CN105158466A - Method for detecting anti-CD19 chimeric antigen receptor T cell effects of inhibiting leukemia cells - Google Patents
Method for detecting anti-CD19 chimeric antigen receptor T cell effects of inhibiting leukemia cells Download PDFInfo
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Abstract
The invention discloses a method for detecting anti-CD19 chimeric antigen receptor T cell effects of inhibiting leukemia cells. The method comprises 1, mixing anti-CD19 chimeric antigen receptor T cells and leukemia cells according to a certain ratio, and transplanting the mixed cells into an immunodeficient mouse model, wherein the anti-CD19 chimeric antigen receptor T cell has an accession number of CCTCC NO: C201503, 2, carrying out immunodeficient mouse model peripheral blood cell dyeing, and 3, detecting the dyed peripheral blood cells by a flow cytometer. Human leukemia cells and leukemia cell inhibition cells are transplanted into an immunodeficient mouse model together so that effects of inhibiting different human leukemia cells can be really shown and high effectiveness and accuracy of the inhibition effect detection method are guaranteed.
Description
Technical field
The invention belongs to biology Chimeric antigen receptor T cell technical field, specifically a kind of Chimeric antigen receptor T cell detecting anti-CD19 is to the method for leukaemia cyto-inhibition.
Background technology
Chimeric antigen receptor T cell (CART cell) is surface expression identification specific antigen and the T cell of the mosaic type acceptor of energy transmission of signal.T cell plays a significant role in antitumor, CART cell is the T cell expressing such molecule: extracellular fragment is that heavy chain of antibody is connected by a peptide section with variable region of light chain, form single-chain variable (ScFv), born of the same parents' inner segment is born of the same parents' inner segment chimera of various signal transduction molecule, comprise CD3zeta, CD28, OX-40,4-1BB etc., cross-film district is then from the cross-film district of other molecules (as CD8, CD4, CD28 and CD3zeta).CART cell is identified by the special molecular of Ag-Ab recognition mode to tumor cell surface, is then undertaken activating, breeding and play cell killing function by the intracellular signaling in its born of the same parents.CART cell having a extensive future in immunotherapy of tumors, marketable value is huge.But CART cellular immunotherapy is at the lagging in development of China in developed country, and clinical practice lacks and strictly regulates.And existing CART cell can not treat all types of tumour, novel C ART cell is the new direction of future tumors immunization therapy.But the safe and reliable sexual needs of novel C ART cell are verified just can carry out clinical testing at human body afterwards.At present, killing and wounding of checking CART cells against tumor cells is mainly tested by co culture system in vitro, but co culture system in vitro experiment truly can not reflect the effect of CART cell in tumor patient body, and the reaction of different tumor patient to same CART cell is also different, the preclinical test therefore carrying out individuation to each patient can be the security of CART cell therapy and validity provides safeguard.
2013, CART cellular immunotherapy was listed in the U.S.'s " science " magazine year ten big science breakthrough umber one.But this immunotherapy security and validity have much room for improvement, add that its medical expense is expensive, therefore only rest on clinical experimental stage at present.Clinical testing specific procedure comprises: (1) isolates T cell from peripheral blood in patients; (2) with CD3, CD28 antibody, stimulation activation and amplification are carried out to T cell; (3) retrovirus or the slow virus that comprise Chimeric antigen receptor gene is built; (4) T cell is made to express Chimeric antigen receptor by virus transfection T cell; (5) by the T cell of expression Chimeric antigen receptor again defeated time sufferers themselves's body; (6) monitor the clinical indices of this therapy effect of every prompting of patient, this is directly the verification method of object with human body, can not realize industrialization.
To sum up, following defect is there is: the inhibiting effect of CART cell for tumour cell can not be reflected really when verifying the inhibiting effect of CART cells against tumor cells in prior art, cannot ensure accuracy, can not realize industrialization, these defects all govern the development of CART cell application.
Summary of the invention
For ensureing the inhibiting accuracy of checking CART cells against tumor cells in prior art, the defect of industrialization can not be realized, the invention provides a kind of anti-CD19 Chimeric antigen receptor T cell to the method for leukaemia cyto-inhibition, its object realized is, obtain a kind of safe, reliable, degree of accuracy is high, can the verification method of commercial application.
