CN107502591B - 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 PDFInfo
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Abstract
The invention discloses a kind of concentration gradient rhIL-2 iNKT methods for cell expansion relied on and its applications.This method includes that (1) extracts peripheral blood mononuclear cells PBMC；Final concentration of 100ng/ml α-GalCer is added in the PBMC cell extracted to step (1) to cultivate 48 hours；RhIL-2 concentration in culture medium is gradually increased in a manner of gradient into step (2) culture cell.The present invention is cultivating the 21st day harvest iNKT cell, and iNKT cell number improves 100,000 times, wherein 90% the above are CD4-iNKT cells, has up-regulation immune response and direct cytotoxicity.The iNKT cell amplification efficiency of method activation and amplification of the invention is higher, selectively expands Th1 sample iNKT cell, strengthens the function of the enhancing of amplification in vitro iNKT cellular immunity, tumour immunity monitoring and killing.
Description
Technical field
The invention belongs to tumour cell immunotherapy fields, more particularly it relates to Tumor cytotoxicity
The methods and applications of Th1 sample iNKT cell rapid, high volume amplification.
Background technique
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 is unique subgroup in T lymphocyte, has class NK (nature
Killer) cell sample adjusts the innate immune function of immune response to the rapid generation cell factor of stimulation and chemotactic factor (CF),
The reaction for originating in raw specificity is fought by T cell receptor (TCR).Different from the TCR of classical T lymphocytes, iNKT cell tool
There is highly conserved TCR α chain, the TCR α chain of the mankind is made of 28 part V α 24-J α, can be with a variety of glycolipid class antigen-reactives.This
Outside, different from the I class or II class of traditional T cell identification antigen presenting cell (antigen presenting cell, APC)
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 class molecule CDld in surface offers.After activation
INKT cell secretes cytokine profiles, directly or indirectly participates in different immune responses.The iNKT of different subgroups or hypotype is thin
Born of the same parents have different immunoloregulation functions.
INKT cell has the function of inducing Immune-enhancing effect and immunosurveillance simultaneously.Under antigenic stimulus, iNKT cell can
To secrete a series of cell factors, comprising: 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 tumor necrosis factor (tumor necrosis factor-alpha, TNF-α).
The inducing dendritic shape cell that iNKT cell passes through CD1d-TCR compound and CD40-CD40L interaction specificity
(Dendritic Cells, DC) is mature, and secretes IL-12.It is more that IL-12 stimulates NK, NKT cell and other T cells to generate
IFN-γ.Both cell factors significantly have activated downstream effect cell mass such as NK cell, CD8 together+T cell and gamma delta T are thin
Born of the same parents.The activation of iNKT cell further promotes the expression of the costimulatory molecules such as DC up-regulation CD70, CD80 and CD86.And DC is expressed
CD70 is CD8+T cell starts necessary to adaptive immunity.The IL-2 inducing memory CD4 of the iNKT cell secretion of activation+T
Helper cell proliferation.CD4+T cell is divided into different t helper cell subgroups under the action of different cytokines.INKT cell
Th1/ proinflammatory (IFN-γ, TNF-α) or Th2/ anti-inflammatory (IL-4,10,13) cell factor, this cascade enhancing can be secreted
Effect excite the innate immunity and acquired immunity, the tumour cell of the induced killer MHC positive simultaneously.It swells to MHC feminine gender
Oncocyte, iNKT cell show NK cell sample MHC independence cell cytotoxic activity after the activation of the glycolipid antigens such as α-GalCer,
Pass through the direct killings such as inducing expression perforin/relevant apoptosis induction ligand of granzyme FasL/Fas or TNF (TRAIL).
To CD1d+Tumour cell (such as acute T lymphocyte leukaemia, monocytic leukemia and other hematologic malignancies),
With Direct Recognition and effective target cell lethal effect can be showed.
