CN103834614A - Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof - Google Patents
Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof Download PDFInfo
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Abstract
The invention provides a culture method for monkshood polysaccharide-induced nature killer T cell (Nature Killer T cell, NKT) proliferation. The method comprises the characteristics as follows: human peripheral blood, marrow or a mononuclear cell with an umbilical cord blood source are continuously cultivated by traditional Chinese medicine monkshood polysaccharide and recombinant human interleukin 2, a lot of nature kill T cells are obtained, NK<1.1+> and CD<8+> are expressed, interferon gamma is secreted, the ratio in lymphocyte at the 14th day is more than 16.15%, the order of magnitude can be up to over 5*10<9>, and the nature kill T cell have effective anti-tumor and anti-virus functions. A clinical application of the NKT cell obtained by the method is also provided by the invention.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of Radix Aconiti Lateralis Preparata polysaccharide induction natural killer T cells propagation.The mononuclearcell that employing human peripheral, marrow or the Cord blood the present invention relates to obtains through in-vitro separation purifying, cultured continuously under Aconitum carmichaeli polysaccharide and people's recombinant interleukin 2 conditions, obtains a large amount of NKT cells easily, expresses NK1.1
+and CD8
+, and secretion of gamma-IFN, there is antitumor and antiviral function, can be used for all kinds of cancers including solid tumor and neoplastic hematologic disorder, and the immunotherapy of multiple courses for the treatment of of multiple virus infection.
Background technology
Immunocyte anti-tumor virus is widely used clinically, particularly cytokine-induced killer cell, and the toxic side effect that does not have current conventional treatment to bring, it is comparatively safe, has ensured patient's quality of the life and dignity.Form in the lymphocyte of body immune system, except T, B, NK extracellular, also have a kind of new clone-NKT cell.This cell not only itself has effect antitumor, antiviral and inhibition autoimmune disorder, and can also stimulate the activity of the immunocyte such as T, NK, strengthens its cellulotoxic effect, suppresses the generation of GVHD.The many course of therapy of NKT cell vaccine are to patient's curative effect and have obvious help lifetime, and approved clinically.
At present, the source that obtains NKT cell is mainly from peripheral blood in patients or marrow isolates mononuclearcell, at cytokine profiles alone or in combination under inducing action, as rhIL-2, recombinant human interleukin 15, recombination human interleukin 4, rHuGM-CSF (GM-CSF), recombinant human interleukin-12, recombinant human interleukin-11 8, or with the effect of α-semi-lactosi schwann's sheath amine alcohol (α-Galcer) combined induction, obtain NK1.1
+with TCR β
+nKT cell, cell count magnitude is 5 × 10
8individual left and right, only meets the clinical treatment of 1-2 time, has affected to a great extent the treatment to patient, and need to again take a blood sample when many course of therapy, gives patient very large stress.
Radix Aconiti Lateralis Preparata polysaccharide has important regulating effect to immunity system, can not only activated T cell, B cell, NK cell, cytotoxic cell and LAK, maturing dendritic cell can be effectively impelled, the generation of the cytokines such as interleukin, tumor necrosis factor alpha, G CFS and Interferon, rabbit can also be promoted.The present invention is by white corpuscle in human peripheral, marrow or Cord blood, and the mononuclearcell obtaining through purification, applies Radix Aconiti Lateralis Preparata polysaccharide and recombinant interleukin 2 combined continuous are cultivated, and obtains easily a large amount of NK1.1
+and CD8
+nKT cell, and secretion of gamma-IFN.So not only can meet many courses for the treatment of of NKT cellular immunization treatment, continue the immunologic function of performance NKT cell, contribute to improve clinical efficacy and extend the survival of patients phase, and application Radix Aconiti Lateralis Preparata polysaccharide can reduce costs greatly.
Summary of the invention
Prior art mainly goes out mononuclearcell by separation and purification from human peripheral, marrow or Cord blood, be generally 20 milliliters to 300 milliliters, at cytokine profiles alone or in combination under inducing action, as rhIL-2, recombinant human interleukin 15, recombination human interleukin 4, rHuGM-CSF (GM-CSF), recombinant human interleukin-12, recombinant human interleukin-11 8, or with the effect of α-semi-lactosi schwann's sheath amine alcohol (α-Galcer) combined induction, cultured continuously obtains NKT cell, its quantity is limited, is only 5 × 10
8individual cell left and right.If a NKT cell vaccine treatment is at least used 5 × 10
8individual cell, multipotency meets the treatment of 1-2 time.In addition, after NKT cell-stimulating, can regulate by secretion IL-4 and IFN-γ the differentiation of Th cell, thereby realize immunne response and autoimmune adjusting.Thereby, in order to realize antitumor, the antiviral immunne response of NKT cell, also need to impel NKT emiocytosis IFN-γ, the cellullar immunologic response effect of performance Th1.
