CN107158367A - Colorectal cancer stem cells vaccine preparation method and application - Google Patents
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Abstract
The invention discloses colorectal cancer stem cells vaccine preparation method and application, CD133 is prepared by multigelation method by using from the postoperative tumor tissue cell of colorectal cancer patients, Human colorectal cancer cells system Lovo or mouse colorectal cancer cell system CT26+Colorectal cancer stem cells vaccine.Gained CD133+Colorectal cancer stem cells vaccine can lower immune mouse serum TGF β levels, improve IFN γ levels, enhancing spleen NK cell killing activities and CDC activity, immunized mice is resisted colorectal cancer cell attack, induction immunocyte target killing CD133+Colorectal cancer stem cells and then the growth for suppressing tumour.
Description
Technical field
The invention belongs to biological product technical field, and in particular to colorectal cancer stem cells vaccine preparation method and application.
Background technology
Colorectal cancer (Colorectal Cancer) is one of common alimentary system malignant tumour, related positioned at cancer
Cause of death second.For the developing country of the Asian-Pacific area, colorectal cancer is more easy to send out in developed countries such as America and Europes
Disease.Reported according to 2017 Chinese city cancers:Colorectal cancer is located at the 5th in male's incidence of disease, and the women incidence of disease is located at the 3rd, about
A 50% colorectal cancer patients cancer return of first 3 years after surgery experience, five-year survival rate is shifted because of cancer to remote organ
Decline obvious.Research is thought, in colorectal cancer evolution process, and the mutation of gene and the change of microenvironment further increase knot
The generation of carcinoma of the rectum malignant phenotype, and cancer stem cell (Cancer Stem Cells, CSCs) theory to the understanding of colon cancer and
Treatment is increasingly becoming the focus of colon cancer research field.Cancer stem cell is that a group has the tumour of self-renewing and differentiation potential thin
Born of the same parents, are widely present in the Several Kinds of Malignancy such as colon cancer, cancer of pancreas, oophoroma, breast cancer, prostate cancer.
Research shows that there is quantity in colorectal cancer cell system seldom can but promote tumour growth, transfer and the one of recurrence
Group's cell, researcher is referred to as colorectal cancer CSCs.CSCs has following biological characteristics：One is that CSCs has self-renewing
Ability, shows as that clone can be formed on soft agar medium, and most of tumour cells can not form clone；Two be CSCs
As normal stem cell, the ability with differentiation can be split into two difference DNA and chromatin, centrosome mixture, egg
The equal cell of white matter content, and promote tumour formation and transfer；Three be that CSCs has side population cell (side
Population cells) ability, the dye Hoechst 33342 and antineoplastic for entering nucleus can be discharged
It is extracellular, so as to help anti-chemotherapeutic and the recurrence of tumour.Four be that CSCs can be expressed and normal cell, tumour cell and stem cell
Related molecular labeling, such as colorectal cancer CSCs can express CD44, CD133, CD166 specific molecular marker, wherein CD133+Phenotype cells are widely regarded as colorectal cancer CSCs.In CSCs various " dryness ", epithelial-mesenchymal conversion
(Epithelial-Mesenchyme Transition, EMT) characteristic makes colorectal cancer CSCs have stronger compared with Common tumors cell
Locomitivity, be more easy to break through surrounding tissue and enter the circulatory system, and in target organ formation far-end transfer, this is probably Colon and rectum
Cancer recurrence transfer, the main cause that can not be effected a radical cure.
Stem cell is the primitive undifferentiated cells that a class has self-replacation, more New function and multi-lineage potential, is divided into
Embryonic stem cell, germline stem cell and adult stem cell.The stem cell of mutation and precursor are probably CSCs source, are swollen
CSCs " dryness " is for maintaining tumor proliferation, resistance, invasion and attack and recurrence to play a decisive role in knurl.Therefore, how to kill swollen
" seed " cell is CSCs in knurl, it is to avoid " revivable " thorny problem occurred in tumor therapeutic procedure, reaches and " cuts grass to remove
Root " cures tumour purpose, is the key problem that should be solved at present.Research shows, after operation, radiotherapy, chemotherapy, colorectal cancer
Stem cell vaccine is increasingly becoming one of effective ways of colorectal cancer biological therapy.