For achieving the above object, the technical solution used in the present invention is, a kind of Chimeric antigen receptor T cell detecting anti-CD19 disclosed by the invention is to the method for leukaemia cyto-inhibition, comprise the steps, (1) be transplanted in immunodeficient mouse model after the Chimeric antigen receptor T cell expressing anti-CD19 being mixed with leukaemia's equal proportion, the preserving number CCTCCNO:C201503 of the Chimeric antigen receptor T cell of anti-CD19, this cyropreservation is in China typical culture collection center (Wuhan), address is Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys (attached primary school of Wuhan University first opposites) in the school, Wuhan University's preservation center, preservation date is on February 13rd, 2015, and this preservation center is detected complete on March 12nd, 2015 to the Chimeric antigen receptor T cell of anti-CD19 of the present invention, general cell equal proportion refers to number of cells equal proportion,
(2) peripheral blood cells of immunodeficient mouse model is gathered, with fluorescently-labeled anti-human CD45 and CD19 antibody human peripheral blood cell dyeing;
(3) cell after step (2) being dyeed adopts flow cytometer to detect, and can detect that the Chimeric antigen receptor T cell of anti-CD19 is to the inhibiting effect of leukaemia according to the quantity of leukaemia.The detection method of the present invention's design, human leukemia cell is implanted in immunodeficient mouse model, the Chimeric antigen receptor T cell of the anti-CD19 of individual difference can be understood to the inhibiting effect of leukaemia, true reflection, to the inhibiting effect of human leukaemia cell, can ensure the high efficiency, the accuracy that detect inhibiting effect method.
As preferably, immunodeficient mouse model is NOD/SCIDIL2Rg-/-immunodeficient mouse, NOD/SCIDIL2Rg-/-immunodeficient mouse refers to the hyperimmunization deficient mice obtained after NOD/SCID mouse knocks out IL-2Rgamma gene, specifically refer to the patent document that publication number is 103409468A, the method for building up of this mouse is also that the applicant's development and Design completes, can be obtained by commercial system, T is lacked in NOD/SCIDIL2Rg-/-immunodeficient mouse body, B and NK tri-kinds is to the vital cell of immunologic function, the biological cells and tissues of people can be grown in this Mice Body, by this mouse model, reflect human internal environment more truly, further guarantee detects the Chimeric antigen receptor T cell of anti-CD19 to the accuracy of leukaemia cyto-inhibition method.
Further, leukaemia is B cell leukemia cell.
Further, marrow, the spleen cell of immunodeficient mouse model in step (2), can also be gathered, then with fluorescently-labeled anti-human CD45 and CD19 antibody, marrow, spleen cell be dyeed.Take marrow, spleen cell can ensure to detect the inhibiting accuracy of Chimeric antigen receptor T cell to leukaemia of anti-CD19.
Further, the method obtaining the Chimeric antigen receptor T cell expressing anti-CD19 comprises the steps, (1) synthesizes the gene of the Chimeric antigen receptor cell of anti-CD19, and the gene of synthesis is designated as CAR19;
(2) structure of slow virus plasmid: the CAR19 gene integration 1. synthesized, in pUC57 plasmid, is designated as pUC57-CAR19 plasmid; 2. with restriction enzyme PmeI and SpeI, double digestion is carried out to pWPXLd-EGFP and pUC57-CAR19, then cut glue recovery extract CAR19 genetic fragment and pWPXLd-EGFP skeleton large fragment by agarose electrophoresis, glue recovery kit MAGEN; 3. CAR19 and pWPXLd-EGFP fragment coupled together with SolutionI, reaction system is that 5ulSolutionI adds 4ulCAR19 and 1ulpWPXLd-EGFP fragment, and 16 DEG C of reaction 2h, connect product conversion to TOP10 competence Escherichia coli; 4. competence bacterium after conversion is coated on ampicilin agar plates, 37 DEG C of incubated overnight; 5. second day picking, 4 cell clones are to 2ml containing in 50ug/ml ampicillin LB nutrient culture media, and 37 DEG C, 250rpm cultivates 12h, extracts the plasmid in 4 pipe bacteriums with the little extraction reagent kit MAGEN of plasmid; 6. identified by PmeI and SpeI double digestion, the plasmid built is designated as pWPXLd-CAR19-EGFP; 7. adopt the large extraction reagent kit of plasmid to extract slow virus envelope plasmid pMD2.G, packaging plasmid psPAX2 and pWPXLd-CAR19-EGFP plasmid is stand-by;
