CN110172449A - A kind of leukaemia cell's excretion body and its preparation method and application - Google Patents
A kind of leukaemia cell's excretion body and its preparation method and application Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001111—Immunoglobulin superfamily
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/80—Vaccine for a specifically defined cancer
- A61K2039/804—Blood cells [leukemia, lymphoma]
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
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- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
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Abstract
The invention discloses a kind of leukaemia cell's excretion body vaccines and its preparation method and application, belong to field of biomedicine.Leukaemia cell's excretion body is that the leukaemia cell for being overexpressed gene modification by CD80 and/or CD86 secretes.Further, the CD80 and/or CD86 overexpression gene modification, which refers to, utilizes the slow virus carrier of load C D80 gene and/or CD86 gene to transfect leukaemia cell.Leukaemia cell's excretion physical efficiency provided by the invention, which is enough in, prepares Antileukemia immunity vaccine, can overcome LEX low expression costimulatory molecules and the low defect of immunogenicity, its immunogenicity is made to be improved significantly.
Description
Technical field
The invention belongs to fields of biomedicine, and in particular, to leukaemia cell's excretion body vaccine and preparation method thereof and
Using.More particularly, to a kind of leukaemia cell's excretion body and preparation method thereof for being overexpressed modification through CD80 and/or CD86
And application.
Background technique
Acute leukemia is common one of the malignant disease of hematological system, depends on high-dose chemotherapy and hematopoiesis for a long time
Stem cell transplantation (HSCT).Nevertheless, high relapse rate is still the treatment problem of the malignant hematologic diseases such as leukaemia.Leukemia relapse
Root essentially consist in the leukaemia cell (MRL) of internal remaining, therefore, removing MRL is the long-term life extended after patient's treatment
Deposit the key even cured.Proud clinical treatment is achieved in recent years for the specific active immunotherapy of leukemia cell antigen
Effect, had both overcome the non-target tropism of classic chemotherapy and hematopoietic stem cell transplantation, while also avoiding receiving traditional Radiotherapy chemotherapy because long-term
And the obvious toxic-side effects of HSCT, thus receive significant attention.
In recent years, based on the anti-tumor immunotherapy of excretion body (exosomes, EXO) in animal model and clinical research
In achieve obvious curative effects.It includes the release of the eukaryocytes such as immunocyte, tumour cell, mescenchymal stem cell that EXO, which is various,
Diameter be 30-100nm more vesica corpusculums.Research confirms that tumour cell includes the excretion body of leukaemia cell's secretion
Load has the antigen of tumor cell specific, therefore can be used as the tumour antigen main source of anti-tumor immunotherapy, with tumour
Anti-tumor vaccine based on the excretion body (tumor cell-derived exosomes, TEX) of cell origin can be small in lotus knurl
Specific for tumour antigen antineoplastic immune is induced in mouse model, and tumour growth is significantly inhibited.K562,L1210
The excretion body (leukemia cell-derived exosomes, LEX) of equal leukaemia cells' release can equally lure in vivo, outside
Lead certain Antileukemia immunity effect.
But the Antileukemia immunity effect that LEX is induced is still very low, it is difficult to which the anti-leukocythemia disease for reaching clinical treatment is strong
Degree, main cause is that LEX immunogenicity is low.Since tumour cell includes the equal low expression of leukaemia cell, or even do not express altogether
Stimulation molecule, this is also the main mechanism of immunosurveillance escape, therefore the LEX also low expression costimulatory molecules of its secretion,
It is lower so as to cause the immunogenicity of LEX, it is difficult to the Antileukemia immunity of inducement efficient.
Summary of the invention
In order to overcome LEX defect, inventor has carried out a large amount of research and probe, it has unexpectedly been found that: up-regulation Ehrlich tumor
The expression of cell surface costimulatory molecules CD80 and/or CD86 molecule can get and be overexpressed by separating and purifying its supernatant
The LEX of CD80 and CD86 molecule, immunogenicity with higher, thereby completing the present invention.
One aspect of the present invention provides a kind of leukaemia cell's excretion body, and leukaemia cell's excretion body is by through CD80
And/or CD86 is overexpressed leukaemia cell's secretion of gene modification.
Second aspect of the present invention provides leukaemia cell's excretion body described in first aspect present invention in preparing cancer vaccine
Application.
In some embodiments of the present invention, it includes leukaemia that the cancer, which is tumour,.
Fourth aspect present invention provides the preparation method of leukaemia cell's excretion body described in first aspect present invention, this method
Include the steps that the carrier of load C D80 gene and/or CD86 gene transfecting leukaemia cell.
In some embodiments of the present invention, the carrier is slow virus carrier.
In some specific embodiments of the invention, the carrier is pLenO-DCE Greenp or LenO-DCE
puro。
In embodiments of the invention, the CD80 gene is using with SEQ ID NO.1 and SEQ ID NO.2 institute
Show what the primer pair amplifies of nucleotide sequence obtained.
In some embodiments of the present invention, the CD80 gene has nucleotide sequence shown in SEQ ID NO.5.
In embodiments of the invention, the CD86 gene is using with SEQ ID NO.3 and SEQ ID NO.4 institute
Show what the primer pair amplifies of nucleotide sequence obtained.
