein the value is about 300 HZ; if the sample is too low or too high, the detection is stopped, and the sample is diluted properly and then measured. And after the detection is finished, storing the data.

(3) Western Blot detection

The screened exosome samples were processed as follows:

protein extraction: adding lysis solution into the screened exosome, lysing for 15 minutes at 4 ℃, then centrifuging for 5 minutes at 4 ℃/12000g, putting into a water bath at 100 ℃ for 20 minutes, and then continuously centrifuging for 2 minutes at 12000g at 4 ℃, and preserving at-20 ℃ for later use.

SDS-PAGE was configured: and (5) washing the glass plate, and drying. Putting the dried glass plate into a manufacturing tool according to the requirement, preparing SDS-PAGE glue according to the size of protein, firstly preparing separation glue, adding 7ml of separation glue into the glass plate, then adding 2ml of absolute ethyl alcohol, preparing concentrated glue after the separation glue is fully solidified after 30min, removing the absolute ethyl alcohol in the glass plate, sucking the residual absolute ethyl alcohol by using filter paper, adding 2ml of concentrated glue, and then inserting the filter paper into comb teeth.

Loading and electrophoresis: after the gel is solidified, the gel is placed in an electrophoresis tank, and sufficient electrophoresis liquid is added to start to prepare for sample loading. The comb was gently pulled upward by holding both ends of the comb with both hands, the loading hole was gently purged by sucking electrophoresis buffer with a 1ml pipette, the prepared samples were loaded, the same total protein amount was taken for each sample, and the samples were slowly added into the loading hole perpendicularly to the loading hole with a 20ul pipette. Adding proper amount of electrophoretic liquid into the lower part of the electrophoretic tank

Electrophoresis: constant current 30mA 2 hours

Immunoblotting: after the electrophoresis is finished, the protein is transferred to a PVDF membrane by using a transfer electrophoresis device and electrotransformation for 120 minutes under the conditions of 4 ℃ and 400mA constant current: pouring 500-800ml of electrotransfer buffer solution into a medical tray, taking out the glass plate from the electrophoresis device, prying the two glass plates at the upper end of the glass plate by using a rubber shovel, slightly cutting off the bottom end of the rubber by using the rubber shovel, slightly supporting the rubber by using the rubber shovel and placing the rubber on filter paper, and placing the rubber in sequence from the negative electrode to the positive electrode: filter paper-glue-PVDF membrane-filter paper, the tool is placed into a transfer electrophoresis device, and 1L of electrotransfer buffer solution is added for membrane transfer.

And (3) immune color development:

and (3) sealing: pouring 40-50ml of blocking solution into the culture dish, putting the transferred PVDF membrane into the culture dish with the front side facing upwards to prevent protein shedding, completely immersing the PVDF membrane in the blocking solution, and blocking at room temperature for 1 hour.

Primary antibody incubation: the sealed PVDF membrane is covered by a PE glove, an antibody diluted by the sealing liquid is added, and the membrane is placed on a mixer to incubate for 12 hours at 4 ℃.

Washing the membrane: the PVDF membrane was removed from the PE glove, placed in a petri dish, added with 40-50ml of TBST solution, gently shaken on a decolorizing shaker, and the membrane was washed 3 times for 10 minutes each time.

And (3) secondary antibody incubation: the corresponding secondary antibody was diluted with blocking solution and the PVDF membrane was incubated at room temperature for 2 hours.

Color development: color development was performed using ECL + plusTM Western blotting system kit from Amersham: the film acquisition of the display strip was performed in a dark room.

The specific steps of ECL adding, exposure, development and fixation are as follows:

placing the PVDF film on a flat preservative film, and mixing the PVDF film with a mixing ratio of 1: 40 to prepare 1ml of total volume, uniformly dripping the mixed solution on a PVDF membrane, and reacting for 5 minutes in a dark place.

