CN105158466B - A kind of method of Chimeric antigen receptor T cell for detecting anti-CD19 to leukaemia cyto-inhibition - Google Patents
A kind of method of Chimeric antigen receptor T cell for detecting anti-CD19 to leukaemia cyto-inhibition Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/57426—Specifically defined cancers leukemia
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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Abstract
A kind of method the invention discloses Chimeric antigen receptor T cell for detecting anti-CD19 to leukaemia cyto-inhibition：(1) it is transplanted to after the Chimeric antigen receptor T cell for expressing anti-CD19 is mixed with leukaemia's equal proportion in immunodeficient mouse model, the preserving number CCTCCNO of anti-CD19 Chimeric antigen receptor T cell:C201503；(2) peripheral blood cells of immunodeficient mouse model are dyed；(3) peripheral blood cells after dyeing are detected using flow cytometer.The advantage of the invention is that, human leukemia cell and the cell co-transplantation that can suppress its development are entered in immunodeficient mouse model, the inhibitory action to different human body leukaemia can truly be reflected, can ensure to detect high efficiency, the accuracy of inhibitory action method.
Description
Technical field
It is specifically a kind of to detect the chimeric of anti-CD19 the invention belongs to biology Chimeric antigen receptor T cell technical field
Method of the antigen receptor T cell to leukaemia cyto-inhibition.
Background technology
Chimeric antigen receptor T cell (CAR T cells) is surface expression identification specific antigen and can transmit the chimeric of signal
The T cell of receptor.T cell plays a significant role in antitumor, and CAR T cells are to express the T cell of such molecule：
Extracellular fragment is that heavy chain of antibody is connected with light chain variable district by a peptide fragment, forms single-stranded variable region (ScFv), intracellular section is
The intracellular section chimera of various signal transduction molecules, including CD3zeta, CD28, OX-40,4-1BB etc., transmembrane region then comes from it
The transmembrane region of his molecule (such as CD8, CD4, CD28 and CD3zeta).CAR T cells are by Ag-Ab recognition mode to tumour
The special molecular of cell surface is identified, and then enters line activating, propagation by the signal transduction of its intracellular and plays cell and kill
Hinder function.CAR T cells having a extensive future in immunotherapy of tumors, market value is huge.However, CAR T cells are exempted from
Epidemic disease treats the lagging in development in China in developed country, and clinical practice shortage is strictly regulated.And existing CAR T cells are not
All types of tumours can be treated, novel C AR T cells are the new directions of future tumors immunization therapy.But novel C AR T cells
Security reliability need be verified after just can human body carry out clinical test.At present, verify that CAR T cells are thin to tumour
The killing of born of the same parents is mainly tested by co culture system in vitro, but co culture system in vitro experiment can not truly reflect CAR T cells swollen
The effect of knurl patient's body, and reaction of the different tumor patients to same CAR T cells is also different, therefore each patient is entered
The preclinical test of row individuation can be that the security of CAR T cells treatment and validity provide safeguard.
2013, CAR T cell immunotherapies were listed in the U.S.《Science》Magazine year, ten big sciences broke through the umber one.But should
Immunotherapy security and validity have much room for improvement, expensive plus its medical expense, therefore only rest on clinical test rank at present
Section.Clinical test specific procedure includes:(1) T cell is isolated from peripheral blood in patients；(2) with CD3, CD28 antibody to T cell
Carry out stimulating activation and amplification；(3) retrovirus of the structure comprising Chimeric antigen receptor gene or slow virus；(4) with virus
Transfecting T cells make T cell express Chimeric antigen receptor；(5) by the T cell of expression Chimeric antigen receptor again defeated time sufferers themselves
In vivo；(6) items for monitoring patient prompt the clinical indices of the therapy effect, this authentication directly using human body as object
Method, industrialization can not be realized.
To sum up, verify CAR T cells to following defect be present during the inhibitory action of tumour cell in the prior art：Can not be true
Real reflection CAR T cells can not ensure accuracy, can not realize industrialization for the inhibitory action of tumour cell, these
Defect all governs the development of CAR T cells application.
The content of the invention
, can not for that can not ensure to verify the accuracy that CAR T cells act on inhibiting tumour cells in the prior art
The defects of realizing industrialization, the present invention provide a kind of anti-CD19 Chimeric antigen receptors T cell to leukaemia cyto-inhibition
Method, its realize purpose be obtain it is a kind of it is safe and reliable, accuracy is high, can commercial application verification method.