In some embodiments of the present invention, the CD86 gene has nucleotide sequence shown in SEQ ID NO.6.
In some embodiments of the present invention, the preparation method of leukaemia cell's excretion body further comprises separation
The step of purifying excretion body.
In one embodiment of the invention, the step of separation excretion body, includes:
1) logarithmic phase L1210 cell is collected, PBS is added, 1,200rpm is centrifuged 10min after piping and druming mixes, and abandons supernatant；
2) adjustment cell density is 5 × 106/ mL is transferred in AIM-V serum free medium, is placed in 37 DEG C, 5%CO2 training
It supports and is incubated for 36h in case, cells and supernatant is collected by centrifugation；
3) the L1210 cell supernatant of free serum culture is collected, 800g low-temperature centrifugation (4 DEG C, similarly hereinafter) 30min removes cell
Take supernatant；
4) 10,000g low-temperature centrifugation 1h removes cell fragment and takes supernatant；
5) 100,000g low temperature ultracentrifugation 1h abandons supernatant；
6) precipitating is suspended in PBS, 100 at 4 DEG C, 000 × g ultracentrifugation 60min, precipitating appropriate PBS weight
It is outstanding, obtain the excretion body in leukaemia cell source, collect simultaneously -80 DEG C freeze it is spare.
Beneficial effects of the present invention
Leukaemia cell's excretion body provided by the invention can overcome the defect of LEX low expression costimulatory molecules, improve it
Immunogenicity.
The present invention prepares the leukaemia cell for being overexpressed CD80 and/or CD86, further separates excretion body, is to improve LEX
The strategy for having very much clinical application potential of Antileukemia immunity vaccine, this strategy equally can widely other tumour excretion bodies
(TEX)。
Detailed description of the invention
Fig. 1 shows the characteristic pattern of LEX: the LEX of the visible electric microscopic observation of Figure 1A meets typical excretion volume morphing, directly
Diameter is 30~100nm, and form is uniform, is round or oval imitated vesicle structure；By being carried out to extracted excretion body sample
Western blot analysis of protein as it can be seen that extracted LEX sample standard deviation expression excretion body characteristic protein CD9, CD63,
CD81, HSP70 (Figure 1B).Fig. 1 C and Fig. 1 D show L1210 leukaemia cell and transfect the slow virus load for carrying costimulatory molecules
After body, the horizontal significant up-regulation of surface C D80 and CD86 developed by molecule.
Fig. 2 shows the LEX new generation vaccines of costimulatory molecules gene modification can more effectively inducing specific CD4+And CD8+
T cell responses, as seen from the figure, the LEX-CD8086 of the LEX of costimulatory molecules gene modification, especially dual-gene modification can more have
Effect ground inducing specific CD4+T cell is proliferated the release of (Fig. 2A) and Th1 cell factor, and it is anti-to generate stronger leukaemia cell
The antileukemie ctl response (Fig. 2 B, C, D) of former specificity.* indicates that p < 0.01, * indicate p < 0.05.
Fig. 3 show costimulatory molecules gene modification LEX new generation vaccine can effectively inducing leukemia cellular antigens it is special
The anti-leukocythemia epidemic prevention effect of property.Fig. 3 A shows the influence for injecting different excretion bodies to mouse tumor volume, and Fig. 3 B shows
Influence of the different excretion bodies of injection to mouse survival is gone out.As seen from the figure, LEX-CD8086 more effectively can protect mouse to exempt from
By tumour attack.
Specific embodiment
In order to which the technical problems, technical solutions and beneficial effects solved by the present invention is more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.
Embodiment
Following example is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under
State the technology disclosed in example represent inventor discovery can be used for implementing technology of the invention, therefore can be considered as implementation this
The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification
Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.
Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention
Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here
Invention specific embodiment many equivalent technologies.These will equally be comprised in claims.
The preparation for the L1210 leukaemia cell source property excretion body that embodiment 1CD80 and CD86 are overexpressed
One, the culture of leukaemia cell
Good 1640 complete mediums containing 10% fetal calf serum are configured, the culture medium includes 100U/mL Benzylpenicillin sodium salt
With 100 μ g/mL streptomycin sulphates, there is the cryopreservation tube of L1210 cell to direct plunge into 37 DEG C of warm water after taking out in liquid nitrogen container for freezing
In, and gently shaking melts it as early as possible, takes out cryopreservation tube, opens after disinfecting in alcohol, and cell suspension, transfer is sucked out with suction pipe
To 15mL conical centrifuge tube, and the culture solution of 10 times of dropwise addition or more, centrifugation 1,200rpm × 10min abandon supernatant, fresh is complete
Culture solution is seeded to culture bottle after being resuspended.
Culture bottle is placed in 37 DEG C, 5%CO2It is incubated in incubator, cell is in suspension growth, changes liquid or passage within every 1~2 day.
Two, the building of slow virus carrier
1. prepared by slow virus carrier
Mouse source CD80 and CD86 gene are expanded with PCR method:
CD80 design of primers are as follows:
Forward primer: 5'-CATAGAAGATTCTAGAATGGCTTGCAATTGTCAGTT-3'(SEQ ID NO.1)；
Reverse primer: 5'-ATTTAAATTCGAATTCCTAAAGGAAGACGGTCTGT-3'(SEQ ID NO.2).
