CN109096386A - A kind of preparation method and application of breast carcinoma stem cell antigen - Google Patents
A kind of preparation method and application of breast carcinoma stem cell antigen Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and application of breast carcinoma stem cell antigen, this method includes culture breast carcinoma stem cell, hypochlorous acid is added in the solution of culture breast carcinoma stem cell, is uniformly mixed, inducing mammary cancer stem cell apoptosis, crack hypochlorous acid treated breast carcinoma stem cell, and collect the step of cell lysate obtains antigen, wherein hypochlorous acid is added in the solution of culture breast carcinoma stem cell, concentration is 20~80 μM, and the hypochlorous acid processing time is 30~60 minutes.The antigen of the method for the present invention preparation can be used for the preparation of tumour DC vaccine.The method that antigen is regathered after handling breast carcinoma stem cell with hypochlorous acid is not only economical, simple, additionally it is possible to which that the more strongly immune response of activation antigen specific T-cells improves the tumor-killing ability of T cell.
Description
Technical field
The present invention relates to bioengineering and biomedicine field, and in particular to a kind of preparation side of breast carcinoma stem cell antigen
Method and application.
Background technique
Breast cancer is the malignant tumour occurred in breast epithelial tissue, and in women, male is only accounted for for 99% generation in breast cancer
1%.Since the last century 90's, Chinese pathogenesis of breast carcinoma growth rate is twice of global average speed, city
Area is especially pronounced.Currently, breast cancer is the highest cancer disease of Chinese women disease incidence, occupy female malignant it is dead the
Five.For breast cancer, life cycle rate is greater than 85% countries and regions about 25 within 5 year, wherein Asian countries's packet
Include Israel, South Korea and 3, Japan.5 year life cycle rate of breast cancer in China is in middle reaches position in Asian countries and area, small
In Israel, South Korea and Japan, it is also slightly smaller than Taiwan Province of China and Hong Kong.
Clinically, Her-2 positive type and three negative Prognosis in Breast Cancer will be worse than luminal (luminal) type, but
Be Her-2 positive type breast cancer application targeted therapy after, available significant improvement.To triple negative breast cancer, tradition
Treat nearly unavailable, recurrence rate after chemotherapy 3 years is 40% -50%, and DISTANT METASTASES IN once occurs, and cure rate is just almost
Zero.And triple negative breast cancer is exactly multiple in the young woman group in Asia, accounts for 15%-the 20% of all breast cancer, because
It is very urgent that this, which finds a kind of more effective clinical modalities,.
DC cell full name Dendritic Cells (Dendritic Cells, DCs), is in recent years by sole duty concerned by people
Antigen presenting cell (Antigen Presenting Cells, APCs), can absorb, processes and present antigen, and starting T cell is situated between
The immune response led, DC are that American scholar Si Tanman (Steinman) had found in mouse lymph nodes for the first time in 1973.DC
Cell is that known in vivo functionality is most strong, can uniquely activate the professional antigen presenting cells of Resting T cells, is starting, regulation and dimension
Hold the key link of immune response.DC is the key enabler of immune system, and DC is distributed in the tissue and organ of whole body, have pair
Ambient enviroment and antigen fast reaction and the ability for causing immune response.DC can by phagocytosis, huge pinocytosis and by
The modes such as the endocytosis that body mediates absorb the antigen of surrounding.The intake of antigen will lead to DC maturation, and DC will increase antigen submission
Molecule (MHC- I and MHC- II), costimulatory molecules (such as CD80 and CD86), cell adhesion molecule (such as CD83 and CD54) with
And the expression of chemokine receptors (such as CCR7), it migrates to neighbouring lymph node, by antigen presenting molecule antigen
Submission is to T cell.By a large amount of external evoked and culture DC cell, fed back when cell reaches certain amount back loading tumour antigen
To patient, inducible body generates strong anti tumor immune response.Clinical research proves that DC vaccine can be used for treating black
The kinds cancers such as plain tumor, lung cancer, liver cancer, gastric cancer, breast cancer and leukaemia.The antigen of DC vaccine can be from the more of synthesis
Peptide, recombinant DNA, tumor tissues RNA or tumor cell lysate, even allow DC cell directly to swallow tumour cell.If
It is the polypeptide for using synthesis or recombinant DNA as antigen, because the deficiency of antigen type may result in the generation of immunologic escape；
If using tumor tissues RNA or Tumor Tissue Lysates as antigen, can there is a problem of antigen levels deficiency；If made
Antigen is prepared with the Tumor cell cultivated, can theoretically obtain a large amount of tumour antigen, but Tumor cell is very
Hardly possible cultivates and is not easy to remove fibroblast during the cultivation process.