Serious iNKT cell quantity reduction and functional disturbance are often accompanied by tumor patient body.Due to oneself expression phosphoric acid sheath
Ammonia alcohol receptor (S1P1R) and a variety of chemokine receptors (C-X-C chemokine receptor) turn iNKT cell selective
Tumor locus is moved on to, causes to recycle in colon cancer, head and neck cancer, breast cancer, clear-cell carcinoma, melanoma and Peripheral Blood of Patients with Hepatocellular Carcinoma
Property iNKT cell quantity substantially reduce, but cell generate IFN-γ ability do not damage.Although body-internal-circulation iNKT cell
Quantity is reduced, but iNKT cell can be proliferated rapidly through α-GalCer stimulation in vitro, and the iNKT cell of in-vitro multiplication is same
Sample shows preferable cellulotoxic effect killing tumor cell.It is a variety of in recent years based on human body iNKT cellular immunotherapy tumour
Scheme is developed, but in the iNKT cell to tumour patient body of clinical selectivity injection Activated in Vitro, remission rate is lower, is immunized
Reaction individual difference is larger, and antitumous effect is ideal not to the utmost.The mankind have CD4+And CD4-INKT cell, according to cytokine-expressing
It is divided into Th1 sample iNKT cell and Th2 sample iNKT cell.Wherein, Th1 sample iNKT cell generates IFN-γ, and function is to mediate directly
It connects tumor-killing and raises the function of immune response and immunosurveillance in tumor microenvironment.The phenotype mark of Th1 sample iNKT cell
Will is great expression CD8 α and considerably less CD8 α β, and there is also CD4-CD8-。CD8+INKT cell ratio CD4+Or CD4-CD8-
INKT cell secretes more IFN-γ, and cytotoxicity is also stronger.Due to CD4 in human peripheral iNKT cell+INKT cell accounts for
Relatively high (~90%), traditional amplification method expand without selectivity Th1 sample iNKT cell, and the iNKT cell after amplification is each
Hypotype ratio is unstable, and it is larger to individual difference is immunoreacted after tumour patient to cause feedback.Therefore, technological difficulties master at present
It concentrates in the amplification of the sufficient amount of functional iNKT cell with tumor-killing and mediated immunity supervisory function bit.
There are proliferation times using the iNKT cell that fixed concentration rhIL-2 joint α-GalCer is obtained for conventional amplification method
After long (~4 weeks), amplification after limited (100 times average), the amplification of iNKT cell proportion relatively low (~20%), growth multiple
INKT cell CD4+It accounts for relatively high (~40%), the problems such as internal killing ability is low, is not able to satisfy clinical demand.The present invention adopts
With the increased mode of rhIL-2 concentration gradient, offer α-GalCer using DC, combine rhIL-15, rhIL-7, CD3 monoclonal resists
Body is by iNKT cell by CD4+It is converted into CD4-.INKT cell can be expanded 100,000 times in 21 days by culture, wherein CD8+INKT cell accounts for
Than > 50%, CD4+INKT cell accounting < 10%.Solve the problems, such as iNKT quantity and purity, and selective amplification Th1 sample
INKT cell can sufficiently meet the needs of clinical treatment.
Summary of the invention
The present invention provides a kind of amplification Activiation method of concentration gradient rhIL-2 iNKT cell relied on and its applications.This
The method of invention is for existing iNKT amplification in vitro culture efficiency is lower, asks Th1 sample, Th2 sample iNKT cell non-selectivity etc.
Topic proposes a kind of more effective and specific amplification Th1 sample iNKT lymphocyte method.
It in the iNKT methods for cell expansion that a kind of concentration gradient rhIL-2 provided by the invention is relied on, cultivates the 0th day, is added
100ng/mlα-GalCer；It cultivates the 0-11 days, increases rhIL-2 concentration in a manner of gradient.
Preferred embodiment provided by the invention are as follows: the time of fixed intervals, rhIL-2 concentration is in equal difference or waits than increasing；Interval
Time it is 1-72 hours preferred, the preferred 10-400IU/ml of rhIL-2 concentration.
The preferred technical solution of the present invention are as follows: the mature DC of same donor Fiber differentiation is added on the 7th day in culture；DC is adding
100ng/ml α-GalCer is first added and is incubated within two hours before entering iNKT cell；It cultivates the 14th day and uses Anti-iNKT
MicroBeads human is sorted, and the iNKT cell sorted is added in the coated culture bottle of OKT3 monoclonal antibody and trains
It supports；CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium；OKT3 MAb concentration is 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 solution of the present invention is as follows:
(1) peripheral blood mononuclear cells PBMC is extracted；
(2) final concentration of 100ng/ml α-GalCer is added in the PBMC cell extracted to step (1) to cultivate 48 hours；
(3) rhIL-2 concentration in culture medium is gradually increased in a manner of gradient into step (2) culture cell.