CD86 design of primers are as follows:
Forward primer: 5'-ATGGACCCCAGATGCACCAT-3'(SEQ ID NO.3)；
Reverse primer are as follows: 5'-TCACTCTGCATTTGGTTTTG-3'(SEQ ID NO.4).
It is (SEQ ID NO.5) as follows to expand obtained CD80 gene nucleotide series:
ATGGCTTGCAATTGTCAGTTGATGCAGGATACACCACTCCTCAAGTTTCCATGTCCAAGGCTCATTCTT
CTCTTTGTGCTGCTGATTCGTCTTTCACAAGTGTCTTCAGATGTTGATGAACAACTGTCCAAGTCAGTGAAAGATAA
GGTATTGCTGCCTTGCCGTTACAACTCTCCTCATGAAGATGAGTCTGAAGACCGAATCTACTGGCAAAAACATGACA
AAGTGGTGCTGTCTGTCATTGCTGGGAAACTAAAAGTGTGGCCCGAGTATAAGAACCGGACTTTATATGACAACACT
ACCTACTCTCTTATCATCCTGGGCCTGGTCCTTTCAGACCGGGGCACATACAGCTGTGTCGTTCAAAAGAAGGAAAG
AGGAACGTATGAAGTTAAACACTTGGCTTTAGTAAAGTTGTCCATCAAAGCTGACTTCTCTACCCCCAACATAACTG
AGTCTGGAAACCCATCTGCAGACACTAAAAGGATTACCTGCTTTGCTTCCGGGGGTTTCCCAAAGCCTCGCTTCTCT
TGGTTGGAAAATGGAAGAGAATTACCTGGCATCAATACGACAATTTCCCAGGATCCTGAATCTGAATTGTACACCAT
TAGTAGCCAACTAGATTTCAATACGACTCGCAACCACACCATTAAGTGTCTCATTAAATATGGAGATGCTCACGTGT
CAGAGGACTTCACCTGGGAAAAACCCCCAGAAGACCCTCCTGATAGCAAGAACACACTTGTGCTCTTTGGGGCAGGA
TTCGGCGCAGTAATAACAGTCGTCGTCATCGTTGTCATCATCAAATGCTTCTGTAAGCACAGAAGCTGTTTCAGAAG
AAATGAGGCAAGCAGAGAAACAAACAACAGCCTTACCTTCGGGCCTGAAGAAGCATTAGCTGAACAGACCGTCTTCC
TTTAG
It is (SEQ ID NO.6) as follows to expand obtained CD86 gene nucleotide series:
ATGGACCCCAGATGCACCATGGGCTTGGCAATCCTTATCTTTGTGACAGTCTTGCTGATCTCAGATGCT
GTTTCCGTGGAGACGCAAGCTTATTTCAATGGGACTGCATATCTGCCGTGCCCATTTACAAAGGCTCAAAACATAAG
CCTGAGTGAGCTGGTAGTATTTTGGCAGGACCAGCAAAAGTTGGTTCTGTACGAGCACTATTTGGGCACAGAGAAAC
TTGATAGTGTGAATGCCAAGTACCTGGGCCGCACGAGCTTTGACAGGAACAACTGGACTCTACGACTTCACAATGTT
CAGATCAAGGACATGGGCTCGTATGATTGTTTTATACAAAAAAAGCCACCCACAGGATCAATTATCCTCCAACAGAC
ATTAACAGAACTGTCAGTGATCGCCAACTTCAGTGAACCTGAAATAAAACTGGCTCAGAATGTAACAGGAAATTCTG
GCATAAATTTGACCTGCACGTCTAAGCAAGGTCACCCGAAACCTAAGAAGATGTATTTTCTGATAACTAATTCAACT
AATGAGTATGGTGATAACATGCAGATATCACAAGATAATGTCACAGAACTGTTCAGTATCTCCAACAGCCTCTCTCT
TTCATTCCCGGATGGTGTGTGGCATATGACCGTTGTGTGTGTTCTGGAAACGGAGTCAATGAAGATTTCCTCCAAAC
CTCTCAATTTCACTCAAGAGTTTCCATCTCCTCAAACGTATTGGAAGGAGATTACAGCTTCAGTTACTGTGGCCCTC
CTCCTTGTGATGCTGCTCATCATTGTATGTCACAAGAAGCCGAATCAGCCTAGCAGGCCCAGCAACACAGCCTCTAA
GTTAGAGCGGGATAGTAACGCTGACAGAGAGACTATCAACCTGAAGGAACTTGAACCCCAAATTGCTTCAGCAAAAC
CAAATGCAGAGTGA
The cDNA segment of the purifying CD80 and CD86 Jing Guo PCR amplification is inserted into pLenO-DCE respectively using double digestion method
EcoR I and the BamH I site of Greenp and LenO-DC Epuro plasmid.