To achieve the above object, the technical solution adopted by the present invention is that a kind of anti-CD19's of detection disclosed by the invention is embedding
Antigen receptor T cell is closed to the method for leukaemia cyto-inhibition, is comprised the following steps, (1) will express the chimeric of anti-CD19
Antigen receptor T cell is transplanted to after being mixed with leukaemia's equal proportion in immunodeficient mouse model, and anti-CD19's is chimeric
The preserving number CCTCCNO of antigen receptor T cell:C201503, the cyropreservation are (military in China typical culture collection center
The Chinese), address is No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road (the first attached primary school of Wuhan University opposite) in the school, military
Chinese university collection, preservation date is on 2 13rd, 2015, and the collection is on March 12nd, 2015 to the present invention
Anti- CD19 Chimeric antigen receptor T cell detection finish；General cell equal proportion refers to number of cells equal proportion；
(2) peripheral blood cells of immunodeficient mouse model are gathered, with the anti-human CD45 and CD19 antibody pair of fluorescence labeling
Peripheral blood cells dye；
(3) cell after step (2) is dyed is detected using flow cytometer, and the quantity according to leukaemia is
It can detect that anti-CD19 inhibitory action of the Chimeric antigen receptor T cell to leukaemia.The detection method that the present invention designs,
Human leukemia cell is implanted into immunodeficient mouse model, it will be appreciated that the anti-CD19 of individual difference chimeric antigen by
Body T cell the true inhibitory action reflected to human leukaemia cell, can ensure to detect to the inhibitory action of leukaemia
High efficiency, the accuracy of inhibitory action method.
Preferably, immunodeficient mouse model is NOD/SCIDIL2Rg-/- immunodeficient mouse, NOD/
SCIDIL2Rg-/- immunodeficient mouse refers to that NOD/SCID mouse knock out the hyperimmunization obtained after IL-2Rgamma genes and lacked
Swaged mouse, Publication No. 103409468A patent document is specifically referred to, the method for building up of the mouse is also that the applicant opens
Hair design is completed, and can be obtained by commercial system, lack in NOD/SCIDIL2Rg-/- immunodeficient mouse body T, B and
Tri- kinds of NK can grow the cell and tissue of people, by the mouse mould to immunologic function vital cell in the Mice Body
Type, more truly reflect human internal environment, be further ensured that the Chimeric antigen receptor T cell for detecting anti-CD19 to leukaemia
The accuracy of inhibitory action method.
Further, leukaemia is B cell leukemia cell.
Further, marrow, the spleen cell of immunodeficient mouse model can also be gathered in step (2), then with glimmering
The anti-human CD45 and CD19 antibody of signal dyes to marrow, spleen cell.Marrow, spleen cell is taken to ensure that detection is anti-
Accuracy of the CD19 Chimeric antigen receptor T cell to the inhibitory action of leukaemia.