Further, in step (3) rhIL-2 concentration be it is increased with Fixed Time Interval, the Fixed Time Interval is
1-72 hours.
Further, it is in that equal ratio or equal difference mode increase that the rhIL-2 concentration, which is 10-400IU/ml,；The 11st of culture the
It when, rhIL-2 concentration increases to 400IU/ml.
Further, it cultivates the 7th day and mature dendritic cell, mature dendron shape is added into step (3) cell culture medium
The quantity ratio of cell and iNKT cell is 1:10.
Further, before iNKT cell is added 100ng/ml α-GalCer is first added in two hours in mature dendritic cell
It is incubated for.
Further, it cultivates the 14th day and is sorted using Anti-iNKT MicroBeads human, sorted
INKT cell is added in the coated culture bottle of CD3 monoclonal antibody and cultivates.
Further, CD3 MAb concentration is 1-5 μ g/ml, preferably 2 μ g/ml.
Further, CD28 antibody, rhIL-15 and rhIL-7 are added when culture；CD28 antibody concentration is 1-5 μ g/ml, excellent
It is selected as 3 μ g/ml；RhIL-15 concentration is 1-10ng/ml, preferably 5ng/ml；RhIL-7 concentration is 1-10ng/ml, preferably
5ng/ml。
The method is cultivating the 21st day harvest iNKT cell, and iNKT cell number improves 100,000 times, wherein 90% the above are
CD4iNKT cell has the function of up-regulation immune response and killing tumour.
The present invention solves the problems, such as the quantity and purity of iNKT cell expansion ex vivo culture in the prior art, cultivates 21 days
INKT cell can be expanded to 100,000 times, and selective amplification Th1 sample iNKT cell, wherein CD8+INKT cell accounting > 50%,
CD4+INKT cell accounting < 10%, can sufficiently meet the needs of clinical treatment.RhIL-2 concentration is gradually increased, can cultivated
Seedling selection expands iNKT cell, in this way, can improve iNKT cell proportion in culture to 6% left side at the 7th day
The right side, iNKT cell number improve 60 times.It is thin in the 7th day addition α-GalCer coated maturation DC, orientable induction Th1 sample iNKT
Born of the same parents' amplification, promotes CD4+INKT cell is to CD4?Conversion.It in this way, can be at the 14th day by iNKT cell proportion in culture
It improves to 16% or so, iNKT cell number and improves 1000 times.The 14th day pure iNKT cell obtained through magnetic bead sorting is cultivated, according to
The T cell characteristic that iNKT cell has is added in the coated culture bottle of CD3 monoclonal antibody and cultivates, and costimulatory molecules are added
CD28 antibody can make the iNKT cell massive amplification of purifying, and rhIL-15 and rhIL-7, which is added, can maintain Th1 sample iNKT cell to increase
It grows.The 21st day harvest iNKT cell is being cultivated, iNKT cell number improves 100,000 times, wherein 90% the above are CD4?INKT cell,
With up-regulation immune response and direct cytotoxicity.The iNKT cell amplification efficiency of present invention amplification and activation is higher, selectivity
Ground expands Th1 sample iNKT cell, strengthens the function of the enhancing of amplification in vitro iNKT cellular immunity and tumour immunity monitoring.
Detailed description of the invention
Fig. 1 .rhIL-2 handles iNKT cellular processes
Fig. 2 difference rhIL-2 E-test handles iNKT cell Flow cytometry figure
Fig. 3 .iNKT cell expansion ex vivo cell quantity linear graph
The experiment of Fig. 4 .iNKT cell killing tumour cell
Fig. 5 .iNKT cell secretion of gamma-IFN capacity experimental
Fig. 6 .iNKT cell kills liver cancer cells experiment in animal body
Specific embodiment
The embodiment of the present invention is described below in detail, the example of illustrated embodiment is shown in the accompanying drawings.In following embodiments
Experimental method, particular technique or condition person is not specified, then the institute such as in " ATCC cell culture handbook " according to conventional laboratory conditions
When stating condition, and having clearly stated Reagent Company's specification in embodiment, then to specifications proposed by condition carry out.Institute
Production firm person is not specified with reagent or instrument, being can be with conventional products that are commercially available.