2. slow virus packaging and titer determination
Then recombinant slow virus (1.7 μ g), pSPAX2 (1.13 μ g) and pMD2G (0.57 μ g) use LipofiterTMTransfection examination
Agent cotransfection 293T cell, 48 and 72h collects viral supernatants twice respectively after transfection, is filtered after collection with 0.45 μm of filter, 4
DEG C, 72,000g/min be centrifuged 120min, resulting viral vectors is named as LV-CD80-GFP and LV-CD86-puro respectively,
Viral pellet is resuspended with fresh medium again, carries out titer determination to obtain virus titer being 2 × 10 by the dilution method of counting8FU/
ML, -80 DEG C save backup.
Three, slow-virus transfection L1210 cell and jamming effectiveness test
Selection grows vigorous, the L1210 cell in logarithmic phase, and adjustment cell concentration is 105/mL.Cell is spread in advance
In 96 orifice plates, 100 holes μ L/, if 3 multiple holes, are then placed in 37 DEG C, 5%CO2Continue to cultivate in incubator.Second day, each
LV-CD80, LV-CD86 are separately added into hole, MOI value is 100.Cell is placed in plate centrifuge after virus is added, with 2,
000g, plate is centrifuged 90min under the conditions of 4 DEG C.It is subsequently placed in 37 DEG C, 5%CO2It is cultivated in incubator and changes liquid afterwards for 24 hours, continue to cultivate
48h then observes intracellular green fluorescence expression under inverted fluorescence microscope.Collect cell, 4 DEG C, be centrifuged under 300g
5min is resuspended with the RPMI-1640 culture solution containing 10%FBS, is continued to expand according to aforementioned cultural method and be cultivated.Pass through streaming
Screening obtains the positive L1210 cell for surely turning CD80 of GFP；It is screened by puromycin, after cellular control unit is completely dead,
Amplification infected group cell is to obtain the L1210 cell for surely turning CD86.By aforesaid infections method, it will surely turn the L1210 cell of CD86
Continue to infect LV-CD80, observe green fluorescence expression, after expanding culture, is screened, that is, obtained by streaming and puromycin
Obtain the L1210 cell for surely turning CD80 and CD86.
Four, the separation of leukaemia cell's excretion body and characterization of CD80 and CD86 gene modification
1. excretion body extracts
1) logarithmic phase L1210 cell is collected, PBS is added, 1,200rpm is centrifuged 10min after piping and druming mixes, and abandons supernatant.
2) adjustment cell density is 5 × 106/ mL is transferred in AIM-V serum free medium, is placed in 37 DEG C, 5%CO2Training
It supports and is incubated for 36h in case, cells and supernatant is collected by centrifugation.
3) the L1210 cell supernatant of free serum culture is collected, 800g low-temperature centrifugation (4 DEG C, similarly hereinafter) 30min removes cell
Take supernatant.
4) 10,000g low-temperature centrifugation 1h removes cell fragment and takes supernatant.
5) 100,000g low temperature ultracentrifugation 1h abandons supernatant.
6) precipitating is suspended in PBS, 100 at 4 DEG C, 000 × g ultracentrifugation 60min, precipitating appropriate PBS weight
It is outstanding, obtain the excretion body in leukaemia cell source, collect simultaneously -80 DEG C freeze it is spare.Will from L1210, L1210-CD80,
The excretion body of L1210-CD86 and L1210-CD8086 leukaemia cell is respectively designated as LEXnull, LEX-CD80, LEX-CD86
And LEX-CD8086.
The protein quantification of 2.LEX measures (BCA method)
1) protein standard substance is diluted to the working solution of 0.5mg/mL, and is added to 96 holes by 0,1,2,4,8,12,16,20 μ L
In plate standard sample wells, every hole is complemented into 20 μ L with standard dilutions.
2) configuration of BCA working solution: A liquid and B liquid press 50:1 proportional arrangement into BCA working solution.
3) ddH will be used220 μ L of sample after 1 times of O dilution adds to the sample well of 96 orifice plates.Each sample does 3 multiple holes.
4) the 200 μ L of BCA working solution that configuration in advance is good and is kept in dark place is added in every hole, dries after being wrapped up with masking foil at 37 DEG C
30min is incubated in case.
5) each hole OD value is measured at wavelength 562nm.
6) standard curve is manufactured, and calculates the average value of sample concentration, protein sample has diluted 1 when due to measurement
Times, thus the sample after calculating is needed multiplied by 2, so that it is determined that protein sample final concentration.
3. excretion body characteristics are identified
It projects Electronic Speculum and observes excretion body sample morphological feature:
1) glass surface is pasted on parafilm film is smooth.
2) by 50 μ L excretion body sample drops on film.
3) copper mesh of film will be supported to be covered on sample drop with formvar, its floating is made (to have that for supporting film to face
Under) 3-10min, it is adsorbed onto sample and supports on film.
4) the phosphotungstic acid dye liquor of 50 μ L 2% is dripped on parafilm film.
5) copper mesh is moved away from sample drop, is placed in copper mesh edge with small pieces filter paper and is inhaled to suck liquid on copper mesh, then by this
Copper mesh with sample covers on 2% phosphotungstic acid dye liquor drop and floats 3min, and filter paper is blotted along copper mesh edge, under incandescent lamp
Dry 10min.
6) it projects and is observed under Electronic Speculum (Phillip CM120).
As shown in Figure 1a, visible LEX is diameter in the plate-like vesica of 40-100nm to electricity under the microscope, meets typical excretion body
Morphological feature.