(3) preparation of CAR19 slow virus: by 293T cell packaging slow virus, concrete operations are as follows: 1. add 10%FBS and 1% dual anti-cultivation 293T cell to 150mm double dish with DMEM high glucose medium, when 293T cell density reaches 80-90%, change into after DMEM high glucose medium adds 1%FBS and 1% dual anti-cultivation 2-6h, with PEI by pWPXLd-CAR19-EGFP or control plasmid pWPXLd-EGFP, pMD2.G and psPAX2 tri-Plastid transformation 293T cells, each 150mm double dish transforms pWPXLd-CAR19-EGFP or the control plasmid pWPXLd-EGFP of 9 μ g, the pMD2.G of 3 μ g and the psPAX2 of 12 μ g, PEI is 72ug, 2. 24h, 48h and 72h after transforming collect three supernatants, supernatant is after 2500g is centrifugal, after 0.45 μm of metre filter, the centrifugal 1.5h of Ultracentrifuge 28000rpm, after removing supernatant immediately, each ultracentrifugation pipe adds 200 μ lPBS, is placed in 4 DEG C and spends the night resuspended, and the PBS containing slow virus collects by next day,
(4) separation and purification of T cell: first isolated the mononuclearcell in blood by Ficoll density gradient method, is removed after red blood cell through erythrocyte cracked liquid cracking, then goes out T cell by MACS magnetic bead sorting;
(5) activation and proliferation of T cell: 1. wrap by AntiCD3 McAb OKT-3 and CD28 antibody in advance: with PBS, AntiCD3 McAb is become 1 μ g/ml and 2 μ g/ml with CD28 antibody dilution, add in six orifice plates, every hole 1ml, cultivates case and bag by 2h for 37 DEG C; 2. T cell is diluted to 2.5x10 with containing 10%FBS and 1% dual anti-RPMI-1640 nutrient culture media
6individual/ml, sops up the coating buffer in 6 orifice plates, and what add every hole 3ml contains T cell nutrient culture media, 3. T cell is placed on 37 DEG C, 5%CO
2in incubator, stimulate T cell 48h;
(6) slow-virus transfection of T cell: by resuspended for post-stimulatory T cell, 300g, 5min is centrifugal remove supernatant after change the fresh RPMI-1640 nutrient culture media containing 8 μ g/mlpolybrene, the slow virus of expressing GFP and CAR19 is added respectively in different hole, according to slow virus titre, MOI is set to 10.37 DEG C, 5%CO
2incubator is cultivated after 24h, re-suspended cell, 300g, 5min are centrifugal remove supernatant after change the fresh RPMI-1640 nutrient culture media containing 300IU/mlIL-2,37 DEG C, 5%CO
2after 24h cultivated by incubator, flow cytometer detection GFP is positive, and namely obtains the Chimeric antigen receptor T cell expressing anti-CD19.
Further, after obtaining expressing the Chimeric antigen receptor T cell of anti-CD19 in step (6), this cell can also be increased: cultivated by the Chimeric antigen receptor T cell of anti-for the expression the obtained CD19 RPMI-1640 nutrient culture media containing concentration being 300IU/mlIL-2, the Chimeric antigen receptor T cell concentration in the medium expressing anti-CD19 is 1-2x10
6individual cell/ml, within 2-3 days, carry out a subculture half amount and change liquid, and T cell is counted, the T cell after amplification can be obtained after cultivating 2 weeks, 100 times can be increased through the Chimeric antigen receptor T cell of said method to the anti-CD19 of expression obtained, be transplanted in immunodeficient mouse model after the more many cells amplified can be used for mixing with leukaemia's equal proportion of different people, suppress the effect of leukaemia to detect to it.
It is pure that the present invention adopts above-mentioned synthetic method to obtain the Chimeric antigen receptor T cell background of anti-CD19, do not have other to affect the factor of test effect, also has simultaneously and expend the advantage such as short, easy and simple to handle low, consuming time.
