CN117384859B - Preparation method and application of exosome from dendritic cells - Google Patents
Preparation method and application of exosome from dendritic cells Download PDFInfo
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- CN117384859B CN117384859B CN202311703663.5A CN202311703663A CN117384859B CN 117384859 B CN117384859 B CN 117384859B CN 202311703663 A CN202311703663 A CN 202311703663A CN 117384859 B CN117384859 B CN 117384859B
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Abstract
The invention provides a preparation method and application of an exosome derived from dendritic cells. The exosome from antigen presenting cell source provided by the invention anchors TRAIL (tumor necrosis factor related apoptosis inducing ligand) molecule with bioactivity on the exosome membrane, and through specific expression of protein CD81 on the exosome membrane surface, the loading rate of TRAIL molecule is enhanced, and the killing efficiency of the exosome on tumor cells is greatly improved. The invention also provides application of the exosome in tumor treatment.
Description
Technical Field
The invention relates to the technical field of biological medicine and oncology, in particular to an anti-tumor engineering exosome, a preparation method and application.
Background
With the progressive development of tumor immunity, tumor vaccines such as Dendritic Cell (DC) vaccines have achieved a pleasing success in recent decadesAnd (5) fruits. However, more problems limit the clinical application of DC vaccines, such as high preparation cost, high storage requirements, immunosuppressive environment inside tumor, screening of tumor antigens, and severe degradation of antigen peptides in vivo, which greatly affect the market of DC cell vaccines. However, exosomes derived from DC cells have many advantages over these problems, and can carry a variety of proteins, RNAs and lipids. More importantly, DC cell-derived exosomes express tumor antigens, MHC molecules and co-stimulatory molecules on their surface, triggering antigen-specific CD4 + And CD8 + Proliferation of T cells. By virtue of the double-layer membrane structure, the DC exosome can be prevented from being degraded, and meanwhile, good biocompatibility and safety are ensured. In addition, the DC exosomes can be stored in vitro for a longer time, reducing production costs. In clinical applications, DC exosomes have been shown to have good antitumor efficacy. Compared with DC vaccine, the DC exosome is a natural nano delivery carrier with wider clinical application space due to higher immunogenicity and stronger resistance to immunosuppression.
Examples of direct tumor killing by exosomes have been reported, such as NK cell-derived exosomes, exosomes engineered to be loaded with antitumor small molecule drugs, and the like. By means of the molecular advantages of the surface of a DC cell membrane, DC cell exosomes are engineered through DC cell indirect genetic engineering, tumor necrosis factor related apoptosis-inducing ligand (TRAIL) molecules are stably expressed on the surface of the membrane through slow viruses, and TRAIL molecules are naturally loaded while exosomes are generated. However, TRAIL loaded in this way has low molecular load and weak killing, and needs to be further improved. The invention provides a preparation method of exosomes for increasing TRAIL load, which can obviously enhance the anti-tumor capability of exosomes.
Disclosure of Invention
In one aspect, provided herein is an exosome comprising a fusion protein, wherein the fusion protein comprises exosome membrane protein CD81 and an anti-tumor factor.
In some embodiments, the anti-tumor factor is from a tumor necrosis factor family member, preferably the anti-tumor factor is TRAIL.
In some embodiments, the fusion protein comprises SEQ ID NO: 6.
In some embodiments, the exosomes are derived from immune cells engineered to be capable of expressing the fusion protein.
In some embodiments, the immune cell is a DC cell, T cell, B cell, or NK cell.
In another aspect, the invention provides a method of preparing an exosome comprising:
1) Introducing into a cell a nucleic acid molecule comprising a coding sequence for a fusion protein, wherein the fusion protein comprises exosome membrane protein CD81 and an anti-tumor factor;
2) Culturing the cells under conditions suitable for expression of the coding sequence and collecting the exosomes secreted thereby.
In some embodiments, the anti-tumor factor is from a tumor necrosis factor family member, preferably the anti-tumor factor is TRAIL.
In some embodiments, the nucleic acid molecule comprises SEQ ID NO:5, and a nucleotide sequence as described in the specification.
