CN107502591A - The iNKT methods for cell expansion and its application that a kind of concentration gradient rhIL 2 is relied on - Google Patents

The iNKT methods for cell expansion and its application that a kind of concentration gradient rhIL 2 is relied on Download PDF

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CN107502591A
CN107502591A CN201710939066.0A CN201710939066A CN107502591A CN 107502591 A CN107502591 A CN 107502591A CN 201710939066 A CN201710939066 A CN 201710939066A CN 107502591 A CN107502591 A CN 107502591A
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inkt
cell
rhil
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concentration
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CN107502591B (en
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闾军
孙文峰
陈辉
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Beijing Gene Qiming Biology Technology Co ltd
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Beijing Gene Qiming Biological Science And Technology Co Ltd
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Abstract

The invention discloses a kind of concentration gradient rhIL 2 iNKT methods for cell expansion relied on and its application.This method includes (1) extraction PMNC PBMC；Final concentration of 100ng/ml α GalCer are added in the PBMC cells extracted to step (1) to cultivate 48 hours；Cultivated to step (2) in cell gradually increases the concentration of rhIL 2 in culture medium in a manner of gradient.The present invention harvests iNKT cells on the 21st day in culture, and iNKT cell numbers improve 100,000 times, wherein more than 90% is CD4-iNKT cells, has up-regulation immune response and direct cytotoxicity.The iNKT cell amplification efficiencies of method activation and the amplification of the present invention are higher, optionally expand Th1 sample iNKT cells, strengthen the function of the enhancing of amplification in vitro iNKT cellular immunities, tumour immunity monitoring and killing.

Description

The iNKT methods for cell expansion and its application that a kind of concentration gradient rhIL-2 is relied on
Technical field
The invention belongs to tumour cell immunotherapy field, more particularly it relates to Tumor cytotoxicity The methods and applications of Th1 sample iNKT cells rapid, high volume amplification.
Background technology
Constant natural killer T (invariant nature killer T, iNKT) cell is a kind of naturally occurring mediation The immunocyte of congenital immunity and acquired immunity, it is subgroup unique in T lymphocytes, there is class NK (nature Killer) cell sample is rapid to stimulation produces cell factor and chemotactic factor (CF) to adjust the innate immune function of immune response, Resisted by φt cell receptor (TCR) and originate in raw specific reaction.Different from the TCR of classical T lymphocytes, iNKT cells tool There are highly conserved TCR α chains, the TCR α chains of the mankind are made up of the parts of V α 24-J α 28, can be with a variety of glycolipid class antigen-reactives.This Outside, the I classes or II classes of antigen presenting cell (antigen presenting cell, APC) are identified different from traditional T cell Major histocompatibility complex molecule (major histocompatibility complex, MHCI or II) offers antigen Peptide, the sugared lipid antigen that the iNKT cell energy specific recognition APC non-classical MHC I quasi-molecules CDld in surface offer.After activation INKT cells secrete cytokine profiles, directly or indirectly participate in different immune responses.The iNKT of different subgroups or hypotype is thin Born of the same parents have different immunoloregulation functions.
INKT cells have the function of induction Immune-enhancing effect and immunosurveillance simultaneously.Under antigenic stimulus, iNKT cells can To secrete a series of cell factors, including：Interferon-γ (interferon-gamma, IFN-γ), interleukin (IL) -2,4, 10,13,17,21,22 and grain-monosystem colony stimulating factor (granulocyte-macrophage colony- Stimulating factor, GM-CSF) and TNF (tumor necrosis factor-alpha, TNF-α).