Further, the method for obtaining the Chimeric antigen receptor T cell for expressing anti-CD19 comprises the following steps that (1) synthesizes
The gene of anti-CD19 Chimeric antigen receptor cell, the gene of synthesis are designated as CAR19；
(2) structure of slow virus plasmid：1. the CAR19 gene integrations synthesized are designated as pUC57- in pUC57 plasmids
CAR19 plasmids；2. carrying out double digestion to pWPXLd-EGFP and pUC57-CAR19 with restriction enzyme PmeI and SpeI, then pass through agar
Sugared electrophoresis, glue reclaim kit MAGEN gel extractions extract CAR19 genetic fragments and pWPXLd-EGFP skeleton large fragments；③
CAR19 and pWPXLd-EGFP fragments are connected with Solution I, reaction system is that 5ul Solution I add 4ul
CAR19 and 1ul pWPXLd-EGFP fragments, 16 DEG C of reaction 2h, connection product are transformed into TOP10 competence Escherichia coli；4. will
Competence bacterium is coated on ampicilin agar plates after conversion, and 37 DEG C are incubated overnight；5. second day picking, 4 cells
It is cloned into 2ml ampicillins containing 50ug/ml LB culture mediums, 37 DEG C, 250rpm culture 12h, with the small extraction reagent kit of plasmid
MAGEN extracts the plasmid in 4 pipe bacteriums；6. being identified by PmeI and SpeI double digestions, the plasmid built is designated as
pWPXLd-CAR19-EGFP；7. using the big extraction reagent kit extraction slow virus envelope plasmid pMD2.G of plasmid, packaging plasmid psPAX2
And pWPXLd-CAR19-EGFP plasmids are stand-by；
(3) preparation of CAR19 slow virus：Slow virus is packed by 293T cells, concrete operations are as follows：1. with the high sugar of DMEM
Culture medium adds the dual anti-culture 293T cells of 10%FBS and 1% to 150mm culture dishes, when 293T cell densities reach 80-90%,
Change into after DMEM high glucose mediums add the dual anti-culture 2-6h of 1%FBS and 1%, with PEI by pWPXLd-CAR19-EGFP or control
Tri- plasmid conversion 293T cells of plasmid pWPXLd-EGFP, pMD2.G and psPAX2, each 150mm culture dishes convert 9 μ g's
PWPXLd-CAR19-EGFP or control plasmid pWPXLd-EGFP, 3 μ g pMD2.G and 12 μ g psPAX2, PEI 72ug；②
24h, 48h and 72h after conversion collect supernatant three times, and supernatant is after 2500g is centrifuged, and after being filtered by 0.45 μm of filter, surpass
Supercentrifuge 28000rpm centrifuges 1.5h, and after removing supernatant immediately, each ultracentrifugation pipe adds 200 μ l PBS, is placed in 4 DEG C of mistakes
Night is resuspended, and next day collects the PBS containing slow virus；
(4) T cell isolates and purifies：The mononuclearcell in blood is isolated by Ficol l density gradient methods first,
Go out T cell after erythrocyte cracked liquid cracks and removes red blood cell, then by MACS magnetic bead sortings；
(5) activation and proliferation of T cell：1. coating AntiCD3 McAb OKT-3 and CD28 antibody in advance：With PBS by AntiCD3 McAb and CD28
Antibody is diluted to 1 μ g/ml and 2 μ g/ml, adds in six orifice plates, and per hole 1ml, 37 DEG C of incubators are coated with 2h；2. T cell is with containing
RPMI-1640 culture mediums dual anti-10%FBS and 1% are diluted to 2.5x106Individual/ml, the coating buffer in 6 orifice plates is sopped up, added
Enter every hole 3ml culture medium containing T cell, T cell is 3. placed on 37 DEG C, 5%CO2In incubator, T cell 48h is stimulated；
(6) slow-virus transfection of T cell：Post-stimulatory T cell is resuspended, 300g, 5min centrifugation are changed after removing supernatant
The upper fresh RPMI-1640 culture mediums containing 8 μ g/ml polybrene, expression GFP and CAR19 is separately added into different holes
Slow virus, according to slow virus titre, MOI is set to 10.37 DEG C, 5%CO2After incubator culture 24h, resuspension cell, 300g,
5min centrifugations remove after supernatant to change the fresh IL-2 containing 300IU/ml RPMI-1640 culture mediums, 37 DEG C, 5%CO2Incubator
After cultivating 24h, flow cytometer detection GFP is positive, that is, has obtained the anti-CD19 of expression Chimeric antigen receptor T cell.
Further, can also be by the cell after the Chimeric antigen receptor T cell for obtaining expressing anti-CD19 in step (6)
Amplification：By the obtained anti-CD19 of expression Chimeric antigen receptor T cell with containing the RPMI-1640 that concentration is 300IU/ml IL-2
Culture medium is cultivated, and it is 1-2x10 to express the concentration of anti-CD19 Chimeric antigen receptor T cell in the medium6Individual cell/
Carry out within ml, 2-3 days the amount of a subculture half and change liquid, and T cell is counted, the T after culture can be expanded after 2 weeks is thin
Born of the same parents, through the above method it is amplifiable to the anti-CD19 of expression of acquisition Chimeric antigen receptor T cell 100 times, amplify it is more thin
Born of the same parents can be used for after mixing with leukaemia's equal proportion of different people being transplanted in immunodeficient mouse model, suppress white to it
The effect of blood disease cell is detected.