Reagent used in the embodiment of the present invention:
AIM V cell culture medium, ThermoFisher company
Lymphoprep separating liquid (instant), Axis-Shield company
CD3 monoclonal antibody, CD28 monoclonal antibody, Biolegend company
IL-2, IL-7, IL-15, Peprotech company
α-GalCer, Enzo Life Sciences company
Anti-iNKT MicroBeads human, Miltenyi Biotec company
Fixation/Permeabilization Solution Kit with BD GolgiPlugTM, BD company
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-γ, Biolegend company
1 peripheral blood mononuclear cells of embodiment (PBMC) is extracted
(1) early morning adopts the anticoagulant venous blood of 20ml.
(2) 15ml Lymphoprep separating liquid is added in 50ml centrifuge tube.
(3) it is slowly added to isometric 0.9% sterile saline gentle inversion in anticoagulant venous blood three times, mixes well,
Blood after being diluted.
(4) blood after dilution is slowly added into Lymphoprep separating liquid surface layer using 2ml sterile dropper, all moved into
After be sure not to shake or reverse.
(5) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:4/0, room temperature centrifugation
30min。
(6) the extra blood plasma in top layer part is sucked out.Buffy coat is gently sucked out with 2ml aseptic straw, moves into new
In 50ml centrifuge tube, the buffy coat in all pipes is sucked in the same 50ml centrifuge tube.0.9% sterile physiological salt is added
Water is mixed well 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 medium that incubation is taken out from 37 DEG C of incubators, takes 2ml to mix well cell precipitation, pays attention to light
Soft operation avoids generating bubble.Remaining culture medium is then added, mixes cell.
(10) cell suspension of mixing is moved into 75cm2Tissue Culture Flask shakes gently mixing, is placed in 37 DEG C of incubators
Middle culture.
2 iNKT cell of embodiment expands for the first time
(1) PBMC extracts the final concentration of 100ng/ml α-GalCer of same day addition and cultivates 48 hours.
(2) concentration that rhIL-2 is gradually increased into culture medium is separated from cell to culture the 11st day, and rhIL-2 concentration is in
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 separation, every 48 hours (remove the 10th day to the 11st day and be spaced 24 hours),
RhIL-2 concentration is in wait to increase than mode.
As shown in Figure 1B, since cell separation, every 72 hours (remove the 10th day to the 11st day and be spaced 48 hours),
RhIL-2 concentration increases in equal difference mode.
The Fiber differentiation of 3 Dendritic Cells of embodiment
(1) PBMC for obtaining density-gradient centrifugation method is added in Tissue Culture Dish, 37 DEG C with the resuspension of AIM V culture medium
5%CO2Culture.
(2) 3 as a child took out culture dish, shaked gently, and suspension cell is sucked out.
(3) the AIM V culture medium of rhIL-4 containing 500IU/ml, 500IU/ml rhGM-CSF are added into culture dish.
(4) respectively after incubation the 3rd day, the 5th day half amount change liquid, fresh rhIL-4 containing 500IU/ml, 500IU/ml is added
The AIM V culture medium of rhGM-CSF.
(5) it cultivates the 6th day and final concentration of 10ng/ml rhTNF- α is added
(6) the 7th days harvest mature dendritic cells.
4 iNKT cell of embodiment stimulates again
(1) in 2 cell of embodiment and 3 cell culture of embodiment the 7th day, it is thin that mature dendron shape in embodiment 3 is collected in centrifugation
Born of the same parents are added final concentration 100ng/ml α-GalCer and are cultivated 2 hours with the resuspension of AIM V culture medium.
(2) the free α-GalCer of centrifugation removal, carries out cell count.With the first amplified production of iNKT cell: DC=10:
1 ratio by step (1) culture obtain DC be added embodiment 2 described in and the first amplified production of iNKT cell 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 cell (CD3+6B11+) ratio gradually increases, and CD4 in iNKT cell+Cell proportion by
It gradually reduces, CD4?CD8?Cell, CD8+Cell proportion is gradually increased.As shown in Fig. 2A, rhIL-2 concentration is selected in example 2
Increase in equal than mode, in the 14th day iNKT cell > 15%, CD8+Cell > 50%, CD4+Cell < 10%.By Fig. 2 B institute
Show, selects rhIL-2 concentration to increase in equal difference mode in embodiment 2, in the 14th day iNKT cell > 4%, CD8+Cell >
40%, CD4+Cell < 10%.In conclusion it is in equal difference mode that a preferred embodiment of the invention, which is rhIL-2 concentration,
Increase, a further preferred embodiment is that rhIL-2 concentration is in wait to increase than mode.