In some embodiments, the fusion protein comprises CD81 and TRAIL.
In some embodiments, the fusion protein comprises SEQ ID NO: 6.
In some embodiments, the cell is an immune cell, preferably a DC cell, T cell, B cell or NK cell.
In another aspect, the invention provides an anti-tumor pharmaceutical composition comprising the exosome described above and a pharmaceutically acceptable carrier.
In another aspect, the invention provides the use of the exosomes described above in the manufacture of a medicament for the treatment of a tumor.
In some embodiments, the tumor is selected from lung cancer, liver cancer, melanoma, fibrosarcoma, cervical cancer, pancreatic cancer, prostate cancer, breast cancer, esophageal cancer, brain glioma, colorectal cancer, renal cancer, multiple myeloma, and hematological tumors, such as chronic myelogenous leukemia.
The exosome from antigen presenting cell provided by the invention anchors TRAIL (tumor necrosis factor related apoptosis inducing ligand) molecule with bioactivity on the exosome membrane, and through specific expression of protein CD81 on the exosome membrane surface, the loading rate of TRAIL molecule is enhanced, and the killing efficiency of the exosome on tumor cells is greatly improved.
Drawings
FIG. 1 shows the results of Western-blot identification of exosome membrane markers of example 1.
Fig. 2 shows the nanoparticle tracking analysis results of eDex of example 1.
Fig. 3 shows the electron microscope observation result of eDex of example 1.
FIG. 4 shows the effect of engineered DC supernatant on killing A375 cells in example 2.
FIG. 5 shows the effect of eDex on killing A375 cells in example 3.
FIG. 6 shows the effect of the engineered DC supernatant on killing Hela cells in example 4.
FIG. 7 shows the effect of eDex on killing Hela cells in example 5.
FIG. 8 shows the effect of engineered DC supernatant on killing A549 cells in example 6.
FIG. 9 shows the effect of eDex on killing A549 cells in example 7.
FIG. 10 shows the effects of eDex on killing RPMI8226 cells in example 8.
FIG. 11 shows the effect of eDex on killing HT1080 cells in example 9.
FIG. 12 shows the effect of eDex on killing HepG2 cells in example 10.
FIG. 13 shows the effect of eDex on killing K562 cells in example 11.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The terms "comprises" or "comprising" are intended to include the recited element, integer or step, but not to exclude any other element, integer or step. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, are also intended to cover the circumstance that the recited elements, integers or steps consist of them.
Reference to a pharmaceutical composition, as used herein, "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, etc., which may be safely administered, and which is suitable for administration to humans and/or animals without undue adverse side effects, while maintaining the viability of the drug or active agent located therein.
An "exosome" is a small vesicle secreted by numerous cells, with a lipid bilayer membrane structure, approximately 10-300 a nm a diameter (other sizes are possible). Exosomes may contain proteins, lipids and nucleic acids specific to their secreting cells, and can be used as mediators of intercellular information and substance communication. Exosomes play an important role in many physiological pathologies, such as antigen presentation in immunity, tumor growth and migration, repair of tissue damage, etc. Exosomes secreted by different cells have different compositions and functions and can be used as biomarkers for disease diagnosis. The exosome has a lipid bilayer membrane structure, can well protect the substance coated by the exosome, and can be modified to target specific cells or tissues, so that the exosome can also be used as a tool for targeted drug delivery.
"fusion protein" refers to a peptide chain comprising at least two interconnected peptide segments, which are not normally interconnected in nature. Methods for producing fusion proteins are known in the art, for example, by joining the nucleic acid sequences encoding the two peptide fragments together by genetic engineering techniques and expressing them in a host cell with the aid of an appropriate expression vector. The two peptide fragments in the fusion protein may be linked by a short peptide linker or, alternatively, directly without affecting the activity of both. In a fusion protein, to express the positional relationship between two peptide fragments, a peptide fragment near the N-terminus may be referred to as an "upstream" peptide fragment, and a peptide fragment near the C-terminus may be referred to as a "downstream" peptide fragment. Among the fusion proteins mentioned herein, including CD81 and TRAIL, TRAIL is preferably located at the C-terminus, but it is not excluded that it may also have a similar effect when it is located at the N-terminus.