Colorectal cancer stem cells vaccine refers to the colorectal cancer CSCs of separation, by physics, chemistry and biological factor
The processing of (viral infection, gene transfer etc.), changes or eliminates its carcinogenicity, retains its immunogenicity, and combines with adjuvant etc.
A kind of new generation vaccine of specific anti-CSCs immune responses is produced using preparation, inducible body.So far, it is straight on knot
The research of intestinal cancer stem cell vaccine is still in the preclinical earlier trials stage.Although colorectal cancer CSCs expresses the immunogene of antigen
Property it is slightly strong relative to common colorectal cancer cell, but because it is that normal cell is developed, the immunogenicity of its antigen is still
It is weaker, it is impossible to induce very strong specific immune response.Therefore scholar reduces the carcinogenic of CSCs by methods such as genetic modifications
Property, its immunogenicity is improved, colorectal cancer stem cells vaccine is prepared, in muroid vivo applications colorectal cancer stem cells vaccine
Receive and substantially suppress tumour growth and the effect of recurrence.But due to human immune system and muroid difference, therefore Colon and rectum
Immune effect of the cancer stem cell vaccine for human body, in addition it is also necessary to further clinical verification.
The content of the invention
The technical problem of solution：, should it is an object of the invention to provide colorectal cancer stem cells vaccine preparation method and application
Colorectal cancer stem cells vaccine can strengthen immune function of mice, trigger good GVT.
Technical scheme：The preparation method of colorectal cancer stem cells vaccine, comprises the following steps：
Step 1, colorectal cancer cell is obtained, Secondary Culture is carried out；
Step 2, using immunological magnetic bead sorting buffer solution suspension colorectal cancer cell；
Step 3, FcR blocking agents and CD133 are added in the cell suspension of step 2+The straight mark monoclonal of marked by magnetic bead resists
Body, 4 DEG C of incubations；
Step 4, using the gained colorectal cancer cell of immunological magnetic bead sorting buffer solution washing step 3；
Step 5, step 4 colorectal cancer cell is resuspended using immunological magnetic bead sorting buffer solution；
Step 6, splitter is put into magnetic field, adds the cell suspension of step 5, collect negative thin with wash buffer
Born of the same parents, i.e. CD133-Colorectal cancer cell；
Step 7, splitter is removed into magnetic field, positive cell, i.e. CD133 is collected with wash buffer+Colorectal cancer is dry thin
Born of the same parents；
Step 8, the CD133 obtained using immunological magnetic bead sorting buffer solution resuspending step 7+Colorectal cancer cell, through repeatedly
Colorectal cancer stem cells vaccine is made in freeze thawing.
Further, it is post operative colo-rectal cancer knurl that the colorectal cancer cell in colorectal cancer cell is obtained described in step 1
Histocyte, Human colorectal cancer cells system Lovo cells or mouse colorectal cancer cell system CT26 cells.
Further, every 1 × 10 in step 28Colorectal cancer cell, adds 300 μ L immunological magnetic bead sortings buffer solutions and suspends
Colorectal cancer cell.
Further, every 1 × 10 in step 38Colorectal cancer cell, adds 100 μ L FcR blocking agents, adds CD133+Magnetic
The straight mark monoclonal antibody of pearl mark.
Further, every 1 × 10 in step 58Colorectal cancer cell, adds 500 μ L immunological magnetic bead sortings buffer solutions and is resuspended
Colorectal cancer cell.
Further, it is by CD133 in step 8+Colorectal cancer stem cells are placed in after -80 DEG C of refrigerator freezing 30min and taken out,
22 DEG C of room temperature is put to dissolve naturally；Colorectal cancer stem cells vaccine is made after 3 inactivations of multigelation.
The colorectal cancer stem cells vaccine that above-mentioned preparation method is prepared.
Application of the above-mentioned colorectal cancer stem cells vaccine in treatment colorectal cancer medicine is prepared.