The iNKT cell massive amplification that embodiment 5 purifies
(1) the coated Tissue Culture Flask of CD3 monoclonal antibody should be made at the 13rd day: 2 μ g/ml CD3 monoclonals of preparation are anti-
Body running liquid (0.9% sterile saline).Prepare two completely new 75cm2Big bottle is each added what 5ml had diluted in big bottle
Antibody working solution.Bottleneck is tightened, body is jiggled, CD3 monoclonal antibody dilution is made sufficiently to infiltrate bottom of bottle.It is sealed with sealed membrane
Good culture bottle nozzle lays flat and saves into 4 DEG C of refrigerators.
(2) it cultivates the 14th day, carries out iNKT cell sorting using Anti-iNKT MicroBeads human, collect thin
Born of the same parents.
(3) it uses and contains 100IU/ml IL-2,3 μ g/ml CD28 antibody, 5ng/mlrhIL-7,5ng/mlrhIL-15's
AIM V culture medium mixes well iNKT cell, pays attention to gentle manipulation, avoids generating bubble, prepare cell concentration not less than 5 ×
106The cell suspension of a/ml mixes cell.
(4) the coated culture bottle of CD3 monoclonal antibody is taken out from 4 DEG C of refrigerators, discards liquid, and 0.9% nothing of 10ml is added
Culture bottle of bacterium brine；The cell suspension of mixing is moved into 75cm2Tissue Culture Flask shakes gently mixing, puts
It is placed in 37 DEG C of incubators and cultivates.
(5) incubation observation cell state and culture medium color can supplement suitable culture medium according to growing state.
(6) cell can be transferred to new culture bottle after stimulating 3 days by CD3 antibody, and supplement the culture medium of corresponding volume.
(7) the 21st day harvest cell is cultivated.
INKT cell expansion ex vivo 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 × 104A (accounting for PBMC total number of cells 0.1% or so), by culture in 21 days available 1 × 109
A iNKT cell.
The experiment of 6 iNKT cell killing tumor cell of liver of embodiment
Effector cell: the iNKT cell for the massive amplification 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
It detects iNKT cell and lactic dehydrogenase (Lactic is used to tumor cell of liver killing capacity experimental
Dehydrogenase, LDH) method for releasing.The specific operation method is as follows:
(1) target cell concentration is adjusted to 1 × 105A/ml.
(2) target cell is transferred in 96 orifice plates according to 100 holes μ l/, three multiple holes of every group of setting.
(3) target cell Spontaneous release hole (negative control) is set.Effector cell is not added and only adds 100 μ l culture solutions.
(4) maximum relief hole (positive control) is set.Effector cell is not added and only adds 100 μ l10%NP40.
(5) 100 μ l of effector cell, effector cell: target cell=0:1 are added in each experimental port；1:1；3:1；10:1；
30:1；50:1.Final concentration of 100ng/ml α-GalCer is added in each hole.
(6) 37 DEG C of 5%CO2Culture collected cell conditioned medium in 6 hours and carries out lactic dehydrogenase (LDH) detection.
(7) culture solution supernatant is drawn, detection LDH numerical value calculates activity.Killing activity (%)=[(experimental group A value-is total certainly
So release A value)/(the total Spontaneous release A value of maximum release group A value -)] × 100%.
Killing experiments grouping are as follows: 1, DC joint iNKT groups of cells；2, iNKT cell independent role group；3, PBMC control group；
4, phosphate buffer (PBS) blank control group.
By Fig. 4 the results show that iNKT cell offers α-GalCer as antigen presenting cell in DC, amplification in vitro can be made
INKT cell acute activation, 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 function.