The invention provides a preparation method and application of an anti-tumor engineering exosome, and the cell is modified in a genetic engineering mode, so that the loading efficiency of anti-tumor molecules and the anti-tumor effect are improved.
The invention relates to an anti-tumor engineering exosome, which is an exosome derived from dendritic cells for specifically expressing CD81-TRAIL.
The exosomes are immune cell-derived exosomes, preferably DC cell-derived exosomes, and may also be NK cell, T cell or B cell-derived exosomes.
The exosomes of the present application are preferably exosomes of human origin.
The invention provides a preparation method of an anti-tumor engineering exosome, which comprises the following steps: culturing cells, engineering the cells, culturing and collecting cell supernatant on a large scale, and separating exosomes.
More specifically, the invention provides an anti-tumor engineering exosome, which is characterized in that the exosome is a membrane protein engineering exosome. In some embodiments, the exosomes are immune cell-derived exosomes. In some embodiments, the exosomes are DC, T, B, or NK cell derived exosomes. In some embodiments, the membrane protein is CD81. In some embodiments, the anti-tumor killing factor used is TRAIL. In some embodiments, the anti-tumor fusion protein used has the structure CD81-TRAIL. The invention also provides application of the engineering exosome in preparing antitumor drugs.
The invention has the beneficial effect that the exosomes (eDex, engineered DC-derived exosomes) provided by the invention can be used as antitumor drugs. The engineered DC cell exosomes eDex have the following properties relative to the native DC cell-derived exosomes Dex:
1. from the large-scale DC generation, large-scale production can be performed.
2. Specific killing of a variety of tumor cells including lung cancer, liver cancer, melanoma, fibrosarcoma, multiple myeloma, chronic myelogenous leukemia, and cervical cancer. Through the test of the tumor cells, the exosomes of the invention can be popularized and extended to other applications of cancer species including pancreatic cancer, prostate cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, renal cancer, hematological tumor and the like.
3. The eDex has various cytokines on natural DC, can activate immune system, and synergistically enhances antitumor effect.
4, only has killing effect on tumor cells, but has no effect on normal cells.
The invention combines the current advantages and defects of exosomes and cellular immunotherapy, reforms the exosomes derived from DC cells, loads CD81-TRAIL on the exosomes, further improves the enrichment of TRAIL on the DC exosomes on the basis of natural loading of TRAIL on the exosomes, and remarkably improves the anti-tumor immunity of the exosomes. By utilizing the inherent molecule CD81 on DC exosomes and the fusion expression of TRAIL, the expression efficiency of TRAIL on exosomes is up-regulated, and the tumor killing effect is greatly enhanced.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the following embodiments specifically describe the technical solutions and practical applications of the present invention with reference to the accompanying drawings. The described embodiments are some, but not all, embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are obtained by a person skilled in the art without making any inventive effort, are within the scope of the invention.
EXAMPLE 1 construction of engineered DCs and extraction of eDex
By utilizing the advantage that mature high-activity dendritic cell engineering DC (see Chinese patent publication CN 108546679A) can be amplified in vitro in a large amount, a large number of dendritic-derived exosomes can be obtained.
1. Designing primers, cloning CD81 and TRAIL full-length genes by PCR, and connecting through BamHI enzyme cutting sites (the CD81 nucleic acid sequence is shown as SEQ ID NO. 1, the CD81 amino acid sequence is shown as SEQ ID NO. 2, the TRAIL nucleic acid sequence is shown as SEQ ID NO. 3, and the TRAIL amino acid sequence is shown as SEQ ID NO. 4), thus constructing the lentiviral vector.
2. Packaging and concentrating lentivirus, and transfecting the lentivirus into the engineering DC cell, wherein the expression of TRAIL is detected to be more than 99% by flow, thus obtaining the DC-TRAIL cell strain.