Taking out the film, draining off redundant ECL substrate reaction liquid, not drying PVDF, keeping PVDF without obvious reaction liquid drops, putting the film into a cassette, laying a preservative film, closing the cassette, and selecting the length of exposure time to make a corresponding mark on the preservative film according to the light emitting degree of ECL. Taking out the X-ray film, putting into the developing solution, taking out after about 1min, rinsing in clear water for a few seconds, and then putting into the fixing solution for 2 min.

Taking the X-ray film out of the fixing solution by using tweezers with gloves, putting the X-ray film into a 65-degree oven for drying and analyzing. And (3) placing the dried X-ray film into a dark box, marking the position of the protein pre-dyeing marker and the name of the sample on the X-ray film by using a marker pen according to the mark made before, and analyzing the result.

FIG. 2B shows electron microscopy of CD 47-siRNA-exosomes (exosomes).

FIG. 3 shows the results of particle size measurement of CD 47-siRNA-Exosome.

FIG. 4 shows the result of detecting CD47-siRNA-Exosome marker protein by Western blot.

Example 7 exosome-siRNA complex preparation

1. Adding a proper amount of siRNA into a 1.5ml centrifuge tube, adding the exosome suspension, and gently mixing. The proportions used are as follows, 10⁹Exosome particles: 1ug siRNA: 400uL of electrotransfer buffer).

2. Neon containing 3ml of electrolytic buffer E^TMThe tubes are inserted into a pipettor rack.

3. The instrument is provided with pulse voltage, pulse width and pulse number.

4. The tip was inserted into a Neon pipette and the exosome mixture was aspirated using a 10. mu.l tip, which had to be bubble free. A neon (tm) pipette with sample was inserted vertically into the neon (tm) tube.

5. The electroporation protocol is selected and the Start key on the touch screen is pressed.

6. After the electric pulse is released, the completion of the display on the touch screen can prompt that the electroporation is completed.

7. The pulsed sample was immediately transferred to a prepared plate containing pre-warmed medium. The culture plate is placed in an incubator for culture.

8. After the experiment is finished, E liquid in the pipettor is poured off, and the electrotransfer instrument is closed.

Through experimental verification, the sequence of the BTFP siRNA is preferably as follows:

CAGGAGAGTTCTCAAGTAGAT(SEQ ID NO.:3)。

example 8 exosome-cell co-incubation experiments

Culturing target cells (U-87-GFP stable strain and U-251-GFP stable strain, purchased from cell bank of Chinese academy of sciences) with good growth state, culturing the target cells in 6-well culture plates in the previous day, and adding exosome particles according to the group of experimental design on the day of co-incubation to perform co-incubation experiment of the target cells. After incubation, GFP expression was observed under a fluorescent microscope.

(1) Cell proliferation assay

The U-87-GFP stable strain and the U-251-GFP stable strain have green fluorescence, cells with the fluorescence can be read and photographed by a Cellomics instrument, and then the number of the cells contained in different groups in the pore plate is calculated by software analysis and processing. After continuous detection for 3-5 days, a cell growth curve graph is drawn, so that the cell growth condition is presented.

a) After the pancreatin digestion of the cells of each experimental group in the logarithmic growth phase, the complete culture medium is re-suspended into cell suspension;

b) counting the cells with a hemocytometer;

c) the plating cell density was determined according to the growth rate of the cells (mostly 2000 cells/well). Each group has 3-5 multiple wells, each well has 100 μ l, and the number of cells added in each well is ensured to be consistent in the plating process;

d) after the plates are paved, the plates are placed in a 37 ℃ 5% CO2 incubator for culture;

e) from the next day after the plate laying, detecting and reading the plate by CELLOMICS once every day, and continuously detecting and reading the plate for 3-5 days;

f) accurately calculating the number of cells with green fluorescence in each scanning pore plate by adjusting the input parameters of the cells arrayscan;

g) the data were statistically plotted and cell proliferation curves were plotted for 5 days.

FIG. 5 shows the results of fluorescence microscopy of exosomes incubated with glioma cells.