It detects iNKT cell IFN-γ secretion ability and uses flow cytometry.The specific operation method is as follows:
(1) it is inoculated with according to killing experiments target cell (HepG2 cell is selected in this experiment) concentration and cell number；
(2) according to effector cell: target cell=50:1 Inoculating efficiency cell.Final concentration of 100ng/ml α-is added in each hole
GalCer。
(3) 2 μ l BD GolgiPlug are added in every holeTM。
(4) 37 DEG C of 5%CO2Cell was collected in culture in 6,12 hours respectively, carried out padding.
(5) rupture of membranes and dye intracellular are carried out according to Fixation/Permeabilization Solution Kit specification
Color.
(6) fixed cell, carries out Flow cytometry and analysis.
INKT cell IFN-γ secretion capacity experimental grouping are as follows: 1, DC joint iNKT groups of cells；2, iNKT cell is individually made
With group；3, PBMC control group；4, phosphate buffer (PBS) blank control group.
By Fig. 5 the results show that iNKT cell is when DC is activated 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 function of In-vitro specificity amplification further illustrates and uses institute of the present invention
The iNKT cell for stating method amplification is mainly Th1 sample iNKT cell, has powerful tumor-killing and immune enhancing function.
Liver cancer killing experiments in embodiment 7.iNKT multicellular animal body
Select the hepatocellular carcinoma cells system HepG2-GFP of GFP fluorescent marker as tumour cell, subcutaneous implantation in 8 week old,
Immunodeficient mouse NOD-SCID back of mice of the weight between 20~30g, every group of 5 NOD-SCID mouse of experimental group.To
After tumour grows up to, temporally puts tail vein and adopt people's iNKT cell, fluorescence imaging observation tumour growth situation and to measure tumour big
It is small.
The specific operation method is as follows:
(1) prepared by iNKT cell: the iNKT cell for the massive amplification that embodiment 5 obtains is suspended in physiological saline before injection
In it is spare；
(2) after iNKT cell infusion frequency is since 14 days 1 times a week, totally 4 weeks, 4 injections, negative control group was note
1% physiological saline group containing albumin is penetrated, positive controls are intraperitoneal injection adriamycin 2mg/kg, are injected into abdominal cavity after 14 days start
Every 5 days 1 time；
(3) inject the determination of iNKT cell dosage: on the basis of the adult of 60kg, iNKT cell is expected to use agent in clinic
Amount is 1 × 109~1 × 1010A cell (about 1.6 × 107~1.6 × 108A cell/kg).With NOD-SCID mouse weight 30g
On the basis of, clinical projected dose is equivalent to 5 × 105~5 × 106A cell/only.We using high dose cell as test dose,
Every mouse adopts 5 × 106A cell；
(4) during injecting, the general symptom of variation, motility, appearance of general state etc. is observed above 3 times a week, and
Whether grasp has dead animal.The volume (mean tumor volume) of tumour: during injection, caliper is used 3 times a week
(calipers) long axis and short axle of tumour are measured, and utilizes calculation formula below, measures the volume of tumour: V (mean
tumor volume,mm3)=AB2/ 2 (A=long axis length, B=minor axis lengths).Every group of 3 NOD-SCID mouse of experimental group.Tool
Body is grouped as follows shown in table.
By Fig. 6 the results show that NOD-SCID mouse subcutaneous implantation HepG2-GFP is after tumour cell 14 days, tail vein is adopted
People's iNKT cell, time point of adopting are respectively the 14th, 21,28,35 day, and hereafter fluorescence imaging observes tumor size weekly, and surveys
It measures gross tumor volume (Fig. 6 A).Such as Fig. 6 B, 6C the results show that iNKT cell is adopted to after tumor-bearing mice, mouse tumor size compared with
Control negative control group is obviously reduced, and iNKT cell is prompted to play the role of apparent 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, those skilled in the art
Member carries out the present invention it is appreciated that without departing substantially from technical scope described in the claims and substantive content
A variety of different modifications and change are possible.The invention also includes these above-mentioned modifications and changes.