3. Designing primer, cloning CD81-TRAIL fusion gene by PCR (nucleic acid sequence is shown as SEQ ID NO:5, amino acid sequence is shown as SEQ ID NO: 6), constructing slow virus vector.
4. Packaging and concentrating lentivirus, and transfecting the lentivirus into the engineering DC cell, wherein the expression of TRAIL is detected to be more than 99% by flow, thus obtaining the DC-CD81-TRAIL cell strain.
CD81 nucleic acid sequence (SEQ ID NO: 1):
ATGGGAGTGGAGGGCTGCACCAAGTGCATCAAGTACCTGCTCTTCGTCTTCAATTTCGTCTTCTGGCTGGCTGGAGGCGTGATCCTGGGTGTGGCCCTGTGGCTCCGCCATGACCCGCAGACCACCAACCTCCTGTATCTGGAGCTGGGAGACAAGCCCGCGCCCAACACCTTCTATGTAGGCATCTACATCCTCATCGCTGTGGGCGCTGTCATGATGTTCGTTGGCTTCCTGGGCTGCTACGGGGCCATCCAGGAATCCCAGTGCCTGCTGGGGACGTTCTTCACCTGCCTGGTCATCCTGTTTGCCTGTGAGGTGGCCGCCGGCATCTGGGGCTTTGTCAACAAGGACCAGATCGCCAAGGATGTGAAGCAGTTCTATGACCAGGCCCTACAGCAGGCCGTGGTGGATGATGACGCCAACAACGCCAAGGCTGTGGTGAAGACCTTCCACGAGACGCTTGACTGCTGTGGCTCCAGCACACTGACTGCTTTGACCACCTCAGTGCTCAAGAACAATTTGTGTCCCTCGGGCAGCAACATCATCAGCAACCTCTTCAAGGAGGACTGCCACCAGAAGATCGATGACCTCTTCTCCGGGAAGCTGTACCTCATCGGCATTGCTGCCATCGTGGTCGCTGTGATCATGATCTTCGAGATGATCCTGAGCATGGTGCTGTGCTGTGGCATCCGGAACAGCTCCGTGTAC
CD81 amino acid sequence (SEQ ID NO: 2)
MGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTLTALTTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSGKLYLIGIAAIVVAVIMIFEMILSMVLCCGIRNSSVY
TRAIL nucleic acid sequence (SEQ ID NO: 3): atggctatgatggaggtccaggggggacccagcctgggacagacctgcgtgctgatcgtgatcttcacagtgctcctgcagtctctctgtgtggctgtaacttacgtgtactttaccaacgagctgaagcagatgcaggacaagtactccaaaagtggcattgcttgtttcttaaaagaagatgacagttattgggaccccaatgacgaagagagtatgaacagcccctgctggcaagtcaagtggcaactccgtcagctcgttagaaagatgattttgagaacctctgaggaaaccatttctacagttcaagaaaagcaacaaaatatttctcccctagtgagagaaagaggtcctcagagagtagcagctcacataactgggaccagaggaagaagcaacacattgtcttctccaaactccaagaatgaaaaggctctgggccgcaaaataaactcctgggaatcatcaaggagtgggcattcattcctgagcaacttgcacttgaggaatggtgaactggtcatccatgaaaaagggttttactacatctattcccaaacatactttcgatttcaggaggaaataaaagaaaacacaaagaacgacaaacaaatggtccaatatatttacaaatacacaagttatcctgaccctatattgttgatgaaaagtgctagaaatagttgttggtctaaagatgcagaatatggactctattccatctatcaagggggaatatttgagcttaaggaaaatgacagaatttttgtttctgtaacaaatgagcacttgatagacatggaccatgaagccagtttttttggggcctttttagttggctaa
TRAIL amino acid sequence (SEQ ID NO: 4):
MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG-
CD81-TRAIL nucleic acid sequence (SEQ ID NO: 5):