Beneficial effect：
1. used in the present invention and derive from the postoperative tumor tissue cell of colorectal cancer patients or Human colorectal cancer cells system Lovo
With mouse colorectal cancer cell system CT26 CD133 is prepared by multigelation method (rather than inaxtivation of drug method)+Colon cancer
Stem cell vaccine, the CD133+Colon cancer stem cell vaccine can lower immune mouse serum TGF-β level in zoopery, carry
High IFN-γ level, enhancing spleen NK cell killing activities and CDC activity, make immunized mice resist colorectal cancer cell attack, induction
Immunocyte target killing CD133+Colorectal cancer stem cells and then the growth for suppressing tumour.Present invention research finds the Colon and rectum
Cancer stem cell vaccine can strengthen immune function of mice, trigger good GVT.
2. compared with inaxtivation of drug method, the present invention prepares colorectal cancer stem cells vaccine using multigelation method, can make cancer
The dominant antigen and " danger signal " of stem cell release are as Hsp70, Hsp 90, DNA, RNA and uric acid etc., easily by internal DC
Surface scavenger receptor-A and TLR-4 are combined, and are more easy to swallow DC and promote its ripe and processing to offer Antigenic Peptide, are effectively lured
Lead CD4+T and CD8+CTL immune responses；In addition the vaccine that prepared by whole oncolysis can be used for all patients, different from swollen
The peptide or protein matter vaccine in knurl source is, it is necessary to consider HLA problems.
Brief description of the drawings
Fig. 1 is Lovo cell line each group vaccine effects in test example 1, and Figure 1A is that different vaccine immunity group nude inoculations are wild
Type Lovo cell challenges anaesthetize execution detection each group tumour gross specimen, the mm of most long radial line≤20 after 35 days；Figure 1B is each vaccine
Immune group nude mice is without knurl time-survivor curve；Fig. 1 C are each vaccine immunity group nude mouse tumor volume growth curve.
Fig. 2 is CT26 cell line each group vaccine effects in test example 1, and Fig. 2A is that different vaccine immunity group BalB/c mouse connect
Plant wild type CT26 cell challenges and execution detection each group tumour gross specimen, most long radial line≤20mm are anaesthetized after 35 days；Fig. 2 B are
Each vaccine immunity group BalB/c mouse are without knurl time-survivor curve；Fig. 2 C are that each vaccine immunity group BalB/c mouse gross tumor volume increases song
Line.
Fig. 3 is obtained in eye socket blood, serum before being put to death for each knurl seedling immune group nude mice anesthesia of Lovo cell lines in test example 2
IFN-γ, TGF-β content detection situation, Fig. 3 A are IFN-γ content in detection each group nude mouse serum, and Fig. 3 B are each group nude mice blood
TGF-β content in clear.
Fig. 4 is each preceding acquisition eye socket blood of knurl seedling immune group BalB/c mouse anesthesia execution of CT26 cell lines, serum in test example 2
Middle IFN-γ, TGF-β content detection situation.Fig. 4 A are IFN-γ content in detection each group BalB/c mouse serum, and Fig. 4 B are each group
TGF-β content in BalB/c mouse serum.
Fig. 5 is each knurl seedling immune group nude mice spleen NK cytotoxicity experiment results of Lovo cell lines in test example 3, and Fig. 5 A are
Different vaccine groups are by the YAC-1 cell fluidic cell peak figures of NK cytotoxic killers；Fig. 5 B are different vaccine groups by NK cell toxicants
Property killing YAC-1 cell block diagrams.
Fig. 6 is each knurl seedling immune group BalB/c mouse spleen NK cytotoxicity experiment results of CT26 cell lines, Fig. 6 A in test example 3
It is different vaccine groups by the YAC-1 cell fluidic cell peak figures of NK cytotoxic killers；Fig. 6 B are different vaccine groups by NK cells
The YAC-1 cell block diagrams of toxicity killing.
Fig. 7 is CD133 in each knurl seedling immune group BalB/c mouse tumor tissues of CT26 cell lines in test example 4+CSCs ratios are examined
Survey, Fig. 7 A are streaming figure, Fig. 7 B are column analysis chart.
Embodiment
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are to be used to illustrate
The present invention and be not limited to limit the scope of the present invention.The bar that the implementation condition used in embodiment can be required according to concrete application
Part does further adjustment, and unreceipted implementation condition is usually the condition in normal experiment.