Claims (11)
1. the iNKT methods for cell expansion that a kind of concentration gradient rhIL-2 is relied on, which is characterized in that culture the 0th day is added
100ng/mlα-GalCer；It cultivates the 0-11 days, increases rhIL-2 concentration in a manner of gradient；The time of fixed intervals, rhIL-
2 concentration are in equal difference or wait than increasing, and interval time is 1-72 hours, and rhIL-2 concentration is 10-400IU/ml；It cultivates the 7th day
The mature DC of same donor Fiber differentiation is added, before iNKT cell is added 100ng/ml α-GalCer is first added in two hours in DC
It is incubated for, culture is sorted on the 14th day using Anti-iNKT MicroBeads human, the iNKT cell sorted
It is added in the coated culture bottle of OKT3 monoclonal antibody and cultivates, CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium:
It is 1-5 μ g/ml, rhIL-15, rhIL-7 concentration is 1- that OKT3 MAb concentration, which is 1-5 μ g/ml, CD28 antibody concentration,
10ng/ml。
2. the method according to claim 1, wherein comprising steps of
(1) peripheral blood mononuclear cells PBMC is extracted；
(2) final concentration of 100ng/ml α-GalCer is added in the PBMC cell extracted to step (1) to cultivate 48 hours；
(3) rhIL-2 concentration in culture medium is gradually increased in a manner of gradient into step (2) culture cell.
3. according to the method described in claim 2, it is characterized in that, the method also includes following steps:
(4) the 7th day of culture, the Dendritic Cells of same donor Fiber differentiation maturation is added into step (3) cell culture medium,
The quantity ratio of mature dendritic cell and iNKT cell is 1:10；The mature dendritic cell the preparation method comprises the following steps: by density level bands
The PBMC that degree centrifugal process obtains is added in Tissue Culture Dish, 37 DEG C of 5%CO with the resuspension of AIMV culture medium2Culture；It 3 hours will training
It supports ware to take out, shake, suspension cell is sucked out；RhIL-4 containing 500IU/ml, 500IU/ml rhGM-CSF are added into culture dish
AIMV culture medium；Respectively after incubation the 3rd day, the 5th day half amount change liquid, fresh rhIL-4 containing 500IU/ml, 500IU/ is added
The AIMV culture medium of ml rhGM-CSF；It cultivates and final concentration of 10ng/ml rhTNF- α is added within the 6th day；7th day harvest adult tree
Prominent shape cell.
4. according to the method described in claim 3, it is characterized in that, step (3) cell culture is being added in mature dendritic cell
Before base, it is resuspended with AIMV culture medium and final concentration 100ng/ml α-GalCer incubation 2 hours is added.
5. according to the method described in claim 2, it is characterized in that, the method further includes following steps:
(5) it cultivates the 14th day, carries out iNKT cell sorting using Anti-iNKT MicroBeads human, sorting is obtained thin
Born of the same parents cultivate in the coated culture bottle of CD3 antibody, and CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium, cultivate in 37 DEG C
It is cultivated in case.
6. according to the method described in claim 5, it is characterized in that, CD3 antibody concentration is 1-5 μ g/m.
7. according to the method described in claim 5, it is characterized in that, CD3 antibody concentration is 2 μ g/ml.
8. according to the method described in claim 5, it is characterized in that, it is 1-5 that CD28 antibody concentration is added in step (5) culture medium
μg/ml；RhIL-15 concentration is 1-10ng/ml；RhIL-7 concentration is 1-10ng/ml.
9. according to the method described in claim 8, it is characterized in that, it is 3 μ that CD28 antibody concentration is added in step (5) culture medium
g/ml；RhIL-15 concentration is 5ng/ml；RhIL-7 concentration is 5ng/ml.
10. according to the method described in claim 5, it is characterized in that, cell is transferred to by step (5) CD3 antibody after stimulating 3 days
New culture bottle, and the culture medium of corresponding volume is supplemented, cultivate the 21st day harvest cell.
11. method described in any claim in -10 according to claim 1, which is characterized in that the method is in culture the 21st
Its harvest iNKT cell, iNKT cell number improves 100,000 times, wherein 90% the above are CD4?INKT cell has up-regulation immune anti-
It should be with the effect of killing tumour.
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| Expression, purification, refolding, and characterization of octreotide-interleukin-2: A chimeric tumor-targeting protein;JING JIANG et al;《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 》;20111231;552页左栏 |
| Generation of Human iNKT Cell Lines;Xiangming Li et al;《Bio Protoc.》;20130331;第4-5页 |
| Innate-Like Effector Differentiation of Human Invariant NKT Cells Driven by IL-7;Claudia de Lalla et al;《J Immunol》;20081231;4416-4422页 |
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