Westernblot detects Hsp70, CD9, CD63 and CD81 expression in separated excretion body:
1) lysate and enzyme inhibitor are mixed with volume ratio 50:1, suitable cracking liquid mixture is taken to be added to excretion body
In sample pellet.
2) after placing 20min on ice, 4 DEG C, 12,000g centrifugation 20min.
3) supernatant, Bradford method determination of protein concentration are collected.
4) sds page (separation gel and concentration glue) is prepared on ice.
5) it configured separation gel will be slowly injected between two glass plates after mixing on ice, isopropanol fluid-tight, room temperature is static
30min is solid to being gelled, and carefully discards isopropanol and wash with distilled water, blots residual.
6) glue glass is concentrated in injection SDS-PAGE, is carefully inserted into stripping fork, empties bubble, is stored at room temperature until glue solidifies completely.
7) it puts glass plate into electrophoresis tank, fills it up with electrophoresis tank with 1 × electrophoretic buffer, gently extract comb, each sample
By 30 μ g loadings, 15 μ L sample loading buffers are added in vacant hole.
8) power on, adjust voltage 80V, after electrophoresis about 40min, when Bromophenol Blue dye enters separation gel from concentration glue,
Voltage is adjusted to 120V, electrophoresis to bromophenol blue, which reaches gel bottom, to be stopped.
9) stripping offset plate carefully pries open glass plate, and glue is immersed in transferring film buffer and balances 10min.Size according to glue is cut
Pvdf membrane is put into methanol and impregnates 10min and be placed in transferring film buffer and balance 2-3min.
10) using transferring film shelf black side the bottom of as, felt pan, 3 layers of filter paper, glue, pvdf membrane, 3 layers of filter paper, felt pan are sequentially placed,
Clean bubble is driven, shelf is clamped, is put into electroporation.
11) addition transferring film buffer, is put into ice chest, constant current 200mA under ice bath, transferring film 60min, takes out after transferring film miscellaneous
Hand over film.
12) pvdf membrane for completing albumen transferring film is placed in the skim milk of 5%TBS configuration in being closed on room temperature shaker
2h。
13) according to antibody specification, primary antibody dilution (anti-Hsp70, anti-CD 63, resists according to 1:1000 dilution primary antibody
CD9, anti-CD81).
14) 4 DEG C of overnight incubations.
15) 1 × TBST room temperature is washed 3 times, each 10min.
16) dilution secondary antibody (1:1000) is incubated at room temperature 1h according to a certain percentage.
17) 1 × TBST room temperature is washed 3 times, each 10min.
18) the luminous developing solution of ECL is prepared, appropriate progress chemiluminescence is added dropwise on film, with Bio-Red ChemiDocTM
XRS+ chemiluminescence imaging system detection image.
19) gray value quantitatively is measured using ImageLab software.
It is as shown in Figure 1B: can be detected in LEXnull, LEX-CD80, LEX-CD86 and LEX-CD8086 CD9,
These significant albumen of excretion body of CD63, CD81 and HSP70, it was demonstrated that inventor's excretion body sample obtained is typical leukaemia
The excretion body of cell origin.
Five, CD4+T cell proliferation experiment and cytokines measurement
1.CD4+T cell proliferation detection
(1) specificity target cell is set by L1210 cell, p388 cell controls target cell receives 4000rad dosage
Irradiation 10 minutes, so that it is lost proliferation activity, but retain its antigenicity.
(2) the mice spleen source property CD4 being immunized through PBS, LEX, LEX-CD80, LEX-CD80 and LEX-CD8086+T cell warp
Above-mentioned steps are resuspended after isolating and purifying with the complete medium containing IL-2 (20IU/ml), with 2 × 105The density bed board in/hole, together
When be added AntiCD3 McAb Ab (1 μ g/mL), be 3 multiple holes.
(3) then, 2 × 10 are separately added into every hole4A L1210 cell or p388 cell through radioinactivation, at 37 DEG C,
72h is co-cultured under 5%CO2 concentration.
(4) every hole is added 0.5 μ Ci's3After H-TdR continues culture 16 hours, cell is adsorbed on glass with cell harvestor
Glass fiber filter paper on piece, then with the abundant washes clean of physiological saline, will be free3H-TdR is removed；5% trichlorine of 1-2mL is added dropwise
Then acetic acid is successively put into equipped with 4mL after drying filter paper with tweezers respectively with fixing cell with dehydrated alcohol dehydration, decoloration
In the plastic tube of scintillation solution, with the cpm value of β liquid scintillation instrument test sample, the mean cpm of background and sample is calculated.
2.ELISA method detects CD4+The Th1 cell factor of T secretion
The cell conditioned medium obtained is collected, ELISA method detection wherein Th1 cell factor, the content of IFN-γ and IL-2 are passed through.