ATGGGAGTGGAGGGCTGCACCAAGTGCATCAAGTACCTGCTCTTCGTCTTCAATTTCGTCTTCTGGCTGGCTGGAGGCGTGATCCTGGGTGTGGCCCTGTGGCTCCGCCATGACCCGCAGACCACCAACCTCCTGTATCTGGAGCTGGGAGACAAGCCCGCGCCCAACACCTTCTATGTAGGCATCTACATCCTCATCGCTGTGGGCGCTGTCATGATGTTCGTTGGCTTCCTGGGCTGCTACGGGGCCATCCAGGAATCCCAGTGCCTGCTGGGGACGTTCTTCACCTGCCTGGTCATCCTGTTTGCCTGTGAGGTGGCCGCCGGCATCTGGGGCTTTGTCAACAAGGACCAGATCGCCAAGGATGTGAAGCAGTTCTATGACCAGGCCCTACAGCAGGCCGTGGTGGATGATGACGCCAACAACGCCAAGGCTGTGGTGAAGACCTTCCACGAGACGCTTGACTGCTGTGGCTCCAGCACACTGACTGCTTTGACCACCTCAGTGCTCAAGAACAATTTGTGTCCCTCGGGCAGCAACATCATCAGCAACCTCTTCAAGGAGGACTGCCACCAGAAGATCGATGACCTCTTCTCCGGGAAGCTGTACCTCATCGGCATTGCTGCCATCGTGGTCGCTGTGATCATGATCTTCGAGATGATCCTGAGCATGGTGCTGTGCTGTGGCATCCGGAACAGCTCCGTGTACGGATCCatggctatgatggaggtccaggggggacccagcctgggacagacctgcgtgctgatcgtgatcttcacagtgctcctgcagtctctctgtgtggctgtaacttacgtgtactttaccaacgagctgaagcagatgcaggacaagtactccaaaagtggcattgcttgtttcttaaaagaagatgacagttattgggaccccaatgacgaagagagtatgaacagcccctgctggcaagtcaagtggcaactccgtcagctcgttagaaagatgattttgagaacctctgaggaaaccatttctacagttcaagaaaagcaacaaaatatttctcccctagtgagagaaagaggtcctcagagagtagcagctcacataactgggaccagaggaagaagcaacacattgtcttctccaaactccaagaatgaaaaggctctgggccgcaaaataaactcctgggaatcatcaaggagtgggcattcattcctgagcaacttgcacttgaggaatggtgaactggtcatccatgaaaaagggttttactacatctattcccaaacatactttcgatttcaggaggaaataaaagaaaacacaaagaacgacaaacaaatggtccaatatatttacaaatacacaagttatcctgaccctatattgttgatgaaaagtgctagaaatagttgttggtctaaagatgcagaatatggactctattccatctatcaagggggaatatttgagcttaaggaaaatgacagaatttttgtttctgtaacaaatgagcacttgatagacatggaccatgaagccagtttttttggggcctttttagttggctaa
CD81-TRAIL amino acid sequence (SEQ ID NO: 6):
MGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVI
LFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTLTALTTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSG
KLYLIGIAAIVVAVIMIFEMILSMVLCCGIRNSSVYGSMAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDD
SYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS
FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTN
EHLIDMDHEASFFGAFLVG-
the engineered DC cells were cultured in exosome-free medium or serum-free medium, and when the cells were in logarithmic growth phase, the supernatant of the DC medium (which could be stored at-80℃for one week) was collected, centrifuged at 4℃for 500g, 5min, and the cells were removed. The supernatant (which was thawed in cold water if taken out of a-80 ℃ C.) was subjected to the following operations: centrifuging at 4deg.C and 10000g for 60min to remove cell debris and large apoptotic nucleosomes; the supernatant was filtered through a 0.22 μm filter to remove microbubbles with larger particle sizes. Add 4 XPEG mother liquor to keep PEG final concentration at 10%, mix by inversion and incubate overnight at 4℃for more than 12 h. The following day is at 4℃and centrifugation is carried out for 60min at 3000 g. Pouring out the supernatant, sucking residual liquid, re-suspending the precipitate with PBS to obtain eDex suspension, filtering with 0.22 μm filter, and packaging and storing in-80 deg.C refrigerator.