Cancer stem cell (cancer stem cells, CSCs), refers to the cancer cell with stem cell properties, that is, has
The cancer cell of self-replacation and differentiation capability.CSC is to the survival of tumour, proliferation, transfer and recurs important role, current
CSC is also equal to cancer initiator cell by research, it is believed that cancer comes from CSC.Ratio CSC shared in tumor tissues is non-
It is often low, but they are mostly in resting stage, it is insensitive to chemotherapeutics, therefore CSC may be to recur after leading to oncotherapy
Root.And gene relevant with drug resistance is repaired in tumor stem cell height expression immunosupress, DNA；Relative to non-Tumor Stem
Cell can cause more strong anti tumor immune response with tumor stem cell antigen.If CSC can be killed effectively,
The clinical cure rate of malignant tumour will be helped to improve.
The tumor stem cell of in vitro culture is the tumour cell purified, without considering there is fibroblast pollution；Tumour
Stem cell can largely expand under serum-free condition, and keep undifferentiated state, tumor stem cell antigen lysate energy
All antigens for relevant antigen occurring to tumour and be personalization are enough provided, so being the antigen of highly desirable DC vaccine
Source.Radiation and chemotherapy energy killing tumor cell, process of the tumour cell in apoptosis (apoptosis) or downright bad (necrosis)
In can generate " danger signal " molecule, these " danger signal " molecules can be heat shock protein (Heat Shock Protein,
HSP), calprotectin (calreticulin) or dyeing qualitative correlation high mobility group protein B 1 (Chromatin-
Associated Protein High-Mobility Group Box 1, HMGB1), these molecules can promote antigen cross to pass
It is in and enhances immune response.It selects suitable antigen and antigen preparation procedure is the pass for improving therapeutic tumor vaccine curative effect
Key.It is thin with ultraviolet light irradiated tumor cell or direct multigelation tumour using the conventional method that tumour cell prepares antigen
Born of the same parents' inducing apoptosis of tumour cell (apoptosis) or downright bad (necrosis) prepare full tumor-cell antigen.
HSP70 is the heat shock protein (Heat Shock Protein 70, HSP70) of molecular weight about 70kD, is heat shock
Important a member in protein family.Important role plays the part of in antigen presentation in HSP70 family.In tumor cell surface
HSP70 can trigger the specific immunity for the tumour cell, and related with DC antitumor action.HSP70 can promote tumour
The growth of cell, drug resistance and the prognosis of tumour etc. have close pass in the generation, development, tumour immunity, oncotherapy with tumour
System.
IL-12 is interleukin 12 (Interleukin-12, IL-12), is mainly generated by cells such as DC, has and lures
The effects of leading NK cell and T cell generates IFN-γ, promoting cytotoxic T cell formation, has been found to participate in cellular immunity, energy
It is cooperateed with NK cell, T lymphocyte etc. and plays anti-tumor effect, also by itself or other cell factors can be induced, such as
IFN-γ plays antitumor action, and the growth and transfer to kinds of tumors (colon cancer, melanoma, oophoroma etc.) have bright
Aobvious inhibiting effect.Host immune competency, tumoricidal immunologic escape function are improved using IL-12 family cell factor
It is the Critical policies of tumor biotherapy.
IFN-γ is interferon-γ (Interferon- γ, IFN-γ), is the unique member of II type interferon, only by living
Change T cell and natural killer cells (NK cell) generates.IFN-γ can inhibit growth of tumour cell in vivo, and anti-curing oncoma turns
It moves and recurs.Both it is thin tumour can directly to have been killed by modes such as the growth cycles of inducing apoptosis of tumour cell and interference tumour cell
Born of the same parents can also inhibit growth of tumour cell by the angiogenic growth of antitumor tissue, can also be by adjusting human immune system
Enhance human body itself to the Scavenging activity of tumour cell.
Hypochlorous acid is a kind of strong sterilization oxidant, is generated in acute inflammation by the neutrophil leucocyte activated.Use hypochlorous acid
Processing tumour cell can cause rapid apoptosis and the necrosis of tumour cell, stimulate intake of the DC to tumour antigen, it is special to increase antigen
The immunocompetence of specific T cell.Hypochlorous acid can also stamp the immunogenicity that aldehyde radical label increases antigen to antigen, make antigen more preferable
Be exposed to T cell.With hypochlorous acid inducing mammary cancer stem cell apoptosis, the tumor stem cell for cracking apoptosis is prepared into DC antigen,
Relatively traditional antigen preparation procedure, the tumour antigen being prepared with hypochlorous acid oxidization method can more strongly activation antigen it is special
The immune response of specific T cell improves the tumor-killing ability of T cell.