Material and instrument
CD133+The straight mark monoclonal antibody of marked by magnetic bead, German Mei Tian Ni companies；
Human colon cancer cell strain Lovo and colon cancer cell line CT26 cell lines are purchased from cell institute of the Shanghai Chinese Academy of Sciences；
BALB/c mouse, nude mice (5-6 week old) are purchased from Yangzhou University's animal center, permit number：SCXK；
Flow cytometer, U.S. company BD.
Embodiment 1
The preparation of colon cancer cell：
Step 1, after sterile acquisition tumor tissues, with the PBS containing dual anti-(100U/mL penicillin and 100U/mL streptomysins)
Tumor tissues are cleaned, slough, connective tissue, blood vessel, fat etc. is rejected, tumor tissues are shredded into 0.5 with scissors~
1mm3Tissue block, plus clostridiopetidase A, DNA enzymatic, digestion enzymolysis filters, prepares single cell suspension；
Step 2, cell suspension is put in blake bottle, after after cell attachment, removes suspension cell, change fresh medium,
Secondary Culture.
The preparation of colorectal cancer stem cells vaccine：
Step 1, obtain and Secondary Culture post operative colo-rectal cancer tumor tissue cell or Human colorectal cancer cells system Lovo cells
Or mouse colorectal cancer cell system CT26 cells, using the RPMI1640 containing 10% calf serum as culture medium, 37 DEG C, 5%CO2
Cultivated under the conditions of saturated humidity, cell length to certain density, after pancreatin digestion is dispelled, cell is collected in 1200rpm, 5min centrifugation
And count；
Step 2, every 1 × 108Colorectal cancer cell, adds 300 μ L immunological magnetic bead sorting buffer solution suspension cells；
Step 3,100 μ L FcR blocking agents are added, 100 μ L CD133 are added+The straight mark monoclonal antibody of marked by magnetic bead, is mixed
4 DEG C of incubation 30min after even；
Step 4,1-2mL immunological magnetic bead sortings buffer solution washing colorectal cancer cell is added, 1200rpm, 10min are centrifuged,
Abandon supernatant；
Step 5, every 1 × 108Colorectal cancer cell, adds 500 μ L immunological magnetic bead sortings buffer solutions and is resuspended；
Step 6, splitter is put into magnetic field, added after buffer solution rinse splitter, add the cell suspension of step 5,
It is CD133 to add wash buffer later and collect negative cells-Colorectal cancer cell；
Step 7, splitter is removed into magnetic field, is put on a suitable collection test tube, is at the uniform velocity rinsed with a small amount of buffer solution,
It is CD133 to collect positive cell+Colorectal cancer stem cells；
Step 8, the CD133 for taking immunological magnetic bead sorting buffer solution suspension sorting step 7 to obtain+Colorectal cancer cell, will
CD133+Colorectal cancer stem cells are placed in after -80 DEG C of refrigerator freezing 30min and taken out, and put 22 DEG C of room temperature and dissolve naturally, multigelation 3
Colorectal cancer stem cells vaccine is used as after secondary inactivation.
Test example 1
Vaccine immunity strategy
Nude mice is respectively adopted and BalB/c mouse experiment in vivo evaluates CSCs vaccine potencies in Lovo cells and CT26 cells.Take
9 Female nude mices and 9 female BalB/c mouse, 5-6 week old, body weight 16-18g, are randomly divided into three groups, every group 3, are respectively
CD133+CSCs vaccine groups (CSC vaccine), Non-CD133+CSCs vaccine groups (Non-CSC vaccine), wild type is thin
Born of the same parents' vaccine group (Lovo cell vaccine).Each group experimental mouse distinguish dorsal sc injection with multigelation method inactivate it is above-mentioned
Cell vaccine (2 × 104) immune three times, per immunization interval 10 days twice, it is subcutaneously injected 2 after last cell vaccine is immune 10 days ×
106Wild type tumor cell challenges.At interval of the tumour formational situation of the every mouse of detection in three days, its most long radial line and most is measured
Minor axis line, and observe the Sulfurless fixative of every mouse.Monitor the general health after every mouse immune, such as behavior simultaneously
Activity, diet situation, changes of weight and chroma of hair.When the most long radial line of tumour is close to 20mm, mouse is euthanized, and is terminated
Experiment.Cell vaccine effect experiment is repeated twice.