INKT cells pass through CD1d-TCR compounds and the specific inducing dendritic shape cell of CD40-CD40L interactions (Dendritic Cells, DC) is ripe, and secretes IL-12.IL-12 stimulates NK, NKT cell and other T cells to produce more IFN-γ.Both cell factors significantly have activated downstream effect cell mass such as NK cells, CD8 together+T cell and gamma delta T are thin Born of the same parents.The activation of iNKT cells further promotes the expression of the costimulatory moleculeses such as DC up-regulations CD70, CD80 and CD86.And DC is expressed CD70 is CD8+T cell starts necessary to adaptive immunity.The IL-2 inducing memories CD4 of the iNKT cells secretion of activation+T Helper cell proliferation.CD4+T cell is divided into different t helper cell subgroups in the presence of different cytokines.INKT cells Th1/ proinflammatory (IFN-γ, TNF-α) or Th2/ anti-inflammatories (IL-4,10,13) cell factor, this cascade enhancing can be secreted Effect excite the innate immunity and acquired immunity simultaneously, tumour cell positive induced killer MHC.MHC feminine genders are swollen Oncocyte, iNKT cells show NK cell sample MHC independence cell cytotoxic activities after the activation of the glycolipid antigens such as α-GalCer, Pass through the direct killings such as apoptosis induction ligand (TRAIL) related induced expression perforin/granzyme FasL/Fas or TNF. To CD1d+Tumour cell (such as acute T lymphocytic leukemias, monocytic leukemia and other hematologic malignancies), With Direct Recognition and effective target cell lethal effect can be showed.
Serious iNKT cell quantities reduction and functional disturbance are often accompanied by tumor patient body.Due to oneself expression phosphoric acid sheath Ammonia alcohol acceptor (S1P1R) and a variety of chemokine receptors (C-X-C chemokine receptor) turn iNKT cell selectives Tumor locus is moved on to, causes to circulate in colon cancer, head and neck cancer, breast cancer, clear-cell carcinoma, melanoma and Peripheral Blood of Patients with Hepatocellular Carcinoma Property iNKT cell quantities substantially reduce, but cell produce IFN-γ ability do not damage.Although body-internal-circulation iNKT cells Quantity is reduced, but iNKT cells stimulate through α-GalCer can breed rapidly in vitro, and the iNKT cells of in-vitro multiplication are same Sample shows preferable cellulotoxic effect killing tumor cell.It is a variety of in recent years based on human body iNKT cellular immunotherapy tumours Scheme is developed, but the iNKT cells of clinical selectivity injection Activated in Vitro, in tumour patient body, remission rate is relatively low, immune Reaction individual difference is larger, and antitumous effect is preferable not to the utmost.The mankind have CD4+And CD4-INKT cells, according to cytokine-expressing It is divided into Th1 sample iNKT cells and Th2 sample iNKT cells.Wherein, Th1 samples iNKT cells produce IFN-γ, and its function is that mediation is straight Connect tumor-killing and the function of immune response and immunosurveillance is raised in tumor microenvironment.The phenotype mark of Th1 sample iNKT cells Will is great expression CD8 α, and considerably less CD8 α β, and there is also CD4-CD8-。CD8+INKT cells compare CD4+Or CD4-CD8- INKT cells secrete more IFN-γs, and cytotoxicity is also stronger.Due to CD4 in human peripheral iNKT cells+INKT cells account for Compare high (~90%), traditional amplification method is expanded to Th1 sample iNKT cells without selectivity, and the iNKT cells after amplification are each Hypotype ratio is unstable, and it is larger to immune response individual difference after tumour patient to cause feedback.Therefore, technological difficulties master at present Concentrate in the amplification of the sufficient amount of feature iNKT cells with tumor-killing and mediated immunity supervisory function bit.
Proliferation time be present using the obtained iNKT cells of fixed concentration rhIL-2 joints α-GalCer in conventional amplification method After long (~4 weeks), amplification after iNKT cell proportions (average 100 times) more limited than relatively low (~20%), growth multiple, amplification INKT cells CD4+Accounting higher (~40%), the problems such as internal killing ability is low, it is impossible to meet clinical demand.The present invention adopts With the increased mode of rhIL-2 concentration gradients, offer α-GalCer using DC, combine rhIL-15, rhIL-7, CD3 monoclonals resist Body is by iNKT cells by CD4+It is converted into CD4-.Culture can expand iNKT cells 100,000 times, wherein CD8 in 21 days+INKT cells account for Than ＞ 50%, CD4+INKT cell accountings ＜ 10%.Solve the problems, such as iNKT quantity and purity, and selective amplification Th1 samples INKT cells, can fully meet the needs of clinical treatment.