The Chimeric antigen receptor T cell background that the present invention obtains anti-CD19 using above-mentioned synthetic method is pure, not other
The factor of test effect is influenceed, while also has the advantages that consuming is low, time-consuming short, easy to operate.
To sum up, beneficial effects of the present invention：By means of immunodeficient mouse model, leukaemia and suppress its development
Cell transplantation enter in immunodeficient mouse model, can truly reflect Chimeric antigen receptor T cell to leukaemia
Inhibitory action, can ensure detect inhibitory action method efficient, accuracy；The anti-CD19 of oneself currently preferred synthesis
Chimeric antigen receptor T cell, the structure link of whole cell can be understood, it is ensured that the cell is true, effectively, after not interfering with
The unnecessary gene expression of continuous verification step, so as to further ensure efficient, the high precision of detection method；To sum up, this hair
It is bright to provide good individuation preclinical detection platform for CAR T cell immunization therapies, it is a kind of safe, reliable, efficient
Detection method.
Brief description of the drawings
Fig. 1 is to suppress leukaemia's effect pair using the inventive method CAR19T groups of cells and control group in embodiment one
Compare Test Drawing；
Fig. 2 is CAR19 slow virus carrier collection of illustrative plates in embodiment two；
Fig. 3 is that the T cell of embodiment two transfects the GFP positive rate schematic diagrames after CAR19 slow virus；
Fig. 4 is to suppress leukaemia's effect pair using the inventive method CAR19T groups of cells and control group in embodiment two
Compare Test Drawing.
Embodiment
Present invention is further illustrated with reference to embodiment, it is necessary to illustrate, in the situation of no specified otherwise
Under, reagent of the present invention and operating method are well known to a person skilled in the art general knowledge, and operating condition is normal
Natural conditions under normal temperature and pressure.
Embodiment one：A kind of Chimeric antigen receptor T cell for detecting anti-CD19 disclosed by the invention presses down to leukaemia
The method of making, by taking B cell leukemia cell as an example, detection method comprises the following steps, (1) will express the chimeric of anti-CD19
Antigen receptor T cell is transplanted in immunodeficient mouse model after being mixed with B cell leukemia cell equal proportion：Cell etc. compares
Example mixing is generally referred to as number of cells equal proportion, such as anti-CD19 Chimeric antigen receptor T cell and B cell leukemia cell
It is respectively 5x106Individual cell, the preserving number CCTCCNO of anti-CD19 Chimeric antigen receptor T cell:C201503, the cyropreservation in
China typical culture collection center (Wuhan), address be No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road in the school
(the first attached primary school of Wuhan University opposite), Wuhan University's collection, preservation date is on 2 13rd, 2015, and the collection
On March 12nd, 2015, the Chimeric antigen receptor T cell detection to the anti-CD19 of the present invention finished；Wherein immunodeficient mouse
Model is the commonly used test material in biological field, can be commercially available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, B is thin
Born of the same parents leukaemia can obtain in hospital, institute of oncology's cell bank；
(2) peripheral blood cells of immunodeficient mouse model are gathered, with the anti-human CD45 and CD19 antibody pair of fluorescence labeling
Peripheral blood cells dye；What CD45CD19 was marked is the cell of people, to determine to whether there is people in immunodeficient mouse model
Leukaemia and leukaemia ratio；
(3) cell after step (2) is dyed is detected using flow cytometer：It will typically be set pair in detection
According to group, the cell of step (2) is designated as experimental group, according to the quantity of control group and the B cell leukemia cell of experimental group, you can inspection
Measure effect of the anti-CD19 Chimeric antigen receptor T cell to B cell leukemia cell, for example, control group be only by it is normal, do not have
The T cell and B cell leukemia cell equal proportion for having transformation are implanted into immunodeficient mouse model, then gather immune deficiency
The peripheral blood cells of mouse, with the anti-human CD45 antibody human peripheral blood cell dyeing of fluorescence labeling, this is cellular control unit, such as with
The result of lower experimental group cell and cellular control unit flow cytomery：Its result as shown in figure 1, can be clear and definite from figure,
In experimental group and control group immunodeficient mouse body in peripheral blood (PB) B cell leukemia cell (CD45+CD19+) ratio：
Have 6.35% B cell leukemia cell in the peripheral blood of control group, experimental group 0.153%, can also further using pair
The detection method is verified according to the B cell leukemia cell in group, the marrow of the immunodeficient mouse of experimental group, spleen cell
Accuracy, the result shown in figure be have in the spleen of control group mice 42.1% B cell leukemia cell, experimental group
For 3.75%；There are 68.4% B cell leukemia cell, experimental group 0.653% in the marrow of control group mice.