As shown in figure 1, Figure 1A is different vaccine immunity group nude inoculation wild type Lovo cell challenges after 35 days at anesthesia
Dead detection each group tumour gross specimen, most long radial line≤20mm；Figure 1B is each vaccine immunity group nude mice without knurl time-survivor curve；
Fig. 1 C are each vaccine immunity group nude mouse tumor volume growth curve.
As shown in Fig. 2 Fig. 2A is that different vaccine immunity group BalB/c mouse inoculation wild type CT26 cell challenges are numb after 35 days
It is liquor-saturated to put to death detection each group tumour gross specimen, most long radial line≤20mm；Fig. 2 B are that each vaccine immunity group BalB/c mouse survive without knurl
Time graph；Fig. 2 C are each vaccine immunity group BalB/c mouse gross tumor volume growth curves.
Test example 2
EUSA (ELISA)
Each group mouse new blood is obtained before anesthesia is put to death.According to commercial ELISA kit (eBioscience,
San Jose, CA, USA) specification detection serum in gamma interferon (IFN-γ) and transforming growth factor-β (TGF-β).Letter
Yan Zhi, plasma sample 1:After 10 dilutions, captured, then examined by the secondary antibody of biomarker by specific initial antibodies per cell factor
Survey.Read plate under ELIASA (Bio-Rad Labs, Hercules, CA, USA) difference 450nm, 570nm wavelength.Sample and standard items
There are three multiple holes, the susceptibility of reagent detection IFN-γ and TGF-βs is 0.1 unit/mL.Kit is applied to detection cell
Cell factor in culture supernatant and blood serum sample.
Each knurl seedling immune group nude mice anesthesia of Lovo cell lines obtains IFN-γ, TGF-β content in eye socket blood, serum before putting to death
Detection case is as shown in Figure 3.Wherein, Fig. 3 A are IFN-γ content in detection each group nude mouse serum：CSC vaccine groups (CSC
Vaccine) compared to Non-CSC vaccine groups (Non-CSC vaccine) (P<0.05), wild type Lovo cell vaccines group
(Lovo cell vaccine)(P<0.01) notable difference is respectively provided with, difference is statistically significant；Fig. 3 B are each group nude mouse serum
Middle TGF-β content：CSC vaccine groups are compared to Non-CSC vaccine groups (P<0.05), wild type Lovo cell vaccines
Group (P<0.01) there is notable difference, difference is statistically significant.
Each knurl seedling immune group BalB/c mouse anesthesia of CT26 cell lines obtains IFN-γ, TGF- in eye socket blood, serum before putting to death
β content detection situations are as shown in Figure 4.Wherein, Fig. 4 A are IFN-γ content in detection each group BalB/c mouse serum：CSC
Vaccine groups (CSC vaccine) are compared to Non-CSC vaccine groups (Non-CSC vaccine) (P<0.05) it is, wild
Type CT26 cell vaccines group (Lovo cell vaccine) (P<0.01) notable difference is respectively provided with, difference is statistically significant；Figure
4B is TGF-β content in each group BalB/c mouse serum：CSC vaccine groups are compared to Non-CSC vaccine groups (P<
0.05), wild type CT26 cell vaccines group (P<0.01) there is notable difference, difference is statistically significant.
Test example 3
Spleen NK cell toxicity tests
Under aseptic condition spleen tissue is obtained from immune mouse.5×106Splenocyte uses concentration for the 5 of 0.5mM
(6)-carboxyl diacetic acid fluorescein succinimide ester (CFSE；20 μ g/mL) 37 DEG C be incubated 20min.Splenocyte, which is used, contains 5%FBS
PBS wash twice to remove unnecessary CFSE.The splenocyte of CFSE marks is seeded in containing fixed qty as effector cell
In 96 orifice plates of YAC-1 target cells, to imitate target ratio as 30:1 inoculation.Then 7-ADD in the negative groups of flow cytomery CFSE
Positive cell ratio.