The content of the invention
The invention provides a kind of amplification Activiation method of concentration gradient rhIL-2 iNKT cells relied on and its application.This The method of invention is for existing iNKT amplification in vitros culture efficiency is relatively low, Th1 samples, Th2 sample iNKT cell non-selectivities etc. are asked Topic, propose a kind of more effective and specific amplification Th1 sample iNKT lymphocytes method.
In the iNKT methods for cell expansion that a kind of concentration gradient rhIL-2 provided by the invention is relied on, cultivate the 0th day, add 100ng/mlα-GalCer；Cultivate the 0-11 days, increase rhIL-2 concentration in a manner of gradient.
Preferred scheme provided by the invention is：The time of fixed intervals, rhIL-2 concentration is in equal difference or waits than increase；Interval Time preferred 1-72 hours, the preferred 10-400IU/ml of rhIL-2 concentrations.
Technical scheme of the invention preferred is：Cultivate the 7th day ripe DC for adding same donor Fiber differentiation；DC is adding First add 100ng/ml α-GalCer and be incubated within two hours before entering iNKT cells；Cultivate the 14th day and use Anti-iNKT MicroBeads human are sorted, and the iNKT cells for sorting to obtain are added in the coated blake bottle of OKT3 monoclonal antibodies and trained Support；CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium；OKT3 MAb concentrations are 1-5 μ g/ml, CD28 antibody Concentration is 1-5 μ g/ml；RhIL-15, rhIL-7 concentration are 1-10ng/ml.
The further preferred technical scheme of the present invention is as follows：
(1) PMNC PBMC is extracted；
(2) final concentration of 100ng/ml α-GalCer are added in the PBMC cells extracted to step (1) to cultivate 48 hours；
(3) cultivated to step (2) in cell gradually increases rhIL-2 concentration in culture medium in a manner of gradient.
Further, rhIL-2 concentration is increased with Fixed Time Interval in step (3), and the Fixed Time Interval is 1-72 hours.
Further, the rhIL-2 concentration is that 10-400IU/ml is in wait ratio or equal difference mode to increase；The 11st of culture the It when, rhIL-2 concentration increases to 400IU/ml.
Further, cultivate the 7th day and mature dendritic cell, ripe dendron shape are added into step (3) cell culture medium The quantity ratio of cell and iNKT cells is 1:10.
Further, mature dendritic cell first adds 100ng/ml α-GalCer in two hours before iNKT cells are added It is incubated.
Further, cultivate the 14th day and sorted using Anti-iNKT MicroBeads human, sort what is obtained INKT cells are added in the coated blake bottle of CD3 monoclonal antibodies and cultivated.
Further, CD3 MAb concentrations are 1-5 μ g/ml, preferably 2 μ g/ml.
Further, CD28 antibody, rhIL-15 and rhIL-7 are added during culture；CD28 antibody concentrations are 1-5 μ g/ml, excellent Elect 3 μ g/ml as；RhIL-15 concentration is 1-10ng/ml, preferably 5ng/ml；RhIL-7 concentration is 1-10ng/ml, is preferably 5ng/ml。
Methods described harvests iNKT cells on the 21st day in culture, and iNKT cell numbers improve 100,000 times, wherein more than 90% is CD4iNKT cells, have the function that to raise immune response and kill tumour.