Summary of the invention
The object of the present invention is to provide a kind of methods for preparing breast carcinoma stem cell antigen using hypochlorous acid oxidization method, not only
Economic, simple, relatively traditional antigen preparation procedure, additionally it is possible to the more strongly immune response of activation antigen specific T-cells,
Improve the tumor-killing ability of T cell.
In order to achieve the above object, the present invention provides a kind of preparation methods of breast carcinoma stem cell antigen, comprising following
Step:
(1) breast carcinoma stem cell is cultivated；
(2) hypochlorous acid is added in the solution of culture breast carcinoma stem cell, is uniformly mixed, inducing mammary cancer stem cell withers
It dies；
(3) cracking hypochlorous acid treated breast carcinoma stem cell, and collect cell lysate and obtain antigen.
Preferably, breast carcinoma stem cell described in step (1) includes to separate and train from fresh breast tumor tissue
Support the breast carcinoma stem cell obtained.
Preferably, concentration of the hypochlorous acid in the solution of the culture breast carcinoma stem cell is 20~80 μM.
Preferably, the time of breast carcinoma stem cell apoptosis described in the hypochlorous acid induction is 30~60 minutes.
Preferably, the cracking in step (3) refers to using the multigelation method cracking hypochlorous acid treated mammary gland
Cancer stem cell.
Preferably, multigelation method is multigelation 4~6 times under the conditions of cell being placed on -80 DEG C and 38 DEG C.
Preferably, the cell lysate is to be centrifuged the supernatant of acquisition after cell is cleaved.
It is that above-mentioned method is prepared the present invention also provides a kind of application of breast carcinoma stem cell antigen, it is described
Breast carcinoma stem cell antigen be used for tumor vaccine preparation.
Preferably, the tumor vaccine is the DC vaccine obtained after the breast carcinoma stem cell antigen sensibilization DC.
Compared with prior art, the side provided by the invention that breast carcinoma stem cell antigen is prepared using hypochlorous acid oxidization method
Method handles breast carcinoma stem cell with hypochlorous acid before cracking breast carcinoma stem cell collects antigen, can rapid induction breast cancer it is dry thin
Born of the same parents' apoptosis makes the antigen obtained have the molecule of more enhancing immune responses；The antigen of preparation can be used for the system of tumour DC vaccine
It is standby, it is not only economical, simple, additionally it is possible to which that the more strongly immune response of activation antigen specific T-cells improves the tumour of T cell
Killing ability.
Detailed description of the invention
Fig. 1 is the aspect graph of the spherical breast carcinoma stem cell clone for the suspension seen under microscope.
Fig. 2 be do not process (direct freeze thawing), ultraviolet treatment with irradiation and hypochlorous acid oxidization processing after cell apoptosis ratio
Example.
Fig. 3 is direct freeze-thaw method, ultraviolet irradiation and the hypochlorous acid oxidization legal system of protein immunoblotting experiment detection
The gene expression abundance result of HSP70 in standby breast carcinoma stem cell antigen.
Fig. 4 is the breast carcinoma stem cell that DC cell is prepared by direct freeze-thaw method, ultraviolet irradiation and hypochlorous acid oxidization method
After antigen load in supernatant IL-12 expression.
The direct freeze-thaw method of Fig. 5, ultraviolet irradiation and hypochlorous acid oxidization method preparation antigen load DC cell after with T cell
It co-cultures, the T cell number of the energy secretion of gamma-IFN of ELISPOT technology detection.
Specific embodiment
Below in conjunction with drawings and examples, the following further describes the technical solution of the present invention.
The invention discloses a kind of preparation methods of breast carcinoma stem cell antigen comprising the steps of:
(1) breast carcinoma stem cell is cultivated；
(2) hypochlorous acid is added in the solution of culture breast carcinoma stem cell, is uniformly mixed, inducing mammary cancer stem cell withers
It dies；
(3) cracking hypochlorous acid treated breast carcinoma stem cell, and collect cell lysate and obtain antigen.