Figure 6 shows the results of the MTT assay with exosomes incubated with glioma cells. The detection result shows that the RNAi of the specific target BPTF gene can obviously inhibit the proliferation of the tumor cells.

(2) Cell clonogenic assay

The tumorigenicity of the lentivirus-infected cells was suggested by their clonogenic capacity on cell culture plates after infection.

a) Digesting the cells of each experimental group in the logarithmic growth phase by pancreatin, and carrying out heavy suspension on a complete culture medium to prepare a cell suspension;

b) counting the cells of the cell suspension by a blood cell counting plate;

c) cell inoculation: inoculating 800 cells/well in each experimental group in a 6-well plate culture plate, wherein each experimental group is provided with 3 multiple wells;

d) continuously culturing the inoculated cells in an incubator for 14 days or until the number of the cells in most single clones is more than 50, changing the liquid every 3day midway and observing the cell state;

e) photographing the cell clone under a fluorescent microscope before the experiment is terminated;

f) cells were washed 1 time with PBS at the end of the experiment;

g) adding 1ml of paraformaldehyde into each hole, and fixing cells for 30-60 min;

h) PBS washed cells 1 time;

i) adding 500 μ L of clean and impurity-free GIEMSA staining solution into each hole, and staining cells for 20 min;

j)ddH₂o washing the cells several times until the plates are washedBackground drying;

k) taking a picture of the monoclonal under a microscope;

l) taking a picture of the whole plate by the digital camera;

m) clone counting.

FIG. 7 shows the results of a clonogenic assay with exosomes incubated with glioma cells. The detection result shows that the exosome carrying the siRNA can effectively inhibit the proliferation of the cell after being endocytosed by the cell after being incubated with the cell.

(3) Cell invasion capacity assay

Invasion from the extracellular matrix is an important step in tumor metastasis, tumor cells begin to invade by adhering to and stretching along the vessel wall, and proteolytic enzymes such as MMP collagenases dissolve the vascular basement membrane to allow cancer cell invasion. BD Biocoat^TM Matrigel^TMInvasion Chamber provides an effective system for detecting tumor cell crossing through a basal membrane model.

a) Taking out the kit from the refrigerator at-20 ℃, disinfecting the forceps by using 70% ethanol, taking out the required number of small chambers to a new 24-pore plate, and standing at room temperature for a period of time to restore the room temperature;

b) adding 500 μ l of incubation (37 ℃) serum-free medium to each of the chamber and the lower chamber, and placing in a 37 ℃ incubator for 2h to rehydrate the Matrigel matrix layer;

c) preparing a cell suspension: digesting each group of cells in logarithmic growth phase by using pancreatin, and carrying out heavy suspension by using a serum-free culture medium to prepare a cell suspension;

d) counting the cells of the cell suspension by a blood cell counting plate;

e) after rehydration in step 3, the chamber was transferred to another well and the medium in the chamber was carefully removed;

f) add 750. mu.l of 30% FBS medium to the lower chamber;

g) add 500. mu.l of the cell suspension prepared in step 4 (cell density adjusted for different cell types, typically 5-10 x 104 cells) to each chamber;

h) culturing at 37 deg.C for 24-48h (adjusted according to cell type);

i) reverse cell on absorbent paper to remove the medium, use cotton swab gently to remove non-invasive cells

j) Add 500. mu.l of staining solution to the wells of the plate;

k) soaking the small chamber in Gimesas staining solution for 30min to stain the lower surface of the membrane to invade cells;

l) preparing a big beaker filled with three-quarter of water, clamping the small chamber with a small forceps, washing back and forth, and airing the small chamber in the air;

m) taking a picture of the whole chamber by using a camera, wherein focusing is important during taking the picture;

n) a microscope photographing film, wherein 100X and 400X are respectively photographed for a plurality of times (more than or equal to 5);

o) adding 200uL 10% acetic acid into the air of a 96-well plate, removing the bottom film by using scissors and tweezers, dissolving in 180uL 10% acetic acid, completely dissolving (sucking and blowing by using a 200uL gun head, uniformly stirring), sucking 100uL into another hole, and detecting by OD 570.