To sum up, beneficial effect of the present invention: by means of immunodeficient mouse model, leukaemia and the cell transplantation that suppresses it to develop are entered in immunodeficient mouse model, the inhibiting effect of Chimeric antigen receptor T cell to leukaemia can be reflected truly, efficient, the accuracy that detect inhibiting effect method can be ensured; The Chimeric antigen receptor T cell of the anti-CD19 of the present invention's oneself synthesis preferred, the structure link of whole cell can be understood, guarantee that this cell is true, effectively, the unnecessary gene expression of subsequent verifying step can not be affected, thus further ensure efficient, the high precision of detection method; To sum up, the present invention can be the clinical front detection platform of individuation that CART cellular immunotherapy provides good, is a kind of safe, reliable, efficient detection method.
Accompanying drawing explanation
Fig. 1 adopts the inventive method CAR19T groups of cells and control group to suppress leukaemia's Piglet s colibacillosis figure in embodiment one;
Fig. 2 is CAR19 slow virus carrier collection of illustrative plates in embodiment two;
Fig. 3 is the GFP positive rate schematic diagram after embodiment two T cell transfection CAR19 slow virus;
Fig. 4 adopts the inventive method CAR19T groups of cells and control group to suppress leukaemia's Piglet s colibacillosis figure in embodiment two.
Embodiment
Content of the present invention is further illustrated below in conjunction with embodiment, it should be noted that, when not having specified otherwise, reagent of the present invention and method of operating are all well known to a person skilled in the art general knowledge, and operating conditions is all the natural conditions under normal temperature and pressure.
Embodiment one: a kind of Chimeric antigen receptor T cell detecting anti-CD19 disclosed by the invention is to the method for leukaemia cyto-inhibition, for B cell leukemia cell, detection method comprises the steps, (1) be transplanted in immunodeficient mouse model by expressing after the Chimeric antigen receptor T cell of anti-CD19 mixes with B cell leukemia cell equal proportion: cell equal proportion mixes and generally refers to number of cells equal proportion, and the Chimeric antigen receptor T cell of such as anti-CD19 and B cell leukemia cell are respectively 5x10
6individual cell, the preserving number CCTCCNO:C201503 of the Chimeric antigen receptor T cell of anti-CD19, this cyropreservation is in China typical culture collection center (Wuhan), address is Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys (attached primary school of Wuhan University first opposites) in the school, Wuhan University's preservation center, preservation date is on February 13rd, 2015, and this preservation center is detected complete on March 12nd, 2015 to the Chimeric antigen receptor T cell of anti-CD19 of the present invention; Wherein immunodeficient mouse model is the test material commonly used in biological field, and can buy from Chinese Academy of Sciences's Shanghai Experimental Animal Center and obtain, B cell leukemia cell can obtain from hospital, institute of oncology's cell bank;
(2) peripheral blood cells of immunodeficient mouse model is gathered, with fluorescently-labeled anti-human CD45 and CD19 antibody human peripheral blood cell dyeing; CD45CD19 mark be the cell of people, in order to determine whether there is the leukaemia of people and the ratio of leukaemia in immunodeficient mouse model;
(3) cell after step (2) being dyeed adopts flow cytometer to detect: generally all will arrange control group when detecting, the cell of step (2) is designated as experimental group, according to the quantity of the B cell leukemia cell of control group and experimental group, can detect that the Chimeric antigen receptor T cell of anti-CD19 is to the effect of B cell leukemia cell, such as, control group is only by normally, the T cell of transformation and B cell leukemia cell equal proportion is not had to be implanted in immunodeficient mouse model, then the peripheral blood cells of immunodeficient mouse is gathered, with fluorescently-labeled anti-human CD45 antibody human peripheral blood cell dyeing, this is cellular control unit, result as following experimental group cell and cellular control unit flow cytomery: its result as shown in Figure 1, can be clear and definite from figure, the ratio of the middle B cell leukemia cell (CD45+CD19+) of experimental group and control group immunodeficient mouse inside and outside week blood (PB): have the B cell leukemia cell of 6.35% in the peripheral blood of control group, experimental group is 0.153%, control group can also be adopted further, the marrow of the immunodeficient mouse of experimental group, B cell leukemia cell in spleen cell verifies the accuracy of this detection method, the result demonstrated in figure is, the B cell leukemia cell of 42.1% is had in the spleen of control group mice, experimental group is 3.75%, have the B cell leukemia cell of 68.4% in the marrow of control group mice, experimental group is 0.653%.