Embodiment 1
Breast carcinoma stem cell culture medium is DMEM/F12 culture medium, and is added: 20ng/mL EGF, 20ng/mL bFGF,
20ng/mL PDGF-AA and 1 × B27.DMEM/F12 culture medium is purchased from match Mo Feishier company；EGF is epidermal growth factor, purchase
From PeproTech company；BFGF is basic fibroblast growth factor, is purchased from PeproTech company；PDGF-AA is a kind of
Platelet derived growth factor；B27 is a kind of stem cell factor, purchased from match Mo Feishier company.
Steps are as follows:
1. dissociating fresh breast tumor tissue, and separate single tumour cell
1.1 fresh breast cancer tissue's blocks are transferred in culture dish with sterile tweezers, with physiological saline cleansing tissue
Block 2 times, lose liquid waste.A small amount of 1640 culture medium of serum-free is added dropwise on tissue block, tissue block is shredded with scissors.?
The tissue block shredded is transferred in the centrifuge tube of a 50mL, and 1500rpm is centrifuged 10 minutes；Waste liquid is siphoned away, in every 5 grams tissues
Digestive juice (5mg/ml Collagenase I, 5mg/ml clostridiopetidase A II, 50U/ml is added in the ratio that 20mL digestive juice is added in the tissue
DNA enzymatic I (DNase I) and the Accutase digestive juice of 1 times of dilution (purchased from match Mo Feishier company)), disappear under conditions of 37 DEG C
Change 1 hour of tissue.
1.2 are digested after single cell when tissue, and the fetal calf serum of 1/10 volume is added toward cell suspension the inside
Neutralize digestive ferment.It blows and beats cell suspension several times, cell suspension is allowed to pass through the filter screen that diameter is 70 μm.With the speed of 1500rpm
Degree centrifugation 10 minutes, removes supernatant；Then cell precipitation is resuspended with 30~40mL physiological saline, cell one is cleaned in centrifugation again
It is secondary, supernatant is abandoned, cell precipitation is resuspended with 20~30mL physiological saline.
1.3 in the centrifuge tube of a 50mL, and 10~20mL lymphocyte separation medium is added, then isometric cell is hanged
Liquid is added slowly to above lymph separating liquid；Centrifuge tube is put into inside centrifuge, turns off the deceleration valve of centrifuge, with 850g
Revolving speed be centrifuged 20 minutes.After centrifugation, there is the white cellular layer of tumour cell to be transferred to another among centrifuge tube
In clean centrifuge tube, cell is resuspended with the physiological saline of 2~3 times of volumes, centrifugation removes supernatant.Again with physiological saline weight
Outstanding cell, cell count, centrifugation.
After 1.4 centrifugations, supernatant is abandoned, tumour cell is tuned into 1 × 10 with breast carcinoma stem cell culture medium6/ mL,
10~15mL cell is added in one T75 culture bottle, then cell is put into incubator and is cultivated.
2. breast carcinoma stem cell culture
Liquid processing cell is partly changed when 2.1 breast cancer cell cultures were by the 5th day, partly changes within every 4 days liquid processing one to cell later
It is secondary.After general 2 weeks, the spherical stem cell clone of suspension can be seen under the microscope.As shown in Figure 1, to see under microscope
Suspension spherical breast carcinoma stem cell clone aspect graph.
2.2 when needing cell to pass on, and uses Accutase digestive juice (purchased from match silent winged generation that under conditions of 37 DEG C
Company) it digests spherical stem cell clone 8 minutes, until tumor stem cell clone is digested single cell, breast cancer is dry thin
The passage ratio of born of the same parents is 1:3.
3. hypochlorous acid is added in the solution of culture breast carcinoma stem cell, it is uniformly mixed, inducing mammary cancer stem cell withers
It dies
3.1 use Accutase digestive juice breast carcinoma stem cell Clone Digestion at single cell, with the speed of 800rpm
Centrifuge cell 7 minutes, remove supernatant；Tumor stem cell is tuned into 1 × 10 with culture medium6A/mL is added 50 μM inside cell
Then hypochlorous acid puts back to cell in incubator and continues culture 40 minutes.
3.2 with speed centrifuge cell 7 minutes of 800rpm, removed supernatant；Again twice with physiology salt washing cell；Use physiology
The concentration of breast carcinoma stem cell is adjusted to 2 × 10 by salt water7/mL。
4. cracking breast carcinoma stem cell, antigen and detectable concentration are collected
4.1 crack breast carcinoma stem cell with multigelation method: cell being placed at -80 DEG C 10 minutes, then cell is put
Cell is allowed to thaw within 2 minutes in 38 DEG C of water-baths, five times repeatedly, breast carcinoma stem cell can crack completely.