FIG. 8 shows the results of testing the invasiveness of exosomes incubated with glioma cells. The detection result shows that the exosome carrying the siRNA can effectively inhibit the invasion capacity of the cell after being endocytosed by the cell after being incubated with the cell.

Example 9 exosome injection tumorigenicity nude mouse experiment

In tumor studies, the most common animal experiment is a nude mouse tumor formation experiment, and most tumor studies use human cells, so that there exists a possibility of xeno-exclusion, and it is necessary to use an immunodeficient mouse as a carrier for a transplantation model, inject tumor cells into the mouse to form tumors, then inject exosome particles, and observe the growth of tumor bodies to judge the biological changes.

a) After pancreatin digestion of tumor cells which are formed by each experiment in logarithmic growth phase, re-suspending a complete culture medium into a cell suspension;

b) counting the cells by using a blood counting plate, and finally re-suspending the cells by using a certain volume of PBS (phosphate buffer solution) to ensure that the concentration of the cell suspension is 1-2 multiplied by 10⁷Individual cells/ml;

c) injecting a certain amount of cell suspension into right armpit of nude mouse by using disposable syringeThe amount of cells of (A) is generally 2X 10⁶(ii) individual cells;

d) feeding the nude mice after injection until the tumor bodies can be seen by naked eyes; (time 2 weeks or so)

e) After injecting exosome PBS solution (100ug/ml,100 ul/mouse) every day and continuing to feed for 4 weeks, ending the experiment and collecting data;

f) photographing (including a photograph of the tumor of the nude mice after sacrifice and a photograph of the tumor body);

g) the data were statistically plotted.

FIG. 9 shows the experimental results of exosome injection tumorigenic nude mice. The detection result shows that the exosome carrying the siRNA can effectively inhibit the proliferation of the tumor through blood circulation after being injected into a nude mouse model.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of exosomes for the preparation of a medicament for the prevention or treatment of tumour; the tumor is brain glioma; the exosome membrane of the exosome contains a CD47 molecule, and the exosome is wrapped by an siRNA molecule or a precursor thereof, and the sequence of the siRNA molecule is shown in SEQ ID NO. 3.

2. The use according to claim 1, wherein the method of preparing said exosomes comprises the steps of:

(1) construction of Lentiviral expression vectors

(2) lentiviral packaging

(3) construction of cell lines

Infecting host cells with the virus particles obtained in step (2), thereby constructing a cell strain stably expressing CD 47;

(4) preparation of exosomes

Culturing the cell strain constructed in the step (3), collecting cell supernatant, and extracting exosomes, thereby obtaining the exosomes;

(5) preparation of exosome-siRNA complexes

Encapsulating the siRNA molecule into the exosome by adopting an electrical transduction mode, thereby preparing the exosome-siRNA compound;

wherein, the siRNA molecule sequence is shown in SEQ ID NO. 3.

3. The use according to claim 2, wherein in step (5), the ratio of the amount of exosome particles to the amount of siRNA used during the electrical transduction is 10⁸～10¹⁰Individual exosome particles: 0.1-10 μ g siRNA.

4. The use according to claim 2, wherein in step (1), the expression vector is a FUGW virus vector as a backbone.

5. The use of claim 2, wherein in step (2) the helper plasmids used in the lentiviral packaging process are a pCMV-dr8.2dvpr vector and a pCMV-VSV-G vector.

6. The use of claim 2, wherein in step (2), the packaging cells are 293T cells.

7. A method for non-therapeutic inhibition of tumor cells in vitro comprising the steps of: culturing the tumor cell in the presence of exosomes, thereby inhibiting the tumor cell; the tumor cell is a glioma cell line; the exosome membrane of the exosome contains a CD47 molecule, and the exosome is wrapped by an siRNA molecule or a precursor thereof, and the sequence of the siRNA molecule is shown in SEQ ID NO. 3.