The present invention solves the problems, such as the quantity and purity of iNKT cell expansion ex vivos culture in the prior art, cultivates 21 days INKT cells can be expanded to 100,000 times, and selective amplification Th1 sample iNKT cells, wherein CD8+INKT cell accountings ＞ 50%, CD4+INKT cell accountings ＜ 10%, can fully meet the needs of clinical treatment.Gradually increase rhIL-2 concentration, can cultivated Seedling selection expands iNKT cells, in this way, can improve iNKT cell proportions in culture to 6% left side at the 7th day The right side, iNKT cell numbers improve 60 times.It is thin that α-GalCer coated ripe DC, orientable induction Th1 samples iNKT were added at the 7th day Born of the same parents expand, and promote CD4+INKT cells are to CD4-Conversion.In this way, can be at the 14th day by iNKT cell proportions in culture Improve to 16% or so, iNKT cell numbers and improve 1000 times.The 14th day pure iNKT cell obtained through magnetic bead sorting is cultivated, according to The T cell characteristic that iNKT cells have, add in the coated blake bottle of CD3 monoclonal antibodies and cultivate, and add costimulatory molecules CD28 antibody, the iNKT cells of purifying can be made largely to expand, adding rhIL-15 and rhIL-7 can maintain Th1 sample iNKT cells to increase Grow.In the 21st day harvest iNKT cell of culture, iNKT cell numbers improve 100,000 times, wherein more than 90% is CD4-INKT cells, With up-regulation immune response and direct cytotoxicity.The iNKT cell amplification efficiencies of present invention amplification and activation are higher, selectivity Ground expands Th1 sample iNKT cells, strengthens the function of the enhancing of amplification in vitro iNKT cellular immunities and tumour immunity monitoring.
Brief description of the drawings
Fig. 1 .rhIL-2 handle iNKT cellular processes
Fig. 2 difference rhIL-2 E-tests handle iNKT cell Flow cytometry figures
Fig. 3 .iNKT cell expansion ex vivo cell quantity linear graphs
Fig. 4 .iNKT cell killings tumour cell is tested
Fig. 5 .iNKT cell secretion of gamma-IFN capacity experimentals
Fig. 6 .iNKT cells kill liver cancer cells experiment in animal body
Embodiment
Embodiments of the invention are described below in detail, the example of illustrated embodiment is shown in the drawings.In following embodiments Experimental method, unreceipted particular technique or condition person, then according to conventional laboratory conditions such as《ATCC cell culture handbooks》Middle institute When stating condition, and Reagent Company's specification has been clearly stated in embodiment, then proposed condition is carried out to specifications.Institute With reagent or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
Reagent used in the embodiment of the present invention：
AIM V cell culture mediums, ThermoFisher companies
Lymphoprep separating liquids (instant), Axis-Shield companies
CD3 monoclonal antibodies, CD28 monoclonal antibodies, Biolegend companies
IL-2, IL-7, IL-15, Peprotech company
α-GalCer, Enzo Life Sciences companies
Anti-iNKT MicroBeads human, Miltenyi Biotec companies
Fixation/Permeabilization Solution Kit with BD GolgiPlugTM, BD companies
FITC anti-human CD3, PE anti-human TCR V α 24-J α 18, PerCP/Cy5.5anti-human CD8, PE/Cy7anti-human CD4, FITC anti-human IFN-γs, Biolegend companies
The PMNC of embodiment 1 (PBMC) extracts
(1) early morning adopts 20ml anti-freezing venous blood.
(2) 15ml Lymphoprep separating liquids are added in 50ml centrifuge tubes.
(3) isometric 0.9% sterile saline gentle inversion is slowly added in anti-freezing venous blood three times, is fully mixed, Blood after being diluted.
(4) blood after dilution is slowly added into Lymphoprep separating liquids top layer using 2ml sterile droppers, all moved into After be sure not to rock or overturn.
(5) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:4/0, room temperature centrifugation 30min。
(6) the unnecessary blood plasma in the superiors part is suctioned out.Buffy coat is gently suctioned out with 2ml aseptic straws, is moved into new In 50ml centrifuge tubes, the buffy coat in all pipes is sucked in same 50ml centrifuge tubes.Add 0.9% sterile physiological salt Water fully mixes to 50ml.
(7) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:9/9 centrifugation, room temperature from Heart 10min.
(8) test tube is taken out, discards liquid completely.
(9) the AIM V culture mediums of incubation are taken out from 37 DEG C of incubators, take 2ml fully to mix cell precipitation, are paid attention to light Soft operation, avoid producing bubble.Remaining culture medium is then added, mixes cell.