Specific step is as follows:
(1) IFN-γ and IL-2ELISA kit are taken out from refrigerator in advance, at room temperature rewarming；
(2) standard curve is established as follows: standard items/sample dilution 1.0mL (1b) is added to the mark of freeze-drying
In quasi- product (1a), after thoroughly dissolving, (concentration 1000pg/mL) is mixed well after standing 15 minutes.First hole is added
200 μ L of standard items after mixing, is precipitated 100 μ L with sample loading gun from the first hole, and the second hole is added, repeatedly two-fold dilution to the
100 μ L are finally sucked out from the 7th hole and discard for 7 holes, and the 8th hole is blank control wells；
(3) from restoring to be taken out into the hermetic bag of room temperature by required lath, in addition to blank well, add respectively in corresponding aperture
Enter corresponding sample (100 hole μ L/), then seal reacting hole with sealing plate gummed paper, reaction plate is placed in 37 DEG C, 5%CO2In incubator
Culture 90 minutes；
(4) board-washing 5 times；
(5) in addition to blank well, every hole adds 100 μ L of biotinylated antibody working solution, then seals reacting hole sealing plate gummed paper
Firmly, in 37 DEG C, 5%CO2It is cultivated 60 minutes in incubator；
(6) continue board-washing 4 times；
(7) in addition to blank well, every hole adds 100 μ L of enzyme conjugates working solution, then seals reacting hole sealing plate gummed paper
Firmly, 37 DEG C, 5%CO2Incubator is incubated for 30 minutes；
(8) continue to wash 4 plates；
(9) color developing agent of 100 μ L is added in every hole, is protected from light and is incubated for 10-15 minutes in 37 DEG C of incubators；
(10) terminate liquid of 100 μ L is added in every hole, measures OD450 value after mixing at once (in 5 minutes).
3 detection CD8+T cell ctl response
Using CytoTox96 Cytotoxic Assay kit, CD8 is detected using LDH method+T cell ctl response intensity.With warp
The CD8 of above-mentioned LEX immune mouse spleen separation+T cell is as effector cell, and the L1210 cell through irradiation inactivation is as special
Anisotropic target cell, p388 cell is then as control target cell.It is 6.25:1,12.5:1,25 that effect/target, which is set as (E/T) than successively:
1 and 50:1.Target cell Spontaneous release is separately set, the release of target cell maximum, effector cell's Spontaneous release hole, every hole sets 3 multiple holes.
Specific steps are as follows:.
(1) CD8 isolated and purified in sensitized mice body+T cell is used after washing 2 times with PBS+1%FBS containing IL-2
The complete medium of (20IU/mL) is resuspended, adjustment cell concentration to 5 × 106/ mL then takes part cell suspension to dilute respectively
To following concentration: 2.5 × 106/ mL, 1.25 × 106/ mL, 0.625 × 106/ mL, 0.3 × 106/mL；
(2) target cell (L1210 cell or p388 cell) is resuspended with PBS+1%FBS, adjustment cell concentration to 1 × 105/
mL；
(3) respectively in 96 hole round bottom culture plates, the effector cell of 50 μ L and target cell, the 100 μ L of 50 μ L is added in every hole
The PBS+1%FBS culture solution of effector cell, 100 μ L target cells and 100 μ L, every hole are all provided with 4 multiple holes；
(4) 300g, at room temperature, after centrifugation 4 minutes, 37 DEG C, 5%CO2It is cultivated 4 hours in incubator；
(5) before centrifugation, (10 ×) 10 μ l lysate is added within 45 minutes in advance into target cell maximum relief hole to crack
Cell；
(6) after terminating culture, 300g is centrifuged plate 4 minutes at room temperature；
(7) it is transferred in a 96 new orifice plates from drawing out 50 μ l supernatants in each hole, and marks；
(8) buffer (Assay Buffer) for melting detection draws 12mL afterwards, adds it to one bottle of Substrate
In Mix powder, gently overturns and rock so that substrate is completely dissolved, masking foil package avoids strong light direct beam substrate, need to make immediately
With；
(9) substrate mixture that 50 μ l are prepared is added into every hole, covered with masking foil or by plate be placed in darkroom with
It is protected from light, in incubation at room temperature 30 minutes；
(10) finally, 50 μ l terminate liquids are added into every hole, avoid occurring bubble in hole as far as possible, if there have bubble to generate to be available
1mL syringe needle is poked.After terminate liquid is added, light absorption value is measured under 490 or 492nm as early as possible；
(11) light absorption value of all experimental ports, effector cell, the spontaneous LDH relief hole of target cell is subtracted into the suction of culture medium background
The mean value of light value is calculated；
(12) light absorption value of target cell maximum LDH release control is subtracted to the mean value of volume correction control light absorption value again；
(13) the corrected value obtained in step 11 and 12 is substituted into following equation, so that calculating each effect target compares institute
The percentage of cytotoxicity of generation.
CD4+T cell plays an important role in antineoplastic immune, thus inventor observes LEX-CD80, LEX-CD80
With LEX-CD8086 to CD4+The influence of the proliferation and its Th1 cytokine release of T cell.As shown in Figure 2 a, compared to PBS with
The CD4 of sensitization in LEXnull, LEX-CD80, LEX-CD86 and LEX-CD8086 body+T cell is equal under L1210 cytositimulation
It can significantly be proliferated.It is wherein the most significant with the proliferation effect of the T cell of LEX-CD8086 sensitization, difference have conspicuousness (p <
0.01).Further to verify LEX-CD8086 to sensitization CD4+The proliferation-inducing effect of T cell has antigentic specificity, invention
People is using another leukemia cell line p388 cell for carrying not synantigen to the CD4 of LEX-CD8086 sensitization+T cell carries out
Stimulation, as the result is shown under p388 cytositimulation, CD4+T cell illustrates what LEX-CD8086 was induced almost without breeder reaction
CD4+T cell proliferation effect has antigentic specificity.