4.2 under the conditions of 4 DEG C, with breast carcinoma stem cell 20 minutes after the speed centrifugation cracking of 12000rpm, in collection
Clearly, the filter screen filtration supernatant for being 0.22 μM with diameter obtains antigen.
4.3 detect the concentration of breast carcinoma stem cell antigen with BCA protein concentration detection kit, dispense antigen, anti-
Original is stored in -80 DEG C.
Comparative example 1 (direct freeze-thaw method)
Direct freeze-thaw method refers to referring to embodiment 1, but in step 3 with no treatment to breast cancer, collects specified
Quantity cell is directly entered step 4, i.e. step 3 is modified are as follows:
1. with Accutase digestive juice breast carcinoma stem cell Clone Digestion at single cell, with the speed of 800rpm from
Core cell 7 minutes, remove supernatant；
2. again twice with physiology salt washing cell；The concentration of breast carcinoma stem cell is adjusted to 2 × 10 with physiological saline7/
mL。
Comparative example 2 (ultraviolet irradiation)
Ultraviolet irradiation refers to referring to embodiment 1, but in step 3, carries out ultraviolet treatment with irradiation to cell, i.e.,
Step 3 modification are as follows:
1. with Accutase digestive juice breast carcinoma stem cell Clone Digestion at single cell, with the speed of 800rpm from
Core cell 7 minutes, remove supernatant；Tumor stem cell is tuned into 1 × 10 with culture medium6A/mL, being added to diameter is 10 centimetres
In culture dish；It allows breast carcinoma stem cell to irradiate 10 minutes under the ultraviolet light of 302nm, then cell is put back in incubator and is cultivated
Overnight.
2. removing supernatant with speed centrifuge cell 7 minutes of 800rpm；Again twice with physiology salt washing cell；Use physiology
The concentration of breast carcinoma stem cell is adjusted to 2 × 10 by salt water7/mL。
The knot of embodiment 1 (hypochlorous acid oxidization method) and comparative example 1 (direct freeze-thaw method) and comparative example 2 (ultraviolet irradiation)
Fruit comparison
One, the Apoptosis ratio before breast carcinoma stem cell is cleaved
Before cracking breast carcinoma stem cell, collects with the processed breast carcinoma stem cell of hypochlorous acid (hypochlorous acid oxidization method) and use
The irradiated breast carcinoma stem cell (ultraviolet irradiation) of ultraviolet light, and collect the breast carcinoma stem cell not processed and (directly freeze
Melt method) as control.Apoptosis ratio is detected using the cell apoptosis detection kit of eBioscience company.
Experimental result is not as shown in Fig. 2, to process (direct freeze thawing), ultraviolet treatment with irradiation and hypochlorous acid oxidization processing
The apoptosis ratio of cell afterwards.
The A of Fig. 2 is the apoptosis ratio for the breast carcinoma stem cell (direct freeze-thaw method) not processed, and apoptosis ratio is
The breast carcinoma stem cell (ultraviolet irradiation) that the B of 4.32%, Fig. 2 are irradiated with ultraviolet light, apoptosis ratio is 36.63%, Fig. 2
C be the processed breast carcinoma stem cell of hypochlorous acid (hypochlorous acid oxidization method), apoptosis ratio is 51.87%.The results show that secondary chlorine
Acid oxidation can rapid induction Apoptosis, the apoptosis ratio of the breast carcinoma stem cell of hypochlorous acid oxidization group irradiates higher than ultraviolet light
Group.
Two, in breast carcinoma stem cell antigen HSP70 gene expression abundance
Compare 3 kinds of hypochlorous acid oxidization method, direct freeze-thaw method and ultraviolet irradiation methods with protein immunoblotting experiment
The gene expression abundance of HSP70 in the breast carcinoma stem cell antigen of preparation.
Steps are as follows:
1. proteantigen after denaturation treatment, takes 50 μ g antigen loading electrophoresis；
2. after electrophoresis, antigen is transferred to polyvinylidene fluoride (Polyvinylidene Fluoride, PVDF) film
On；
3.PVDF film is incubated overnight under the conditions of 4 DEG C with HSP70 and GAPDH primary antibody；
4. washing away primary antibody, pvdf membrane and secondary antibody are incubated for；
5. washing away secondary antibody, antigenic expression is detected with hypersensitive luminescent solution.