(10) cell suspension of mixing is moved into 75cm2Tissue Culture Flask, mixing is gently rocked, be positioned over 37 DEG C of incubators Middle culture.
The iNKT cells of embodiment 2 expand for the first time
(1) the PBMC extractions same day adds final concentration of 100ng/ml α-GalCer and cultivated 48 hours.
(2) the gradual increase rhIL-2 concentration into culture medium, is separated to culture the 11st day, rhIL-2 concentration is in from cell Increase Deng ratio or equal difference mode.
(3) at the 11st day of culture, rhIL-2 concentration increases to 400IU/ml.
As shown in Figure 1A, since cell separate, every 48 hours (except the 10th day to the 11st day be spaced 24 hours), RhIL-2 concentration is in wait to increase than mode.
As shown in Figure 1B, since cell separate, every 72 hours (except the 10th day to the 11st day be spaced 48 hours), RhIL-2 concentration increases in equal difference mode.
The Fiber differentiation of the BMDC of embodiment 3
(1) PBMC for obtaining density-gradient centrifugation method is resuspended with AIM V culture mediums, is added in Tissue Culture Dish, 37 DEG C 5%CO2Culture.
(2) 3 as a child took out culture dish, gently rocked, and suctioned out suspension cell.
(3) rhIL-4 containing 500IU/ml, 500IU/ml rhGM-CSF AIM V culture mediums are added into culture dish.
(4) respectively after incubation the 3rd day, the 5th day half amount change liquid, add fresh rhIL-4 containing 500IU/ml, 500IU/ml RhGM-CSF AIM V culture mediums.
(5) cultivate the 6th day and add final concentration of 10ng/ml rhTNF- α
(6) the 7th days harvest mature dendritic cells.
The iNKT cells of embodiment 4 stimulate again
(1) in the cell of embodiment 2 and the cell culture of embodiment 3 the 7th day, it is thin that ripe dendron shape in embodiment 3 is collected in centrifugation Born of the same parents, it is resuspended with AIM V culture mediums, adds final concentration 100ng/ml α-GalCer and cultivate 2 hours.
(2) centrifugation removes free α-GalCer, carries out cell count.With the first amplified production of iNKT cells:DC=10: 1 ratio by step (1) cultivate obtain DC add embodiment 2 described in and the first amplified production of iNKT cells in.
Through the isolated PBMC of venous blood flow cytometry is carried out respectively within the 0th day, the 7th day, the 14th day in culture respectively Detection.As seen from Figure 2, iNKT cells (CD3+6B11+) ratio gradually increases, and CD4 in iNKT cells+Cell proportion by Gradually reduce, CD4-CD8-Cell, CD8+Cell proportion gradually steps up.As shown in Fig. 2A, in example 2 from rhIL-2 concentration Increase in waiting than mode, in the 14th day iNKT cells ＞ 15%, CD8+Cell ＞ 50%, CD4+Cell ＜ 10%.By Fig. 2 B institutes Show, increased in embodiment 2 from rhIL-2 concentration in equal difference mode, in the 14th day iNKT cells ＞ 4%, CD8+Cell ＞ 40%, CD4+Cell ＜ 10%.In summary, a preferred embodiment of the invention is that rhIL-2 concentration is in equal difference mode Increase, a further preferred embodiment are that rhIL-2 concentration is in wait to increase than mode.
The iNKT cells that embodiment 5 purifies largely expand
(1) the coated Tissue Culture Flask of CD3 monoclonal antibodies should be made at the 13rd day：2 μ g/ml CD3 monoclonals are prepared to resist Body running liquid (0.9% sterile saline).Prepare two brand-new 75cm2Big bottle, each add what 5ml had diluted in big bottle Antibody working solution.Bottleneck is tightened, jiggles body, CD3 monoclonal antibody dilutions is fully infiltrated bottom of bottle.Sealed with sealed membrane The good blake bottle mouth of pipe, keep flat and preserved into 4 DEG C of refrigerators.