The detection method enumerated from embodiment, the detection method that the present invention designs, human leukemia cell is implanted into
In immunodeficient mouse model, finally it can detect that anti-CD19 Chimeric antigen receptor T is thin according to the quantity of leukaemia
Born of the same parents are easy, clear to the inhibitory action of leukaemia, method, it will be appreciated that the anti-CD19 of individual difference Chimeric antigen receptor
T cell the true inhibitory action reflected with human leukaemia cell, can ensure detection suppression to the inhibitory action of leukaemia
High efficiency, the accuracy of action method processed, while the detection data obtained according to this detection method are also the research for leukaemia
Substantial amounts of authentic data is provided to support.
Embodiment two：A kind of Chimeric antigen receptor T cell for detecting anti-CD19 disclosed by the invention presses down to leukaemia
The method of making, by taking B cell leukemia cell as an example, detection method comprises the following steps, (1) will express the chimeric of anti-CD19
Antigen receptor T cell is transplanted in immunodeficient mouse model after being mixed with B cell leukemia cell equal proportion：Cell etc. compares
Example mixing be generally referred to as number of cells equal proportion, such as anti-CD19 Chimeric antigen receptor T cell and B cell leukemia it is thin
Born of the same parents are respectively 5x106Individual cell, the preserving number CCTCCNO of anti-CD19 Chimeric antigen receptor T cell:C201503, it is preserved in China
Type Tissue Collection (Wuhan), address are No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road (Wuhan in the school
The first attached primary school of university opposite), Wuhan University's collection, preservation date be on 2 13rd, 2015, and the collection in
On March 12nd, 2015, the Chimeric antigen receptor T cell detection to the anti-CD19 of the present invention finished；Wherein immunodeficient mouse model
It is the commonly used test material in biological field, can be commercially available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, B cell is white
Blood disease cell can obtain in hospital, institute of oncology's cell bank；
(2) peripheral blood cells of immunodeficient mouse model are gathered, in order to ensure the accuracy of testing result, can also be adopted
Collect marrow, the anti-human CD45 and CD19 antibody human peripheral blood cell dyeing of spleen cell fluorescence labeling of immunodeficient mouse；
(3) cell after step (2) is dyed is detected using flow cytometer.
Preferably, immunodeficient mouse model is NOD/SCIDIL2Rg-/- immunodeficient mouse in step (1), NOD/
SCIDIL2Rg-/- immunodeficient mouse refers to that NOD/SCID mouse knock out the hyperimmunization obtained after IL-2Rgamma genes and lacked
Swaged mouse, Publication No. 103409468A patent document is specifically referred to, the method for building up of the mouse is also that the applicant opens
Hair design is completed, and tri- kinds of T, B and NK are lacked in NOD/SCIDIL2Rg-/- immunodeficient mouse body to immunologic function to closing weight
The cell wanted, the cell and tissue of people can be grown in the Mice Body, by the mouse model, truly reflect human body inner ring
Border, it is further ensured that the accuracy for detecting anti-CD19 Chimeric antigen receptor T cell to leukaemia cyto-inhibition method.