Experimental result is as shown in figure 3, detect direct freeze-thaw method, ultraviolet irradiation and secondary for protein immunoblotting experiment
The gene expression abundance result of HSP70 in the breast carcinoma stem cell antigen of chloric acid oxidizing process preparation.
From the figure 3, it may be seen that it is dry thin that hypochlorous acid oxidization method can induce breast cancer relative to direct freeze-thaw method and ultraviolet irradiation
The more HSP70 molecules of cellular expression.
Three, breast carcinoma stem cell antigen sensibilization DC cell, the IL-12 expression of the DC vaccine of acquisition
Steps are as follows:
1. extracting the 50mL peripheral blood of patient, 20~25mL lymphocyte separation medium is added in each 50mL centrifuge tube,
Then isometric blood is slowly added to above lymphocyte separation medium.Centrifuge tube is put into a centrifuge, centrifugal speed is
500g, centrifugation time is 20 minutes, and turns off reduction of speed valve.The single core of peripheral blood that middle layer white is drawn from centrifuge tube is thin
Born of the same parents (Peripheral Blood Mononuclear Cells, PBMCs), are added in new 50mL centrifuge tube.Piping and druming is uniform,
It is settled to 40~50mL with physiological saline, 600g is centrifuged 10 minutes.Supernatant is abandoned after centrifugation, 40 are settled to physiological saline~
50mL, piping and druming cell, again with 500g centrifugation 10 minutes, abandon supernatant and obtain PBMCs to mixing.With German Mei Tian Ni company
Cell, while cell count is resuspended in DendriMACS GMP rank culture medium；With 500g centrifugation 10 minutes, supernatant is abandoned.
2. every 5 × 107Sorting buffer, 100 μ L Fc receptor blocking pharmacons and the 100 μ L biologies that a PBMC adds 500 μ L to be pre-chilled
Elementization antibody mixed liquor after mixing, is placed in 4 DEG C of refrigerators, takes out after twenty minutes.
3. 4mL, which is added, sorts buffer, cell mixing several times, is centrifuged 8 minutes at 4 DEG C with the speed of 350g, in removal
Clearly.Cell is resuspended with 500 μ L sorting buffer, 500 μ L magnetic beads are added, is placed in 4 DEG C of refrigerators, is taken out after 15 minutes.Again plus
Enter 4mL sorting buffer, gently blows and beats cell more than ten times.
4. the pipe equipped with cell, which is placed on magnet stand, stands 2 minutes, supernatant is transferred to another clean pipe
The inside；Then 4mL is added in former centrifuge tube and sorts buffer, gently blow and beat cell more than ten times, rest on magnet stand 2 points
Clock shifts supernatant；Two parts of supernatants are merged, monocyte is included in inside supernatant.In the supernatant containing monocyte
The serum-free basal medium of 2 times of volumes is added in the inside, and the speed of 350g is centrifuged 8 minutes, loses supernatant.
5. adjusting monocyte with serum-free basal medium, make its concentration 1 × 106A/mL, is added in 6 orifice plates, often
Hole 2.5~3mL culture medium；Then toward addition 1000U/mL granulocyte-macrophage colony stimutaing factor inside basal medium
(Granulocyte-Macrophage Colony Stimulating Factor, GM-CSF) and 500U/mL interleukin-4
(IL-4), 6 orifice plates are put into 37 DEG C of incubator.Monocyte can be divided under the action of GM-CSF and interleukin-4
Jejune DC cell.
6. when the 4th day, the breast carcinoma stem cell antigen of 100 μ g/mL is added in DC cell.
7. when the 5th day, 1000U/mL IFN-γ, 1000U/mL tumor necrosis factor-alpha are added in DC cell
(TNF-a), 100U/mL interferon-a (IFN-a) and 100ng/mL lipopolysaccharides.
8. when the 6th day, collecting the culture supernatant of the mature DC cell and DC cell of suspension.
9. with the IL-12 expression in the ELISA kit detection DC supernatant of IL-12.
Experimental result for DC cell by direct freeze-thaw method, ultraviolet irradiation and hypochlorous acid oxidization method as shown in figure 4, prepared
Breast carcinoma stem cell antigen load after IL-12 in supernatant expression.