The detection method enumerated from embodiment, the detection method of the present invention's design, human leukemia cell is implanted in immunodeficient mouse model, finally can detect that the Chimeric antigen receptor T cell of anti-CD19 is to the inhibiting effect of leukaemia according to the quantity of leukaemia, method is easy, understand, the Chimeric antigen receptor T cell of the anti-CD19 of individual difference can be understood to the inhibiting effect of leukaemia, true reflection is with the inhibiting effect of human leukaemia cell, the high efficiency detecting inhibiting effect method can be ensured, accuracy, the detection data simultaneously obtained according to this detection method are also for leukemic research provides a large amount of authentic data supports.
Embodiment two: a kind of Chimeric antigen receptor T cell detecting anti-CD19 disclosed by the invention is to the method for leukaemia cyto-inhibition, for B cell leukemia cell, detection method comprises the steps, (1) be transplanted in immunodeficient mouse model by expressing after the Chimeric antigen receptor T cell of anti-CD19 mixes with B cell leukemia cell equal proportion: cell equal proportion mixes and generally refers to number of cells equal proportion, and the Chimeric antigen receptor T cell of such as anti-CD19 and B cell leukemia cell are respectively 5x10
6individual cell, the preserving number CCTCCNO:C201503 of the Chimeric antigen receptor T cell of anti-CD19, be preserved in China typical culture collection center (Wuhan), address is Wuchang District, Wuhan City, Hubei Province Bayi Road No. 299 Wuhan Universitys (attached primary school of Wuhan University first opposites) in the school, Wuhan University's preservation center, preservation date is on February 13rd, 2015, and this preservation center is detected complete on March 12nd, 2015 to the Chimeric antigen receptor T cell of anti-CD19 of the present invention; Wherein immunodeficient mouse model is the test material commonly used in biological field, and can buy from Chinese Academy of Sciences's Shanghai Experimental Animal Center and obtain, B cell leukemia cell can obtain from hospital, institute of oncology's cell bank;
(2) gathering the peripheral blood cells of immunodeficient mouse model, in order to ensure the accuracy of testing result, the marrow of immunodeficient mouse, fluorescently-labeled anti-human CD45 and the CD19 antibody human peripheral blood cell dyeing of spleen cell can also be gathered;
(3) cell after step (2) being dyeed adopts flow cytometer to detect.
As preferred in step (1), immunodeficient mouse model is NOD/SCIDIL2Rg-/-immunodeficient mouse, NOD/SCIDIL2Rg-/-immunodeficient mouse refers to the hyperimmunization deficient mice obtained after NOD/SCID mouse knocks out IL-2Rgamma gene, specifically refer to the patent document that publication number is 103409468A, the method for building up of this mouse is also that the applicant's development and Design completes, T is lacked in NOD/SCIDIL2Rg-/-immunodeficient mouse body, B and NK tri-kinds is to the vital cell of immunologic function, the biological cells and tissues of people can be grown in this Mice Body, by this mouse model, reflect human internal environment truly, further guarantee detects the Chimeric antigen receptor T cell of anti-CD19 to the accuracy of leukaemia cyto-inhibition method.
Further, the method obtaining the Chimeric antigen receptor T cell expressing anti-CD19 comprises the steps, (1) gene of the Chimeric antigen receptor cell of anti-CD19 is synthesized, the gene of synthesis is designated as CAR19: the gene order searching the Chimeric antigen receptor FMC63-28Zreceptorprotein of anti-CD19 from ncbi database, its gene order is shown in sequence table, the head end of the gene order of the Chimeric antigen receptor FMC63-28Zreceptorprotein of anti-CD19 adds PmeI restriction enzyme site gttaaacg, end adds SpeI restriction enzyme site actagt, Jin Sirui company is transferred to synthesize,