(2) cultivate the 14th day, iNKT cell sortings are carried out using Anti-iNKT MicroBeads human, collect thin Born of the same parents.
(3) use and contain 100IU/ml IL-2,3 μ g/ml CD28 antibody, 5ng/mlrhIL-7,5ng/mlrhIL-15's AIM V culture mediums fully mix iNKT cells, pay attention to gentle manipulation, avoid producing bubble, prepare cell concentration not less than 5 × 106Individual/ml cell suspension, mix cell.
(4) the coated blake bottle of CD3 monoclonal antibodies is taken out from 4 DEG C of refrigerators, discards liquid, adds the nothings of 10ml 0.9% Blake bottle of bacterium brine；The cell suspension of mixing is moved into 75cm2Tissue Culture Flask, mixing is gently rocked, is put It is placed in 37 DEG C of incubators and cultivates.
(5) incubation observation cell state and culture medium color, appropriate culture medium can be supplemented according to growing state.
(6) cell can be transferred to new blake bottle by CD3 antibody after stimulating 3 days, and supplement the culture medium of corresponding volume.
(7) the 21st day harvesting is cultivated.
INKT cell expansion ex vivos cell quantity linear graph as seen from Figure 3.PBMC is separated from peripheral blood and is cultivated just The 0th day iNKT cell number that begin is 1 × 104Individual (accounting for PBMC TCSs 0.1% or so), by culture in 21 days available 1 × 109 Individual iNKT cells.
The iNKT cell killings tumor cell of liver of embodiment 6 is tested
Effector cell：The iNKT cells for a large amount of amplifications that embodiment 5 obtains
Target cell：Hepatoblastoma cell line HepG2, people differentiated liver cancer cell lines Huh7, height transfer liver cancer cells It is LM3
Detection iNKT cells use lactic dehydrogenase (Lactic to tumor cell of liver killing capacity experimental Dehydrogenase, LDH) method for releasing.Concrete operation method is as follows:
(1) target cell concentration is adjusted to 1 × 105Individual/ml.
(2) target cell is transferred in 96 orifice plates according to 100 μ l/ holes, three multiple holes of every group of setting.
(3) target cell Spontaneous release hole (negative control) is set.It is not added with effector cell and only adds 100 μ l nutrient solutions.
(4) maximum release aperture (positive control) is set.It is not added with effector cell and only adds 100 μ l10%NP40.
(5) the μ l of effector cell 100, effector cell are added in each experimental port：Target cell=0:1；1:1；3:1；10:1； 30:1；50:1.Each hole adds final concentration of 100ng/ml α-GalCer.
(6) 37 DEG C of 5%CO2Culture, cell conditioned medium was collected in 6 hours and carries out lactic dehydrogenase (LDH) detection.
(7) nutrient solution supernatant, detection LDH numerical computations activity are drawn.Killing activity (%)=[(experimental group A values-total from So release A values)/(maximum release group A values-total Spontaneous release A values)] × 100%.
Killing experiments are grouped into:1st, DC combines iNKT groups of cells；2nd, iNKT cells independent role group；3rd, PBMC control groups； 4th, phosphate buffer (PBS) blank control group.
Shown by Fig. 4 results, iNKT cells offer α-GalCer in DC as antigen presenting cell, can make amplification in vitro INKT cell acute activations, to different hepatoma cell lines can specific dissolution, and in the iNKT of nonantigenic presenting cells Stimulation of the cell independent role group due to lacking strong activation signal, when effector cell's number is less, to target cell killing activity It is weaker, it was demonstrated that the validity of amplification in vitro iNKT cell functions.
Detection iNKT cell IFN-γ secretions ability uses flow cytometry.Concrete operation method is as follows：
(1) it is inoculated with according to killing experiments target cell (HepG2 cells are selected in this experiment) concentration and cell number；
(2) according to effector cell：Target cell=50:1 Inoculating efficiency cell.Each hole add final concentration of 100ng/ml α- GalCer。
(3) 2 μ l BD GolgiPlug are added per holeTM。
(4) 37 DEG C of 5%CO2Culture, collect cell respectively in 6,12 hours, carry out padding.