Further, the method for obtaining the Chimeric antigen receptor T cell for expressing anti-CD19 comprises the following steps that (1) synthesizes
The gene of anti-CD19 Chimeric antigen receptor cell, the gene of synthesis are designated as CAR19：Anti- CD19 is searched from ncbi database
Chimeric antigen receptor FMC63-28Z receptor protein gene order, its gene order is shown in sequence table, anti-CD19
The Chimeric antigen receptor FMC63-28Z receptor protein head end of gene order add PmeI restriction enzyme sites
Gttaaacg, end add SpeI restriction enzyme site actagt, transfer to Jin Sirui companies to synthesize；
(2) structure of slow virus plasmid, is shown in Fig. 2, and its concrete operations is：1. the CAR19 gene integrations synthesized are in pUC57 matter
In grain, pUC57-CAR19 plasmids are designated as；2. pWPXLd-EGFP and pUC57-CAR19 is carried out with restriction enzyme PmeI and SpeI double
Digestion, then CAR19 genetic fragments and pWPXLd- are extracted by agarose electrophoresis, glue reclaim kit MAGEN gel extractions
EGFP skeleton large fragments；3. CAR19 and pWPXLd-EGFP fragments are connected with Solut ion I, reaction system 5ul
Solution I add 4ul CAR19 and 1ul pWPXLd-EGFP fragments, 16 DEG C of reaction 2h, and connection product is transformed into TOP10 impressions
State Escherichia coli；4. competence bacterium after conversion is coated on ampicilin agar plates, 37 DEG C are incubated overnight；5. second
4 cell clones of its picking are into 2ml ampicillins containing 50ug/ml LB culture mediums, 37 DEG C, 250rpm culture 12h, use plasmid
Small extraction reagent kit MAGEN extracts the plasmid in 4 pipe bacteriums；6. identified by PmeI and SpeI double digestions, the matter built
Grain is designated as pWPXLd-CAR19-EGFP；7. using the big extraction reagent kit extraction slow virus envelope plasmid pMD2.G of plasmid, packaging plasmid
PsPAX2 and pWPXLd-CAR19-EGFP plasmids are stand-by；
(3) preparation of CAR19 slow virus：Slow virus is packed by 293T cells, concrete operations are as follows：1. with the high sugar of DMEM
Culture medium adds the dual anti-culture 293T cells of 10%FBS and 1% to 150mm culture dishes, when 293T cell densities reach 80-90%,
Change into after DMEM high glucose mediums add the dual anti-culture 2-6h of 1%FBS and 1%, with PEI by pWPXLd-CAR19-EGFP or control
Tri- plasmid conversion 293T cells of plasmid pWPXLd-EGFP, pMD2.G and psPAX2, each 150mm culture dishes convert 9 μ g's
PWPXLd-CAR19-EGFP or control plasmid pWPXLd-EGFP, 3 μ g pMD2.G and 12 μ g psPAX2, PEI 72ug；②
24h, 48h and 72h after conversion collect supernatant three times, and supernatant is after 2500g is centrifuged, and after being filtered by 0.45 μm of filter, surpass
Supercentrifuge 28000rpm centrifuges 1.5h, and after removing supernatant immediately, each ultracentrifugation pipe adds 200 μ l PBS, is placed in 4 DEG C of mistakes
Night is resuspended, and next day collects the PBS containing slow virus；
(4) T cell isolates and purifies：The mononuclearcell in blood is isolated by Ficoll density gradient methods first,
T cell is sub-elected after erythrocyte cracked liquid cracks and removes red blood cell, then by MACS magnetic beads (U.S. day Ni)；
(5) activation and proliferation of T cell：1. coating AntiCD3 McAb OKT-3 and CD28 antibody in advance：With PBS by AntiCD3 McAb and CD28
Antibody is diluted to 1 μ g/ml and 2 μ g/ml, adds in six orifice plates, and per hole 1ml, 37 DEG C of incubators are coated with 2h；2. T cell is with containing
RPMI-1640 culture mediums dual anti-10%FBS and 1% are diluted to 2.5x106Individual/ml, the coating buffer in 6 orifice plates is sopped up, added
Enter every hole 3ml culture medium containing T cell, T cell is 3. placed on 37 DEG C, 5%CO2In incubator, T cell 48h is stimulated；
(6) slow-virus transfection of T cell：Post-stimulatory T cell is resuspended, 300g, 5min centrifugation are changed after removing supernatant
The upper fresh RPMI-1640 culture mediums containing 8 μ g/ml polybrene, expression GFP and CAR19 is separately added into different holes
Slow virus, according to slow virus titre, MOI is set to 10.37 DEG C, 5%CO2After incubator culture 24h, resuspension cell, 300g,
5min centrifugations remove after supernatant to change the fresh RPMI-1640 culture mediums contained, 37 DEG C, 5%CO2After incubator culture 24h, streaming
Detection GFP is positive, that is, has obtained the anti-CD19 of expression Chimeric antigen receptor T cell, seen Fig. 3, the T of untransfected slow virus
Cell (WT) GFP is feminine gender, and the T cell for having transfected CAR19 slow virus then has 22.6% cell positive for GFP.