As shown in Figure 4, relative to Direct Pyrolysis method and ultraviolet irradiation, the breast cancer of hypochlorous acid oxidization method preparation is dry thin
The DC cell of extracellular antigen sensitization can secrete more IL-12, wherein compared with Direct Pyrolysis method and ultraviolet irradiation, p <
0.01。
Four, breast carcinoma stem cell antigen sensibilization DC cell-stimulating T cells (T cell), compare IFN-γ
Expression
Steps are as follows:
(1) cytotoxic T cell of breast carcinoma stem cell antigentic specificity is prepared
1. preparing T cells while preparing DC.Every 10740 μ L buffers (buffer), 10 μ are added in a PBMC
L T cells Biotin-Antibody mixed liquor (Pan T Cell Biotin-Antibody Cocktail) and 10 μ L
Anti- Anti-TCR gamma/delta biotin (Anti-TCR gamma/delta-Biotin) mixes PBMCs and antibody, and 4 DEG C are placed 5 minutes.
2. every 107Be added in a PBMC 30 μ L buffers and 20 μ L T cells magnetic bead mixed liquors (Pan T
Cell MicroBead Cocktail), PBMCs and magnetic bead are mixed, 4 DEG C are placed 10 minutes.
3. LS pillar (German U.S. day Ni) is placed on above magnet stand, 3mL buffer is added, after waiting buffers to drain off completely,
Cell is added to inside pillar, the cell flow down is collected；Then wash that pillar is primary, and collection is eluted with 3mL buffer again
Cell.
4. counting all T cells being collected into, then T cell is centrifuged 8 minutes under the speed of 350g；Supernatant is abandoned, X is used
T cell is resuspended in VIVO-15 culture medium, and the density of T cell is adjusted to 1 × 106/mL。
5. 5mL T cell is added, 20U/mL IL-2 and 20U/mL IL-7 is added, T in the culture bottle of a T25
Cell is put back in incubator and is cultivated.The culture medium of 1/2 volume is added when third day, and adds 20U/mL IL-2 and 20U/
mL IL-7。
6. when the 5th day, collecting T cell while collecting DC.It is thin with X VIVO-15 culture medium adjustment DC and T respectively
The density of born of the same parents, the density of T cell are 2 × 106The density of/mL, DC are 1 × 105/mL.T cell and DC cell are mixed in equal volume
It is inoculated into culture bottle, is used to prepare antigentic specificity cytotoxic T cell, people AB serum, the 20U/ of 5% inactivation are added in culture medium
ML IL-2 and 20U/mL IL-7.The DC and T cell crossed by antigen sensibilization is co-cultured, and as experimental group, does not pass through antigen
The DC and T cell that sensitization is crossed are co-cultured, as a control group.
(2) elisa detection (ELISPOT) experiment:
1. the X VIVO-15 culture medium of 200 μ L serum-frees is added inside each hole of the pre-coated PVDF plate of ELISPOT,
It is stored at room temperature and culture medium is sucked out after five minutes.
2. the antigentic specificity cytotoxic T cell prepared is diluted to 1 × 10 with culture medium6/ mL is added in each hole
100 μ L T cell suspensions；Use AntiCD3 McAb/CD28 magnetic bead activation T cells as positive control.
3. a T cell puts back to 20 hours of culture in incubator.
4. second day, cell and culture medium in pouring aperture, with ice-cold physiological saline hypotonic lysis cell；It outwells in hole
Liquid, cleaned plank 5~6 times with cleaning buffer solution.
5. antibody is added inside plank, finally colour developing and color development stopping.
6. reading spot with elisa automatic image analyzer and statisticalling analyze.
Experimental result is as shown in figure 5, the antigen for direct freeze-thaw method, ultraviolet irradiation and the preparation of hypochlorous acid oxidization method is negative
It is co-cultured after carrying DC cell with T cell, the T cell number of the energy secretion of gamma-IFN of ELISPOT technology detection.
As shown in Figure 5, relative to Direct Pyrolysis method and ultraviolet irradiation, the T cells of hypochlorous acid oxidization method preparation
More IFN-γ can be secreted, wherein compared with Direct Pyrolysis method and ultraviolet irradiation, p < 0.01.
In conclusion hypochlorous acid oxidization method is compared with direct freeze-thaw method and ultraviolet irradiation:
(1) breast carcinoma stem cell is higher by Apoptosis ratio of the hypochlorous acid after apoptosis-induced；
(2) gene expression abundance of HSP70 is higher in the breast carcinoma stem cell antigen of hypochlorous acid oxidization method preparation；
(3) the breast carcinoma stem cell antigen sensibilization DC cell of hypochlorous acid oxidization method preparation, the IL-12 table of the DC vaccine of acquisition
Up to higher level；
(4) the breast carcinoma stem cell antigen sensibilization DC cell of hypochlorous acid oxidization method preparation, the T cells secretion of activation
IFN-γ expression is higher.