(5) carry out rupture of membranes according to Fixation/Permeabilization Solution Kit specifications and intracellular contaminates Color.
(6) fixed cell, carries out Flow cytometry and analysis.
INKT cell IFN-γ secretion capacity experimentals are grouped into:1st, DC combines iNKT groups of cells；2nd, iNKT cells are individually made With group；3rd, PBMC control groups；4th, phosphate buffer (PBS) blank control group.
Shown by Fig. 5 results, for iNKT cells when DC activates as antigen presenting cell, the ability of secretion of gamma-IFN is obvious Higher than other groups, it was demonstrated that the validity of the iNKT cell functions of In-vitro specificity amplification, further illustrate and use institute of the present invention The iNKT cells for stating method amplification are mainly Th1 sample iNKT cells, have powerful tumor-killing and immune enhancing function.
Liver cancer killing experiments in embodiment 7.iNKT multicellular animal bodies
From GFP fluorescence labelings hepatocellular carcinoma cells system HepG2-GFP as tumour cell, subcutaneous implantation in 8 week old, Immunodeficient mouse NOD-SCID mouse back of the body weight between 20~30g, every group of 5 NOD-SCID mouse of experiment packet.Treat After tumour grows up to, temporally put tail vein and adopt people's iNKT cells, fluorescence imaging observation tumour growth situation and to measure tumour big It is small.
Concrete operation method is as follows:
(1) prepared by iNKT cells：The iNKT cells for a large amount of amplifications that embodiment 5 obtains, physiological saline is suspended in before injection In it is standby；
(2) after iNKT cell infusions frequency is since 14 days 1 times a week, totally 4 weeks, 4 injections, negative control group is noted 1% physiological saline group containing albumin is penetrated, positive controls are intraperitoneal injection adriamycin 2mg/kg, and abdominal cavity is expelled to after 14 days start Every 5 days 1 time；
(3) determination of iNKT cell dosages is injected：On the basis of 60kg adult, iNKT cells use agent in clinic expection Measure as 1 × 109~1 × 1010Individual cell (about 1.6 × 107~1.6 × 108Individual cell/kg).With NOD-SCID mouse weights 30g On the basis of, its clinical projected dose is equivalent to 5 × 105~5 × 106Individual cell/only.We using high dose cell as test dose, Every mouse adopts 5 × 106Individual cell；
(4) during injecting, 3 times a week more than the observation change of general state, motility, outward appearance etc. general symptom, and Whether grasp has dead animal.The volume (mean tumor volume) of tumour：During injection, 3 times a week using caliper (calipers) major axis and short axle of tumour are measured, and utilizes following calculation formula, measures the volume of tumour：V(mean tumor volume,mm3)=AB2/ 2 (A=long axis lengths, B=minor axis lengths).Every group of 3 NOD-SCID mouse of experiment packet.Tool Body is grouped as follows shown in table.
Shown by Fig. 6 results, NOD-SCID mouse subcutaneous implantation HepG2-GFP tumour cells after 14 days, adopt by tail vein People's iNKT cells, time point of adopting are respectively the 14th, 21,28,35 day, hereafter fluorescence imaging observation tumor size, and surveying weekly Measure gross tumor volume (Fig. 6 A).As Fig. 6 B, 6C results are shown, iNKT cells are adopted to after tumor-bearing mice, mouse tumor size compared with Control negative control groups are obviously reduced, and prompt iNKT cells to play the role of obvious anti-liver cancer and anti-tumour cell in vivo.
The content for the publication listed in all this specification is included in this specification.In addition, people in the art Member is carried out it is appreciated that in the case of without departing substantially from the technical scope described in claims and substantive content to the present invention A variety of different modifications and change are possible.Present invention additionally comprises these above-mentioned modifications and change.

Claims (12)

1. iNKT methods for cell expansion and its application that a kind of concentration gradient rhIL-2 is relied on, it is characterised in that culture the 0th day, Add 100ng/ml α-GalCer；Cultivate the 0-11 days, increase rhIL-2 concentration in a manner of gradient.