(2) structure of slow virus plasmid, is shown in Fig. 2, and its concrete operations are: the CAR19 gene integration 1. synthesized, in pUC57 plasmid, is designated as pUC57-CAR19 plasmid; 2. with restriction enzyme PmeI and SpeI, double digestion is carried out to pWPXLd-EGFP and pUC57-CAR19, then cut glue recovery extract CAR19 genetic fragment and pWPXLd-EGFP skeleton large fragment by agarose electrophoresis, glue recovery kit MAGEN; 3. CAR19 and pWPXLd-EGFP fragment coupled together with SolutionI, reaction system is that 5ulSolutionI adds 4ulCAR19 and 1ulpWPXLd-EGFP fragment, and 16 DEG C of reaction 2h, connect product conversion to TOP10 competence Escherichia coli; 4. competence bacterium after conversion is coated on ampicilin agar plates, 37 DEG C of incubated overnight; 5. second day picking, 4 cell clones are to 2ml containing in 50ug/ml ampicillin LB nutrient culture media, and 37 DEG C, 250rpm cultivates 12h, extracts the plasmid in 4 pipe bacteriums with the little extraction reagent kit MAGEN of plasmid; 6. identified by PmeI and SpeI double digestion, the plasmid built is designated as pWPXLd-CAR19-EGFP; 7. adopt the large extraction reagent kit of plasmid to extract slow virus envelope plasmid pMD2.G, packaging plasmid psPAX2 and pWPXLd-CAR19-EGFP plasmid is stand-by;
(3) preparation of CAR19 slow virus: by 293T cell packaging slow virus, concrete operations are as follows: 1. add 10%FBS and 1% dual anti-cultivation 293T cell to 150mm double dish with DMEM high glucose medium, when 293T cell density reaches 80-90%, change into after DMEM high glucose medium adds 1%FBS and 1% dual anti-cultivation 2-6h, with PEI by pWPXLd-CAR19-EGFP or control plasmid pWPXLd-EGFP, pMD2.G and psPAX2 tri-Plastid transformation 293T cells, each 150mm double dish transforms pWPXLd-CAR19-EGFP or the control plasmid pWPXLd-EGFP of 9 μ g, the pMD2.G of 3 μ g and the psPAX2 of 12 μ g, PEI is 72ug, 2. 24h, 48h and 72h after transforming collect three supernatants, supernatant is after 2500g is centrifugal, after 0.45 μm of metre filter, the centrifugal 1.5h of Ultracentrifuge 28000rpm, after removing supernatant immediately, each ultracentrifugation pipe adds 200 μ lPBS, is placed in 4 DEG C and spends the night resuspended, and the PBS containing slow virus collects by next day,
(4) separation and purification of T cell: first isolated the mononuclearcell in blood by Ficoll density gradient method, is removed after red blood cell through erythrocyte cracked liquid cracking, then sub-elects T cell by MACS magnetic bead (U.S. sky Ni);
(5) activation and proliferation of T cell: 1. wrap by AntiCD3 McAb OKT-3 and CD28 antibody in advance: with PBS, AntiCD3 McAb is become 1 μ g/ml and 2 μ g/ml with CD28 antibody dilution, add in six orifice plates, every hole 1ml, cultivates case and bag by 2h for 37 DEG C; 2. T cell is diluted to 2.5x10 with containing 10%FBS and 1% dual anti-RPMI-1640 nutrient culture media
6individual/ml, sops up the coating buffer in 6 orifice plates, and what add every hole 3ml contains T cell nutrient culture media, 3. T cell is placed on 37 DEG C, 5%CO
2in incubator, stimulate T cell 48h;
(6) slow-virus transfection of T cell: by resuspended for post-stimulatory T cell, 300g, 5min is centrifugal remove supernatant after change the fresh RPMI-1640 nutrient culture media containing 8 μ g/mlpolybrene, the slow virus of expressing GFP and CAR19 is added respectively in different hole, according to slow virus titre, MOI is set to 10.37 DEG C, 5%CO
2incubator is cultivated after 24h, re-suspended cell, 300g, 5min are centrifugal remove supernatant after change the fresh RPMI-1640 nutrient culture media contained, 37 DEG C, 5%CO
2after 24h cultivated by incubator, flow cytometer detection GFP is positive, and namely obtains the Chimeric antigen receptor T cell expressing anti-CD19, sees Fig. 3, T cell (WT) GFP of untransfected slow virus is negative, and the T cell of transfection CAR19 slow virus then has the cell of 22.6% to be that GFP is positive.
After obtaining expressing the Chimeric antigen receptor T cell of anti-CD19 in step (6), this cell can also be increased: the Chimeric antigen receptor T cell of anti-for the expression the obtained CD19 RPMI-1640 nutrient culture media containing concentration being 300IU/mlIL-2 is cultivated, the Chimeric antigen receptor T cell concentration in the medium expressing anti-CD19 is 1-2x106 cell/ml, within 2-3 days, carry out a subculture half amount and change liquid, and T cell is counted, the T cell after amplification can be obtained after cultivating 2 weeks.