Protein quantification is carried out on the exosome isolate by adopting a BCA protein quantification kit, a standard curve is drawn, and the exosome concentration is calculated. Western blot was used to identify eDex surface markers CD81 and Alix (FIG. 1).
Detecting the concentration of eDex particles by a nanoparticle tracking analyzer (Nanosight), wherein the result of the quantitative analysis shows that the concentration of eDex is 3×10, as shown in FIG. 2, the particle size of eDex is mainly distributed at about 100-130nm 10 And each mL. eDex was observed under electron microscopy as a typical cup holder-like structure (FIG. 3).
Exosomes from DC, DC-TRAIL and DC-CD81-TRAIL cells, designated DCexo, TRAIL-exo and CD81-TRAIL-exo, respectively, used in the examples below were prepared in a similar manner.
EXAMPLE 2 antitumor Activity of CD 81-TRAIL-derived cell supernatant against melanoma A375
A375-luc cells (expression of luciferases luciferase after infection of melanoma A375 cells with lentiviruses) were trypsinized, resuspended in RPMI-1640 medium, and counted according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 2.5 hours. Adding three cell-derived culture supernatants (cell densities of 1×10) of DC control, DC-TRAIL and DC-CD81-TRAIL to cells 6 cells/mL, harvested by incubation for 24 h), after 6 hours or 24 hours of treatment, respectively, the cell supernatant was removed from the 24 well plate, washed 3 times with PBS, 100. Mu.L of cell lysate was added to each well, and incubated for 10min at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The absorbance at 635nm was measured immediately after aspiration of 10. Mu.L to 96-well white plates, addition of 50. Mu.L/well of luciferase substrate in the dark, and cell viability was calculated as a percentage of untreated control cells. The results are shown in FIG. 4, relative to TRAIL groupSupernatant containing CD81-TRAIL can significantly kill A375 cells.
EXAMPLE 3 anti-tumor Activity of PEG-precipitated CD81-TRAIL exosomes against melanoma A375
After pancreatin digestion of A375-luc cells, they were resuspended in RPMI-1640 medium and counted, according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 2.5 hours. The EDEX (DC control group, DC-TRAIL group and DC-CD81-TRAIL group) obtained by PEG precipitation with different dosages is respectively added into a 24-well plate, after 12 hours of treatment, the cell supernatant in the 24-well plate is removed, PBS is used for washing 3 times, 100 mu L of cell lysate is added into each well, and the mixture is incubated for 10 minutes at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results, as shown in figure 5, showed that CD81-TRAIL-exo significantly killed a375 cells relative to TRAIL-exo group, which further demonstrated that the engineered exosome CD81-TRAIL-exo was more killing against melanoma than TRAIL-exo.
EXAMPLE 4 antitumor Activity of CD81-TRAIL-derived cell supernatant against cervical cancer cell Hela
HeLa-luc cells (cervical cancer HeLa cells express luciferase after lentivirus infection) were digested with pancreatin, resuspended in RPMI-1640 medium, and counted according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 4 hours. Adding three cell-derived culture supernatants (cell densities of 1×10) of DC control, DC-TRAIL and DC-CD81-TRAIL to cells 6 cells/mL, harvested by incubation for 24 h), after 6 hours or 12 hours of treatment, respectively, the cell supernatant was removed from the 24 well plate, washed 3 times with PBS, 100. Mu.L of cell lysate was added to each well, and incubated for 10min at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. Aspirate 10. Mu.L into 96 well plates,the luciferase substrate was added at 50. Mu.L/well in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as a percentage of untreated control cells. The results are shown in FIG. 6, where the supernatant containing CD81-TRAIL significantly killed Hela cells relative to the TRAIL group.