2. according to the method for claim 1, it is characterised in that the time of fixed intervals, rhIL-2 concentration is in equal difference or waits Than increase；Interlude is 1-72 hours, and rhIL-2 concentration is 10-400IU/ml.
3. according to the method described in claim 1-2, it is characterised in that culture add within the 7th day same donor Fiber differentiation into Ripe DC；DC first adds 100ng/ml α-GalCer in two hours before iNKT cells are added and is incubated；Cultivate the 14th day and use Anti-iNKT MicroBeads human are sorted, and it is coated that the iNKT cells for sorting to obtain add OKT3 monoclonal antibodies Cultivated in blake bottle；CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium；OKT3 MAb concentrations are 1-5 μ g/ Ml, CD28 antibody concentration are 1-5 μ g/ml；RhIL-15, rhIL-7 concentration are 1-10ng/ml.
4. according to the method for claim 1, it is characterised in that step is：
(1) PMNC PBMC is extracted；
(2) final concentration of 100ng/ml α-GalCer are added in the PBMC cells extracted to step (1) to cultivate 48 hours；
(3) cultivated to step (2) in cell gradually increases rhIL-2 concentration in culture medium in a manner of gradient.
5. according to the method for claim 4, it is characterised in that methods described is further comprising the steps of：
(4) the 7th day of culture, the ripe BMDC of same donor Fiber differentiation is added into step (3) cell culture medium, The quantity ratio of mature dendritic cell and iNKT cells is 1:10；
The preparation method of the mature dendritic cell is：The PBMC that density-gradient centrifugation method is obtained is resuspended with AIM V culture mediums, Add in Tissue Culture Dish, 37 DEG C of 5%CO2Culture；Culture dish was taken out in 3 hours, rocked, suction out suspension cell；To culture dish Middle addition rhIL-4 containing 500IU/ml, 500IU/ml rhGM-CSF AIM V culture mediums；Respectively after incubation the 3rd day, the 5th Its half amount changes liquid, adds fresh rhIL-4 containing 500IU/ml, 500IU/ml rhGM-CSF AIM V culture mediums；Cultivate the 6th day Add final concentration of 10ng/ml rhTNF- α；7th day harvest mature dendritic cell.
6. according to the method for claim 5, it is characterised in that mature dendritic cell is adding step (3) cell culture Before base, it is resuspended with AIM V culture mediums and adds final concentration 100ng/ml α-GalCer incubations 2 hours.
7. according to the method for claim 4, it is characterised in that methods described also comprises the steps of：
(5) cultivate the 14th day, iNKT cell sortings are carried out using Anti-iNKT MicroBeads human, sorting is obtained thin Born of the same parents are cultivated in the coated blake bottle of CD3 antibody, and CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium, in 37 DEG C of cultures Cultivated in case.
8. according to the method for claim 7, it is characterised in that CD3 antibody concentrations are 1-5 μ g/m, preferably 2 μ g/ml.
9. according to the method for claim 7, it is characterised in that it is 1-5 that CD28 antibody concentrations are added in step (5) culture medium μ g/ml, preferably 3 μ g/ml；RhIL-15 concentration is 1-10ng/ml, preferably 5ng/ml；RhIL-7 concentration is 1- 10ng/ml, preferably 5ng/ml.
10. according to the method for claim 7, it is characterised in that cell is transferred to by step (5) CD3 antibody after stimulating 3 days New blake bottle, and the culture medium of corresponding volume is supplemented, cultivate the 21st day harvesting.
11. according to the method described in any claim in claim 1-10, it is characterised in that methods described is in culture the 21st Its harvest iNKT cell, iNKT cell numbers improve 100,000 times, wherein more than 90% is CD4-INKT cells, have up-regulation immune anti- Should be with the effect of killing tumour.
12. antineoplastic is being prepared according to the iNKT lymphocytes that method any one of claim 1-11 prepares In purposes.
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