Each knurl seedling immune group nude mice spleen NK cytotoxicity experiments of Lovo cell lines：It is thin that nude mice anesthesia takes spleen to obtain spleen after putting to death
Born of the same parents' suspension, mark CFSE, as target cell, is 30 in effect target ratio as effector cell, YAC-1:Detect that CFSE is negative under the conditions of 1
7-AAD positive cells in cell mass, are by the cell of spleen NK cytotoxic killers.Fig. 5 A are different vaccine groups by NK cell toxicants
Property killing YAC-1 cell fluidic cell peak figures, be grouped as follows：Lovo cancer stem cells vaccine group (Lovo CSC vaccine
Group), wild type Lovo cell vaccines group (Lovo cell vaccine group), the non-cancer stem cell vaccine groups of Lovo
(Lovo Non-CSC vaccine group)；Fig. 5 B are different vaccine groups by the YAC-1 cell columns of NK cytotoxic killers
Figure, CSC vaccine groups (CSC vaccine) are compared to Non-CSC vaccine groups (Non-CSC vaccine) (P<
0.05), wild type Lovo cell vaccines group (Lovo cell vaccine) (P<0.01) there is notable difference, difference has statistics
Learn meaning.
Each knurl seedling immune group BalB/c mouse spleen NK cytotoxicity experiments of CT26 cell lines：The anesthesia of BalB/c mouse takes spleen after putting to death
Splenocyte suspension is obtained, mark CFSE, as target cell, is 30 in effect target ratio as effector cell, YAC-1:Detected under the conditions of 1
7-AAD positive cells in CFSE negative cells group, are by the cell of spleen NK cytotoxic killers.Fig. 6 A are different vaccine group quilts
The YAC-1 cell fluidic cell peak figures of NK cytotoxic killers, are grouped as follows：The non-cancer stem cell vaccine group (CT26Non- of CT26
CSC vaccine group), wild type CT26 cell vaccines group (CT26cell vaccine group), CT26 cancer stem cells
Vaccine group (CT26CSC vaccine group)；Fig. 6 B are different vaccine groups by the YAC-1 cell columns of NK cytotoxic killers
Figure, CSC vaccine groups (CSC vaccine) are compared to Non-CSC vaccine groups (Non-CSC vaccine) (P<
0.05), wild type CT26 cell vaccines group (Lovo cell vaccine) (P<0.01) there is notable difference, difference has statistics
Learn meaning.
Test example 4
CD133 in tumor tissues+CSCs population analysis
Each vaccine immunity group mouse tumor is obtained, single cell suspension, flow cytomery CD133 is made+CSCs cells
Group.In other words, 2 × 105Tumour cell be suspended in PBS, mark anti-Human CD133 phycoerythrin (PE) antibody
(eBioscience,CA,USA)。
CD133 in each knurl seedling immune group BalB/c mouse tumor tissues of CT26 cell lines+CSCs ratios are detected.Fig. 7 A are streaming
Figure, is grouped as follows：Control group (Control group), non-cancer stem cell vaccine group (Non-CSC vaccine group), open country
Raw type CT26 cell vaccines group (CT26cell vaccine group), cancer stem cell vaccine group (CSC vaccine
group)；Control groups are the undyed blank control of mouse tumor cell, CD133+Cell proportion 0.306%；CD133+
CD133 in CSCs vaccine group tumour cells+Cell proportion 5.22%； Non-CD133+CD133 in CSCs vaccine group tumour cells+
Cell proportion 19.1%；CD133 in wild type CT26 cell vaccine group tumour cells+Cell proportion 13.7%；Fig. 7 B are column
Analysis chart, statistics finds CD133+CD133 in the tumour cell of CSCs vaccine groups (CSC vaccine)+Cell proportion, with
Control groups (P<0.01)、Non-CD133+CSC vaccine groups (Non-CSC vaccine) (P<0.05), wild type CT26 is thin
Born of the same parents' vaccine group (CT26cell vaccine) (P<0.01) it is variant, and difference has statistical significance.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are not it should be appreciated that the present invention is limited by examples detailed above, and the simply explanation described in examples detailed above and specification is of the invention
Principle, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these change and
Improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its is equal
Thing is defined.