Inventor further has detected the CD4 of LEX-CD8086 sensitization+The level of T secretion Th1 cell factor.Such as Fig. 2 B, C
Shown, after L1210 cell is as targeting antigen stimulation, LEX-CD80, LEX-CD86, LEX-CD8086 can be effectively facilitated
CD4+T cell secretes Th1 cytokines (IFN-γ and IL-2).Four groups of CD4+T cell (LEXnull, LEX-CD80, LEX-
CD86, LEX-CD8086) secretion IFN-γ be respectively 45.36 ± 5.37pg/mL, 76.13 ± 3.35pg/mL, 77.12 ±
3.57pg/mL, 116.75 ± 8.97pg/mL, IL-2 are respectively 56.60 ± 4.36pg/mL, 64.81 ± 3.76pg/mL, 69.62
± 3.42pg/mL, 92.15 ± 8.57pg/mL, wherein LEX-CD80, LEX-CD86 compare (p < 0.05) with LEXnull respectively,
Wherein LEX-CD8086 group cytokine secretion is most and LEXnull compares (p < 0.01), illustrates costimulatory molecules gene modification
LEX-CD80 and LEX-CD86 and LEX-CD8086 can be effectively facilitated Th1 cytokine secretion, wherein dual-gene modification
LEX-CD8086 stimulation it is most strong.And after heterogenetic antigen p388 cytositimulation, the CD4 of LEX-CD8086 sensitization+T
The secretion level of cell secretion of gamma-IFN and IL-2 are lower, substantially less than horizontal after the stimulation of L1210 specific antigen, illustrate altogether
The Th1 cytokine secretion that the LEX of costimulatory molecule gene modification is induced has antigentic specificity.
For research LEX-CD80, LEX-CD86 and LEX-CD8086 vaccine could effectively inducing antigen-specific resist white blood
Sick CTL effect, inventor compare under different effect target ratios, the vaccine-induced cell-specific ctl response intensity of each group.Such as Fig. 2 D
Shown, statistical analysis is visible: when imitating target ratio is 12.5:1 or less, equal no difference of science of statistics between each group.When effect target ratio be etc.
When improving to 25:1, the cell killing rate of LEX-CD86 and LEX-CD8086 group is considerably higher than LEXnull group (p < 0.05),
And as effect target is than improving, four groups of vaccine-induced CTL killing percentages are consequently increased.And LEX-CD8086/L1210 group exists
Effect target ratio is 25:1, and when 50:1, the CTL cell toxicant killing rate induced is higher than LEX-CD80, LEX-CD86 group significantly.
Illustrate LEX (LEX-CD8086) induced CD8 of double costimulatory molecules gene modifications+T CTL lethal effect is most strong.It is further
Confirm the CD8 that LEX is induced+T cell CTL immune response has an antigentic specificity, and inventor's comparative analysis simultaneously LEX is right/
L1210 cell and killing rate to p388 cell.As a result for explanation when effect target ratio is 6.25,12.5,25,50, LEX is thin to p388
Born of the same parents do not show killing activity.Illustrate the CD8 that LEX-CD8086 vaccine is induced+CTL effect has antigentic specificity.
Embodiment 2LEX, LEX-CD80, LEX-CD86 and LEX-CD8086 are to mouse tumor method for preventing and protecting
1) the DBA/2 female mice of 6-8 week old is randomly divided into 5 groups, every group 10, at the 0th day respectively in left side thigh
100 μ LPBS solution of inside subcutaneous injection/only, LEX, LEX-CD80, LEX-CD86 and LEX-CD8086 of 10 μ g.
2) hereafter at interval of 7 days, i.e., the 7th day, each group mouse row is inoculated in the same manner within the 14th day, co-injection 3
It is secondary.
3) 7 days after final injection, L1210 cell 5 × 10 is inoculated in the ipsilateral big leg outer side of each group mouse5/ only.
4) hereafter every 2 days, mouse state is observed, and records tumor-free mice quantity in each group, survival time of mice, and with swimming
Mark calliper to measure mouse leg diameter of tumor.
Fig. 3 is seen as the result is shown, injects the mouse of LEX-CD80 or LEX-CD86, relative to the mouse of injection LEXnull, is swollen
Tumor growth becomes slowly, wherein the mouse of injection LEX-CD8086, tumour growth slows down degree very significantly (Fig. 3 A).Injection
The mouse of LEX-CD80 or LEX-CD86, relative to the mouse of injection LEXnull, the time-to-live is also improved significantly, wherein
The mouse of LEX-CD8086 is injected, time-to-live longest can reach 50 days (Fig. 3 B).
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.