After the Chimeric antigen receptor T cell for obtaining expressing anti-CD19 in step (6), the cell can also be expanded：Will
To the anti-CD19 of expression Chimeric antigen receptor T cell carried out with containing the RPMI-1640 culture mediums that concentration is 300IU/ml IL-2
Culture, it is 1-2x106 cell/ml to express the concentration of anti-CD19 Chimeric antigen receptor T cell in the medium, is entered within 2-3 days
The subculture of row one half, which is measured, changes liquid, and T cell is counted, the T cell after being expanded after cultivating 2 weeks.
Using the above method, anti-CD19 Chimeric antigen receptor T cell is detected to leukaemia cyto-inhibition, is being detected
When control group will be typically set, the cell of step (2) is designated as experimental group, according to control group and the B cell leukemia of experimental group
The quantity of cell, you can effect of the anti-CD19 Chimeric antigen receptor T cell to B cell leukemia cell is detected, for example, right
According to group be only by it is normal, without transform T cell and B cell leukemia cell equal proportion be implanted into immunodeficient mouse model
In, the peripheral blood cells of immunodeficient mouse are then gathered, are contaminated with the anti-human CD45 antibody human peripheral blood cell of fluorescence labeling
Color, this is cellular control unit, such as the result of following experimental group cell and cellular control unit flow cytomery：As shown in figure 4,
The ratio of B cell leukemia cell is that 2.42%, CAR19T cells treatment group is 0.301% in control group peripheral blood, control group
B cell leukemia cell proportion in spleen and marrow is respectively 25.5% and 48.8%, and the ratio of CAR19T cell treatment groups
Example is respectively 5.11% and 3.67%.
It is described above, not present invention is imposed any restrictions, it is every according to repairing in present invention technical spirit
Change, in the protection domain for still falling within present invention technical scheme.
Claims (3)
1. a kind of Chimeric antigen receptor T cell for detecting anti-CD19 is to the method for leukaemia cyto-inhibition, it is characterised in that
This method comprises the following steps, after (1) mixes the Chimeric antigen receptor T cell for expressing anti-CD19 with leukaemia's equal proportion
It is transplanted in immunodeficient mouse model, the preserving number CCTCCNO of anti-CD19 Chimeric antigen receptor T cell:C201503；
(2) peripheral blood cells of immunodeficient mouse model are gathered, with the anti-human CD45 and CD19 antibody of fluorescence labeling to periphery
Blood cell staining；
(3) cell after step (2) is dyed is detected using flow cytometer, can be examined according to the quantity of leukaemia
Measure anti-CD19 inhibitory action of the Chimeric antigen receptor T cell to leukaemia；
The leukaemia is B cell leukemia cell, and B cell leukemia cell is the leukaemia of different people, and/or
B cell leukemia obtains in hospital, institute of oncology's cell bank；
The immunodeficient mouse model is NOD/SCIDIL2Rg-/- immunodeficient mouse；
The method for obtaining the Chimeric antigen receptor T cell for expressing anti-CD19 comprises the following steps,
A, the gene of anti-CD19 Chimeric antigen receptor cell is synthesized, the gene of synthesis is designated as CAR19；
B, the structure of slow virus plasmid：1. the CAR19 gene integrations synthesized are designated as pUC57-CAR19 matter in pUC57 plasmids
Grain；2. carry out double digestion to pWPXLd-EGFP and pUC57-CAR19 with restriction enzyme PmeI and SpeI, then by agarose electrophoresis,
Glue reclaim kit MAGEN gel extractions extract CAR19 genetic fragments and pWPXLd-EGFP skeleton large fragments；3. use
SolutionI connects CAR19 and pWPXLd-EGFP fragments, and reaction system is that 5ul Solution I add 4ul CAR19
With 1ul pWPXLd-EGFP fragments, 16 DEG C of reaction 2h, connection product is transformed into TOP10 competence Escherichia coli；4. after converting
Competence bacterium is coated on ampicilin agar plates, and 37 DEG C are incubated overnight；5. second day picking, 4 cell clones are extremely