Adopt said method, detect the Chimeric antigen receptor T cell of anti-CD19 to leukaemia cyto-inhibition, generally all will control group be set when detecting, the cell of step (2) is designated as experimental group, according to the quantity of the B cell leukemia cell of control group and experimental group, can detect that the Chimeric antigen receptor T cell of anti-CD19 is to the effect of B cell leukemia cell, such as, control group is only by normally, the T cell of transformation and B cell leukemia cell equal proportion is not had to be implanted in immunodeficient mouse model, then the peripheral blood cells of immunodeficient mouse is gathered, with fluorescently-labeled anti-human CD45 antibody human peripheral blood cell dyeing, this is cellular control unit, result as following experimental group cell and cellular control unit flow cytomery: as shown in Figure 4, in control group peripheral blood, the ratio of B cell leukemia cell is 2.42%, CAR19T cell processed group is 0.301%, B cell leukemia cell proportion in control group spleen and marrow is respectively 25.5% and 48.8%, and the ratio of CAR19T cell processed group is respectively 5.11% and 3.67%.
The above, not impose any restrictions content of the present invention, every according to the substantial amendment of content technologies of the present invention, all still belongs in the protection domain of content technologies scheme of the present invention.
Claims (6)
1. the Chimeric antigen receptor T cell detecting anti-CD19 is to the method for leukaemia cyto-inhibition, it is characterized in that, the method comprises the steps, (1) be transplanted in immunodeficient mouse model after the Chimeric antigen receptor T cell expressing anti-CD19 being mixed with leukaemia's equal proportion, the preserving number CCTCCNO:C201503 of the Chimeric antigen receptor T cell of anti-CD19;
(2) peripheral blood cells of immunodeficient mouse model is gathered, with fluorescently-labeled anti-human CD45 and CD19 antibody human peripheral blood cell dyeing;
(3) cell after step (2) being dyeed adopts flow cytometer to detect, and can detect that the Chimeric antigen receptor T cell of anti-CD19 is to the inhibiting effect of leukaemia according to the quantity of leukaemia.
2. the Chimeric antigen receptor T cell of the anti-CD19 of detection according to claim 1 is to the method for leukaemia cyto-inhibition, it is characterized in that, immunodeficient mouse model is NOD/SCIDIL2Rg-/-immunodeficient mouse.
3. the Chimeric antigen receptor T cell detecting anti-CD19 according to claim 1, to the method for leukaemia cyto-inhibition, is characterized in that, leukaemia is B cell leukemia cell.
4. detect the Chimeric antigen receptor T cell of anti-CD19 according to claim 1 to the method for leukaemia cyto-inhibition, it is characterized in that, marrow, the spleen cell of immunodeficient mouse model can also be gathered in step (2), then with fluorescently-labeled anti-human CD45 and CD19 antibody, marrow, spleen cell are dyeed.
5. the Chimeric antigen receptor T cell of the anti-CD19 of detection according to claim 1 is to the method for leukaemia cyto-inhibition, it is characterized in that, the method obtaining the Chimeric antigen receptor T cell expressing anti-CD19 comprises the steps,
(1) synthesize the gene of the Chimeric antigen receptor cell of anti-CD19, the gene of synthesis is designated as CAR19;
(2) structure of slow virus plasmid: the CAR19 gene integration 1. synthesized, in pUC57 plasmid, is designated as pUC57-CAR19 plasmid; 2. with restriction enzyme PmeI and SpeI, double digestion is carried out to pWPXLd-EGFP and pUC57-CAR19, then cut glue recovery extract CAR19 genetic fragment and pWPXLd-EGFP skeleton large fragment by agarose electrophoresis, glue recovery kit MAGEN; 3. CAR19 and pWPXLd-EGFP fragment coupled together with SolutionI, reaction system is that 5ulSolutionI adds 4ulCAR19 and 1ulpWPXLd-EGFP fragment, and 16 DEG C of reaction 2h, connect product conversion to TOP10 competence Escherichia coli; 4. competence bacterium after conversion is coated on ampicilin agar plates, 37 DEG C of incubated overnight; 5. second day picking, 4 cell clones are to 2ml containing in 50ug/ml ampicillin LB nutrient culture media, and 37 DEG C, 250rpm cultivates 12h, extracts the plasmid in 4 pipe bacteriums with the little extraction reagent kit MAGEN of plasmid; 6. identified by PmeI and SpeI double digestion, the plasmid built is designated as pWPXLd-CAR19-EGFP; 7. adopt the large extraction reagent kit of plasmid to extract slow virus envelope plasmid pMD2.G, packaging plasmid psPAX2 and pWPXLd-CAR19-EGFP plasmid is stand-by;