EXAMPLE 5 anti-tumor Activity of PEG-precipitated CD81-TRAIL exosomes against cervical cancer cell Hela
After pancreatin digestion of Hela-luc cells, they were resuspended in RPMI-1640 medium and counted, according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 4 hours. The EDEX (DC control group, DC-TRAIL group and DC-CD81-TRAIL group) obtained by PEG precipitation with different dosages is respectively added into a 24-well plate, after 12 hours of treatment, the cell supernatant in the 24-well plate is removed, PBS is used for washing 3 times, 100 mu L of cell lysate is added into each well, and the mixture is incubated for 10 minutes at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results, as shown in FIG. 7, showed that CD81-TRAIL-exo significantly killed Hela cells relative to the TRAIL-exo group, which further demonstrated that the engineered exosome CD81-TRAIL-exo was more killing against cervical cancer than TRAIL-exo.
EXAMPLE 6 antitumor Activity of CD 81-TRAIL-derived cell supernatant against lung cancer cell A549
A549-luc cells (lung cancer cells A549 express luciferases following lentiviral infection) were trypsinized, resuspended in RPMI-1640 medium, and counted according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 4 hours. Adding three cell-derived culture supernatants (cell densities of 1×10) of DC control, DC-TRAIL and DC-CD81-TRAIL to cells 6 cells/mL, cultured for 24h harvest), after 12 hours or 24 hours of treatment, respectively, the cell supernatant was removed from the 24-well plate, PBS was washed 3 times, 100 μl of cell lysate was added to each well and incubated at 4 ℃ for 10min. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results are shown in FIG. 8, where the supernatant containing CD81-TRAIL significantly killed A549 cells relative to the TRAIL group.
EXAMPLE 7 anti-tumor Activity of PEG-precipitated CD81-TRAIL exosomes against lung cancer cell A549
After pancreatin digestion of A549-luc cells, they were resuspended in RPMI-1640 medium, counted and counted according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 4 hours. The EDEX (DC control group, DC-TRAIL group and DC-CD81-TRAIL group) obtained by PEG precipitation with different dosages is respectively added into a 24-well plate, after 12 hours of treatment, the cell supernatant in the 24-well plate is removed, PBS is used for washing 3 times, 100 mu L of cell lysate is added into each well, and the mixture is incubated for 10 minutes at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results, as shown in figure 9, showed that CD81-TRAIL-exo significantly killed a549 cells relative to TRAIL-exo group, which further demonstrated that the engineered exosome CD81-TRAIL-exo had greater killing of lung cancer than TRAIL-exo.
EXAMPLE 8 PEG-precipitated CD81-TRAIL exosomes against human multiple myeloma peripheral blood B lymphocytes
Antitumor Activity of RPMI8226
RPMI8226-luc cells were cultured in RPMI-1640 medium, and the cell count was measured according to 6X 10 4 cells/well were seeded in 24-well plates. The eDex (DC control group, DC-TRAIL group and DC-CD81-TRAIL group) obtained by adding different doses of PEG precipitation into 24-well plates respectively, and collecting cells from each well after 24 hours of treatmentEP tube was washed 3 times with PBS, 100. Mu.L of cell lysate was added to each tube, vortexed and mixed well and incubated at 4℃for 10min.12000g, and centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results, as shown in figure 10, showed that CD81-TRAIL-exo significantly killed RPMI8226 cells relative to TRAIL-exo group, which further demonstrated that the engineered exosome CD81-TRAIL-exo was more killing against human multiple myeloma than TRAIL-exo.
Example 9 antitumor Activity of PEG-precipitated CD81-TRAIL exosomes against fibrosarcoma HT1080
HT1080-luc cells were trypsinized, resuspended in RPMI-1640 medium, and counted, according to 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 4 hours. The EDEX (DC control group, DC-TRAIL group and DC-CD81-TRAIL group) obtained by PEG precipitation with different dosages is respectively added into a 24-well plate, after 12 hours of treatment, the cell supernatant in the 24-well plate is removed, PBS is used for washing 3 times, 100 mu L of cell lysate is added into each well, and the mixture is incubated for 10 minutes at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The absorbance at 635nm was measured immediately after aspiration of 10 μl to 96 well plates, addition of 50 μl of luciferase substrate per well in the dark, and mixing, and cell viability was calculated as a percentage of untreated control cells. The results, as shown in figure 11, showed that CD81-TRAIL-exo significantly killed HT1080 cells relative to TRAIL-exo group, and this result further demonstrated that the engineered exosome CD81-TRAIL-exo was more damaging to fibrosarcoma than TRAIL-exo.