In 2ml ampicillins containing 50ug/ml LB culture mediums, 37 DEG C, 250rpm culture 12h, extracted with the small extraction reagent kit MAGEN of plasmid
Plasmid in 4 pipe bacteriums；6. being identified by PmeI and SpeI double digestions, the plasmid built is designated as pWPXLd-CAR19-
EGFP；7. using the big extraction reagent kit extraction slow virus envelope plasmid pMD2.G of plasmid, packaging plasmid psPAX2 and pWPXLd-
CAR19-EGFP plasmids are stand-by；
C, the preparation of CAR19 slow virus：Slow virus is packed by 293T cells, concrete operations are as follows：1. with the high sugar cultures of DMEM
Base adds the dual anti-culture 293T cells of 10%FBS and 1% when 293T cell densities are up to 80~90%, to be changed into 150mm culture dishes
After DMEM high glucose mediums add 2~6h of the dual anti-cultures of 1%FBS and 1%, with PEI by pWPXLd-CAR19-EGFP or control plasmid
Tri- plasmid conversion 293T cells of pWPXLd-EGFP, pMD2.G and psPAX2, each 150mm culture dishes convert 9 μ g's
PWPXLd-CAR19-EGFP or control plasmid pWPXLd-EGFP, 3 μ g pMD2.G and 12 μ g psPAX2, PEI 72ug；②
24h, 48h and 72h after conversion collect supernatant three times, and supernatant is after 2500g is centrifuged, and after being filtered by 0.45 μm of filter, surpass
Supercentrifuge 28000rpm centrifuges 1.5h, and after removing supernatant immediately, each ultracentrifugation pipe adds 200 μ l PBS, is placed in 4 DEG C of mistakes
Night is resuspended, and next day collects the PBS containing slow virus；
D, T cell isolates and purifies：The mononuclearcell in blood is isolated by Ficoll density gradient methods first, through red thin
After the cracking of cellular lysate liquid removes red blood cell, then T cell gone out by MACS magnetic bead sortings；E, the activation and proliferation of T cell：1. bag in advance
By AntiCD3 McAb OKT-3 and CD28 antibody：AntiCD3 McAb and CD28 antibody are diluted to 1 μ g/ml and 2 μ g/ml with PBS, add six orifice plates
In, per hole 1ml, 37 DEG C of incubators are coated with 2h；2. T cell is diluted with containing 10%FBS and 1% dual anti-RPMI-1640 culture mediums
To 2.5x106Individual/ml, the coating buffer in 6 orifice plates is sopped up, add the culture medium containing T cell per hole 3ml, be 3. placed on T cell
37 DEG C, 5%CO2In incubator, T cell 48h is stimulated；
F, the slow-virus transfection of T cell：Post-stimulatory T cell is resuspended, 300g, 5min centrifugation are gone after supernatant to change fresh
The RPMI-1640 culture mediums containing 8 μ g/ml polybrene, be separately added into different holes expression GFP and CAR19 slow disease
Poison, according to slow virus titre, MOI is set to 10,37 DEG C, 5%CO2After incubator culture 24h, be resuspended cell, 300g, 5min from
The heart go after supernatant to change it is fresh containing the RPMI-1640 culture mediums that concentration is 300IU/ml IL-2,37 DEG C, 5%CO2Incubator
After cultivating 24h, flow cytometer detection GFP is positive, that is, has obtained the anti-CD19 of expression Chimeric antigen receptor T cell.
2. anti-CD19 side of the Chimeric antigen receptor T cell to leukaemia cyto-inhibition is detected according to claim 1
Method, it is characterised in that marrow, the spleen cell of immunodeficient mouse model can also be gathered in step (2), then with fluorescence mark
The anti-human CD45 and CD19 antibody of note dyes to marrow, spleen cell.
3. the anti-CD19 of detection according to claim 1 Chimeric antigen receptor T cell is to leukaemia cyto-inhibition
Method, it is characterised in that, can also be by the cell after the Chimeric antigen receptor T cell for obtaining expressing anti-CD19 in the step f
Amplification：By the obtained anti-CD19 of expression Chimeric antigen receptor T cell with containing the RPMI-1640 that concentration is 300IU/ml IL-2
Culture medium is cultivated, and it is 1-2x10 to express the concentration of anti-CD19 Chimeric antigen receptor T cell in the medium6Individual cell/
Ml, carry out within 2~3 days the amount of a subculture half and change liquid, and T cell is counted, the T after culture can be expanded after 2 weeks is thin
Born of the same parents.
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