Therefore, the method that hypochlorous acid oxidization method provided by the invention prepares breast carcinoma stem cell antigen, not only economic, letter
It is single, additionally it is possible to which that the more strongly immune response of activation antigen specific T-cells improves the tumor-killing ability of T cell.
It is discussed in detail although the contents of the present invention have passed through above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read above content, for of the invention
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (9)
1. a kind of preparation method of breast carcinoma stem cell antigen, which is characterized in that comprise the steps of:
(1) breast carcinoma stem cell is cultivated；
(2) hypochlorous acid is added in the solution of culture breast carcinoma stem cell, is uniformly mixed, inducing mammary cancer stem cell apoptosis；
(3) cracking hypochlorous acid treated breast carcinoma stem cell, and collect cell lysate and obtain antigen.
2. the preparation method of breast carcinoma stem cell antigen as described in claim 1, which is characterized in that cream described in step (1)
Gland cancer stem cell includes to separate from fresh breast tumor tissue and cultivate the breast carcinoma stem cell obtained.
3. the preparation method of breast carcinoma stem cell antigen as described in claim 1, which is characterized in that the hypochlorous acid is in institute
Concentration in the solution for the culture breast carcinoma stem cell stated is 20~80 μM.
4. the preparation method of breast carcinoma stem cell antigen as described in claim 1, which is characterized in that the hypochlorous acid induction
The time of the breast carcinoma stem cell apoptosis is 30~60 minutes.
5. the preparation method of breast carcinoma stem cell antigen as described in claim 1, which is characterized in that the cracking in step (3)
Refer to using the multigelation method cracking hypochlorous acid treated breast carcinoma stem cell.
6. the preparation method of breast carcinoma stem cell antigen as claimed in claim 5, which is characterized in that multigelation method is thin
Multigelation 4~6 times under the conditions of born of the same parents are placed on -80 DEG C and 38 DEG C.
7. the preparation method of breast carcinoma stem cell antigen as described in claim 1, which is characterized in that the cell lysate
It is to be centrifuged the supernatant of acquisition after cell is cleaved.
8. a kind of application of breast carcinoma stem cell antigen, which is characterized in that the breast carcinoma stem cell antigen is by claim 1-7
Described in any item methods are prepared, which is used for the preparation of tumor vaccine.
9. the application of breast carcinoma stem cell antigen as claimed in claim 8, which is characterized in that the tumor vaccine is described
Breast carcinoma stem cell antigen sensibilization DC after the DC vaccine that obtains.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110124021A (en) * | 2019-05-15 | 2019-08-16 | 迪亚思生命科技(武汉)有限公司 | A kind of preparation method of novel tumor vaccine |
| CN115607661A (en) * | 2022-10-31 | 2023-01-17 | 徐州医科大学 | Application of DC vaccine prepared from whole tumor lysate in tumor treatment |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108291205A (en) * | 2015-09-26 | 2018-07-17 | 普莱瓦克斯免疫肿瘤学公司 | Composition and method for producing dendritic cell |
-
2018
- 2018-08-31 CN CN201811009702.0A patent/CN109096386A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108291205A (en) * | 2015-09-26 | 2018-07-17 | 普莱瓦克斯免疫肿瘤学公司 | Composition and method for producing dendritic cell |
Non-Patent Citations (3)
| Title |
|---|
| CHIANG CLL等: "Adjuvants for Enhancing the Immunogenicity of Whole Tumor Cell Vaccines", 《INTERNATIONAL REVIEWS OF IMMUNOLOGY》 * |
| CHIANG CLL等: "Hypochlorous acid enhances immunogenicity and uptake of allogeneic ovarian tumor cells by dendritic cells to cross-prime tumor-specific T cells", 《CANCER IMMUNOLOGY IMMUNOTHERAPY》 * |
| CHIANG CL等: "A dendritic cell vaccine pulsed with autologous hypochlorous acid-oxidized ovarian cancer lysate primes effective broad antitumor immunity: from bench to bedside", 《CLIN CANCER RES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110124021A (en) * | 2019-05-15 | 2019-08-16 | 迪亚思生命科技(武汉)有限公司 | A kind of preparation method of novel tumor vaccine |
| CN115607661A (en) * | 2022-10-31 | 2023-01-17 | 徐州医科大学 | Application of DC vaccine prepared from whole tumor lysate in tumor treatment |
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Application publication date: 20181228 |