Claims (8)
1. the preparation method of colorectal cancer stem cells vaccine, it is characterised in that：Comprise the following steps：
Step 1, colorectal cancer cell is obtained, Secondary Culture is carried out；
Step 2, using immunological magnetic bead sorting buffer solution suspension colorectal cancer cell；
Step 3, FcR blocking agents and CD133 are added in the cell suspension of step 2+The straight mark monoclonal antibody of marked by magnetic bead, 4 DEG C
It is incubated；
Step 4, using the gained colorectal cancer cell of immunological magnetic bead sorting buffer solution washing step 3；
Step 5, step 4 gained colorectal cancer cell is resuspended using immunological magnetic bead sorting buffer solution；
Step 6, splitter is put into magnetic field, adds the cell suspension of step 5, collect negative cells with wash buffer, i.e.,
CD133-Colorectal cancer cell；
Step 7, splitter is removed into magnetic field, positive cell, i.e. CD133 is collected with wash buffer+Colorectal cancer stem cells；
Step 8, the CD133 obtained using immunological magnetic bead sorting buffer solution resuspending step 7+Colorectal cancer cell, through multigelation system
Obtain colorectal cancer stem cells vaccine.
2. the preparation method of colorectal cancer stem cells vaccine according to claim 1, it is characterised in that：Described in step 1
It is post operative colo-rectal cancer tumor tissue cell, Human colorectal cancer cells system Lovo to obtain the colorectal cancer cell in colorectal cancer cell
Cell or mouse colorectal cancer cell system CT26 cells.
3. the preparation method of colorectal cancer stem cells vaccine according to claim 1, it is characterised in that：Every 1 in step 2 ×
108Colorectal cancer cell, adds 300 μ L immunological magnetic bead sorting buffer solution suspension colorectal cancer cells.
4. the preparation method of colorectal cancer stem cells vaccine according to claim 1, it is characterised in that：Every 1 in step 3 ×
108Colorectal cancer cell, adds 100 μ L FcR blocking agents, adds 100 μ L CD133+The straight mark monoclonal antibody of marked by magnetic bead.
5. the preparation method of colorectal cancer stem cells vaccine according to claim 1, it is characterised in that：Every 1 in step 5 ×
108Colorectal cancer cell, adds 500 μ L immunological magnetic bead sortings buffer solutions and colorectal cancer cell is resuspended.
6. the preparation method of colorectal cancer stem cells vaccine according to claim 1, it is characterised in that：Be in step 8 by
CD133+Colorectal cancer stem cells are placed in after -80 DEG C of refrigerator freezing 30min and taken out, and put 22 DEG C of dissolvings naturally；Multigelation goes out for 3 times
Colorectal cancer stem cells vaccine is made after work.
7. the colorectal cancer stem cells that the preparation method of the colorectal cancer stem cells vaccine described in claim 1 to 6 is prepared
Vaccine.
8. the colorectal cancer stem cells vaccine described in right 7 prepares the application in treatment colorectal cancer medicine.
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| CN (1) | CN107158367A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108379566A (en) * | 2018-02-01 | 2018-08-10 | 郑州大学 | Anti-tumor Human umbilical vein endothelial cells vaccine and its application in anticancer |
| CN109999187A (en) * | 2019-05-09 | 2019-07-12 | 英诺激光科技股份有限公司 | A method of cancer cell vaccine is prepared using laser |
| CN111363009A (en) * | 2020-03-18 | 2020-07-03 | 北京鼎成肽源生物技术有限公司 | Rectal cancer target antigen, CTL cell cultured by rectal cancer target antigen stimulation and application thereof |
-
2017
- 2017-04-18 CN CN201710252773.2A patent/CN107158367A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| LIN LU ET AL.: "Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells", 《ONCOIMMUNOLOGY》 * |
| 黄睿等: "结直肠癌肿瘤干细胞研究进展", 《中华结直肠疾病电子杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108379566A (en) * | 2018-02-01 | 2018-08-10 | 郑州大学 | Anti-tumor Human umbilical vein endothelial cells vaccine and its application in anticancer |
| CN109999187A (en) * | 2019-05-09 | 2019-07-12 | 英诺激光科技股份有限公司 | A method of cancer cell vaccine is prepared using laser |
| CN111363009A (en) * | 2020-03-18 | 2020-07-03 | 北京鼎成肽源生物技术有限公司 | Rectal cancer target antigen, CTL cell cultured by rectal cancer target antigen stimulation and application thereof |
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Application publication date: 20170915 |