(3) preparation of CAR19 slow virus: by 293T cell packaging slow virus, concrete operations are as follows: 1. add 10%FBS and 1% dual anti-cultivation 293T cell to 150mm double dish with DMEM high glucose medium, when 293T cell density reaches 80-90%, change into after DMEM high glucose medium adds 1%FBS and 1% dual anti-cultivation 2-6h, with PEI by pWPXLd-CAR19-EGFP or control plasmid pWPXLd-EGFP, pMD2.G and psPAX2 tri-Plastid transformation 293T cells, each 150mm double dish transforms pWPXLd-CAR19-EGFP or the control plasmid pWPXLd-EGFP of 9 μ g, the pMD2.G of 3 μ g and the psPAX2 of 12 μ g, PEI is 72ug, 2. 24h, 48h and 72h after transforming collect three supernatants, supernatant is after 2500g is centrifugal, after 0.45 μm of metre filter, the centrifugal 1.5h of Ultracentrifuge 28000rpm, after removing supernatant immediately, each ultracentrifugation pipe adds 200 μ lPBS, is placed in 4 DEG C and spends the night resuspended, and the PBS containing slow virus collects by next day,
(4) separation and purification of T cell: first isolated the mononuclearcell in blood by Ficoll density gradient method, is removed after red blood cell through erythrocyte cracked liquid cracking, then goes out T cell by MACS magnetic bead sorting;
(5) activation and proliferation of T cell: 1. wrap by AntiCD3 McAb OKT-3 and CD28 antibody in advance: with PBS, AntiCD3 McAb is become 1 μ g/ml and 2 μ g/ml with CD28 antibody dilution, add in six orifice plates, every hole 1ml, cultivates case and bag by 2h for 37 DEG C; 2. T cell is diluted to 2.5x10 with containing 10%FBS and 1% dual anti-RPMI-1640 nutrient culture media
6individual/ml, sops up the coating buffer in 6 orifice plates, and what add every hole 3ml contains T cell nutrient culture media, 3. T cell is placed on 37 DEG C, 5%CO
2in incubator, stimulate T cell 48h;
(6) slow-virus transfection of T cell: by resuspended for post-stimulatory T cell, 300g, 5min is centrifugal remove supernatant after change the fresh RPMI-1640 nutrient culture media containing 8 μ g/mlpolybrene, the slow virus of expressing GFP and CAR19 is added respectively in different hole, according to slow virus titre, MOI is set to 10.37 DEG C, 5%CO
2incubator is cultivated after 24h, re-suspended cell, 300g, 5min are centrifugal remove supernatant after to change fresh be the RPMI-1640 nutrient culture media of 300IU/mlIL-2 containing concentration, 37 DEG C, 5%CO
2after 24h cultivated by incubator, flow cytometer detection GFP is positive, and namely obtains the Chimeric antigen receptor T cell expressing anti-CD19.
6. the Chimeric antigen receptor T cell of the anti-CD19 of detection according to claim 5 is to the method for leukaemia cyto-inhibition, it is characterized in that, after obtaining expressing the Chimeric antigen receptor T cell of anti-CD19 in step (6), this cell can also be increased: cultivated by the Chimeric antigen receptor T cell of anti-for the expression the obtained CD19 RPMI-1640 nutrient culture media containing concentration being 300IU/mlIL-2, the Chimeric antigen receptor T cell concentration in the medium expressing anti-CD19 is 1-2x10
6individual cell/ml, 2-3 days carries out a subculture half amount and changes liquid, and counts T cell, can obtain the T cell after amplification after cultivating 2 weeks.
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Inventor after: Li Peng Inventor after: Lai Yunxin Inventor after: Xu Bing Inventor after: Lin Simiao Inventor after: Yao Yao Inventor after: Qin Le Inventor after: Wei Xinru Inventor after: Li Baiheng Inventor before: Li Peng Inventor before: Lai Yunxin Inventor before: Lin Simiao Inventor before: Yao Yao Inventor before: Qin Le Inventor before: Wei Xinru Inventor before: Li Boheng |