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atggcttgca attgtcagtt gatgcaggat acaccactcc tcaagtttcc atgtccaagg 60
ctcattcttc tctttgtgct gctgattcgt ctttcacaag tgtcttcaga tgttgatgaa 120
caactgtcca agtcagtgaa agataaggta ttgctgcctt gccgttacaa ctctcctcat 180
gaagatgagt ctgaagaccg aatctactgg caaaaacatg acaaagtggt gctgtctgtc 240
attgctggga aactaaaagt gtggcccgag tataagaacc ggactttata tgacaacact 300
acctactctc ttatcatcct gggcctggtc ctttcagacc ggggcacata cagctgtgtc 360
gttcaaaaga aggaaagagg aacgtatgaa gttaaacact tggctttagt aaagttgtcc 420
atcaaagctg acttctctac ccccaacata actgagtctg gaaacccatc tgcagacact 480
aaaaggatta cctgctttgc ttccgggggt ttcccaaagc ctcgcttctc ttggttggaa 540
aatggaagag aattacctgg catcaatacg acaatttccc aggatcctga atctgaattg 600
tacaccatta gtagccaact agatttcaat acgactcgca accacaccat taagtgtctc 660
attaaatatg gagatgctca cgtgtcagag gacttcacct gggaaaaacc cccagaagac 720
cctcctgata gcaagaacac acttgtgctc tttggggcag gattcggcgc agtaataaca 780
gtcgtcgtca tcgttgtcat catcaaatgc ttctgtaagc acagaagctg tttcagaaga 840
aatgaggcaa gcagagaaac aaacaacagc cttaccttcg ggcctgaaga agcattagct 900
gaacagaccg tcttccttta g 921
<210> 6
<211> 930
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
atggacccca gatgcaccat gggcttggca atccttatct ttgtgacagt cttgctgatc 60
tcagatgctg tttccgtgga gacgcaagct tatttcaatg ggactgcata tctgccgtgc 120
ccatttacaa aggctcaaaa cataagcctg agtgagctgg tagtattttg gcaggaccag 180
caaaagttgg ttctgtacga gcactatttg ggcacagaga aacttgatag tgtgaatgcc 240
aagtacctgg gccgcacgag ctttgacagg aacaactgga ctctacgact tcacaatgtt 300
cagatcaagg acatgggctc gtatgattgt tttatacaaa aaaagccacc cacaggatca 360
attatcctcc aacagacatt aacagaactg tcagtgatcg ccaacttcag tgaacctgaa 420
ataaaactgg ctcagaatgt aacaggaaat tctggcataa atttgacctg cacgtctaag 480
caaggtcacc cgaaacctaa gaagatgtat tttctgataa ctaattcaac taatgagtat 540
ggtgataaca tgcagatatc acaagataat gtcacagaac tgttcagtat ctccaacagc 600
ctctctcttt cattcccgga tggtgtgtgg catatgaccg ttgtgtgtgt tctggaaacg 660
gagtcaatga agatttcctc caaacctctc aatttcactc aagagtttcc atctcctcaa 720
acgtattgga aggagattac agcttcagtt actgtggccc tcctccttgt gatgctgctc 780
atcattgtat gtcacaagaa gccgaatcag cctagcaggc ccagcaacac agcctctaag 840
ttagagcggg atagtaacgc tgacagagag actatcaacc tgaaggaact tgaaccccaa 900
attgcttcag caaaaccaaa tgcagagtga 930
Claims (10)
1. a kind of leukaemia cell's excretion body, which is characterized in that leukaemia cell's excretion body is by through CD80 and/or CD86
It is overexpressed leukaemia cell's secretion of gene modification.
2. leukaemia cell's excretion body described in claim 1 is preparing the application in antileukemic vaccination.
3. application according to claim 2, which is characterized in that the cancer is tumour.
4. the preparation method of leukaemia cell's excretion body described in claim 1, which is characterized in that including by load C D80 gene
And/or the carrier of CD86 gene transfects the step of leukaemia cell.
5. the preparation method according to claim 4, which is characterized in that the carrier is slow virus carrier.
6. according to any preparation method of claim 4 or 5, which is characterized in that the CD80 gene is using with SEQ
What the primer pair amplifies of nucleotide sequence shown in ID NO.1 and SEQ ID NO.2 obtained.
7. preparation method according to claim 6, which is characterized in that the CD80 gene has shown in SEQ ID NO.5
Nucleotide sequence.
8. according to any preparation method of claim 4 or 5, which is characterized in that the CD86 gene is using with SEQ
What the primer pair amplifies of nucleotide sequence shown in ID NO.3 and SEQ ID NO.4 obtained.
9. preparation method according to claim 8, which is characterized in that the CD86 gene has shown in SEQ ID NO.6
Nucleotide sequence.
10. according to any preparation method of claim 4 or 5, which is characterized in that further comprise the step for separating excretion body
Suddenly.
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Cited By (2)
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| CN111778212A (en) * | 2020-07-08 | 2020-10-16 | 因诺伟(北京)生物医疗科技有限公司 | Preparation method and application of mobilized hematopoietic stem cell plasma exosome |
| CN112439058A (en) * | 2020-11-25 | 2021-03-05 | 深圳市第二人民医院(深圳市转化医学研究院) | Recombinant novel coronavirus nano vaccine method based on exosome as vector |
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| CN112439058A (en) * | 2020-11-25 | 2021-03-05 | 深圳市第二人民医院(深圳市转化医学研究院) | Recombinant novel coronavirus nano vaccine method based on exosome as vector |
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