EXAMPLE 10 anti-tumor Activity of PEG-precipitated CD81-TRAIL exosomes against liver cancer HepG2
HepG2-luc cells were resuspended in RPMI-1640 medium after pancreatin digestion, and the cell count was calculated to be 6X 10 4 cells/well were seeded in 24-well plates and continued at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 4 hours. 24-holeThe plates were separately added with different doses of PEG for precipitation to obtain eDex (DC control, DC-TRAIL and DC-CD 81-TRAIL), after 12 hours of treatment, the cell supernatants from the 24-well plates were removed, washed 3 times with PBS, 100. Mu.L of cell lysate was added to each well, and incubated for 10min at 4 ℃. After being blown and evenly mixed by a pipetting gun, lysates of all holes are collected into a centrifuge tube, 12000g is centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results are shown in FIG. 12, which further demonstrate that the engineered exosome CD81-TRAIL-exo has a greater killing power against liver cancer than TRAIL-exo, as compared to TRAIL-exo, which can significantly kill HepG2 cells.
EXAMPLE 11 PEG-precipitated CD81-TRAIL exosomes against human chronic myelogenous leukemia cell K562
Activity(s)
K562-luc cells were cultured with RPMI-1640 medium, counted, and cultured according to 6X 10 4 cells/well were seeded in 24-well plates. The Edex (DC control group, DC-TRAIL group and DC-CD81-TRAIL group) obtained by PEG precipitation with different dosages is respectively added into a 24-well plate, after 48 hours of treatment, cells of each well are collected into an EP tube, the EP tube is washed 3 times by PBS, 100 mu L of cell lysate is added into each tube, vortex shaking is carried out, the mixture is fully mixed, and the mixture is incubated for 10 minutes at 4 ℃.12000g, and centrifuged for 3min. The cells were aspirated into 10. Mu.L to 96 well plates, 50. Mu.L/well of luciferase substrate was added in the dark, absorbance at 635nm was measured immediately after mixing, and cell viability was calculated as percentage of untreated control cells. The results, as shown in figure 13, showed that CD81-TRAIL-exo significantly killed K562 cells relative to TRAIL-exo group, which further demonstrated that the engineered exosome CD81-TRAIL-exo had greater killing against human chronic myelogenous leukemia than TRAIL-exo.
The foregoing is merely illustrative of the present invention and is not intended to limit the scope of the invention, which can be modified and varied, without departing from the principles of the invention.
Claims (5)
1. An exosome comprising a fusion protein, wherein the fusion protein consists of an exosome membrane protein CD81 and an anti-tumor factor TRAIL, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:6, and the exosomes are derived from DC cells engineered to be capable of expressing the fusion protein.
2. A method for preparing exosomes comprising
1) Introducing into a DC cell a nucleic acid molecule comprising a coding sequence for a fusion protein, wherein the fusion protein consists of an exosome membrane protein CD81 and an anti-tumor factor TRAIL, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:6 is shown in the figure;
2) Culturing the DC cells under conditions suitable for expression of the coding sequence and collecting the exosomes secreted thereby.
3. The method of claim 2, wherein the fusion protein has a coding sequence as set forth in SEQ ID NO: shown at 5.
4. An anti-tumor pharmaceutical composition comprising the exosome of claim 1 and a pharmaceutically acceptable carrier, wherein the tumor is selected from the group consisting of melanoma, cervical cancer, lung cancer, multiple myeloma, fibrosarcoma, liver cancer, and chronic myelogenous leukemia.
5. Use of the exosome of claim 1 in the manufacture of a medicament for treating a tumor, wherein the tumor is selected from the group consisting of melanoma, cervical cancer, lung cancer, multiple myeloma, fibrosarcoma, liver cancer, and chronic myelogenous leukemia.
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