The tumour cell of NKT cell energy exogenous fast breeding kills and wounds, and NKT cell need to be strengthened constantly, maintains the tumour cell of body " nonego " is killed and wounded, effectively the recurrence of inhibition tumor cell and transfer.Thereby NKT cell vaccine needs repeatedly to treat in clinical application, improve better patient clinical curative effect and extend lifetime.The NKT cell quantity obtaining by prior art, cannot complete patient's treatment repeatedly and guarantee patient's long-term efficacy.
Radix Aconiti Lateralis Preparata polysaccharide has the lymphopoietic effect of B, the T of promotion, and impels human leukocyte secretion interferon-gamma, tumor necrosis factor alpha etc., impel lymphocyte to Th1 cytodifferentiation, and mediated immunity is replied.The technology of the present invention obtains mononuclearcell by human peripheral, marrow and Cord blood through purification, and through Radix Aconiti Lateralis Preparata polysaccharide and the effect of IL-2 Co stituation, propagation obtains a large amount of NKT cells easily, and the order of magnitude can be greater than 5 × 10
9above, while treatment, use 0.5 × 10 at every turn
9nKT cell, can be used for nearly more than 5 times.NKT cell can be expressed NK1.1 simultaneously
+and CD8
+, secretion of gamma-IFN, can produce the Th1 cellullar immunologic response to tumour cell.
The present invention relates to a kind of preparation method of Aconitum carmichaeli polysaccharide induced natural killer T cells propagation, it is characterized in that, comprise following operation: the mononuclearcell of human peripheral, marrow or Cord Blood-Derived, cultured continuously under Aconitum carmichaeli polysaccharide and people's recombinant interleukin 2 conditions, obtain a large amount of natural killer T cells, express NK1.1
+and CD8
+, and secrete interferon-gamma, and in the time of the 14th day, accounting for lymphocytic ratio more than 12.61%, enzyme linked immunological spot detection interferon-gamma is more than 30, the order of magnitude can reach 5 × 10
9above; There is effective antitumour and antiviral Th1 cellullar immunologic response function.
The approximately 50mL human peripheral gathering carries out in-vitro separation purifying, remove red corpuscle, thrombocyte and granulocyte etc., obtain mononuclearcell, mononuclearcell is resuspended with RPMI1640 substratum, tongue is expected blue dyeing counting, using RPMI1640 perfect medium (containing 1ug/mL-1000ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 1IU-400IU/mL IL-2,10% (v/v) foetal calf serum) to adjust to cell concn is 1-5 × 10
6/ mL, is placed into 75cm by cell culture fluid
2in culturing bottle, every bottle of 30ml, in 37 DEG C, 5%CO
2in incubator, cultivate;
(contain 1ug/mL-1000ug/mL Radix Aconiti Lateralis Preparata polysaccharide in the 3rd, 6,9,11,13,15,17 days long-pending fresh RPMI1640 perfect mediums of supplementary monoploid, 1IU-400IU/mLIL-2,10% (v/v) foetal calf serum), cultivate the 14th, 16 and 18 days results NKT cells;
Preferably, the method for the invention is: the RPMI1640 perfect medium that inducing culture NKT cell uses is preferably and contains 10ug/mL-500ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 5IU-200IU/mL IL-2,10% (v/v) foetal calf serum.
Described NKT cell is carried out to Cell viability, cell phenotype and interferon-gamma enzyme linked immunological spot detection, and Cell viability reaches more than 98%; It expresses NK1.1
+and CD8
+molecule, wherein, NK1.1
+and CD8
+cell is for being greater than 12.61%, and interferon-gamma enzyme linked immunological spot detection is more than 30.
Cultivation all can be used for frozen to the NKT cell of 7-12 days results, be about to obtain NKT cell tongue and expect blue counting, 5 × 10
71 milliliter of individual NKT cell cryopreservation, cryopreservation tube is placed in program temperature reduction box, puts into-80 DEG C of Ultralow Temperature Freezers and spends the night, and within second day, goes to liquid nitrogen cryopreservation.NKT can recover when needed and carry out multiplication culture, still can keep same Cell viability, cell phenotype and secretion interferon-gamma, impels the function of Th1 cellullar immunologic response.
Preferably, the method for the invention is: the NKT cell of cultivating the 7th day results all can be used for frozen.
Wherein, preferred Cell viability detection method is: calculate the viable count of cultivating the 14th day.Get the nutrient solution that 1mL cultivates, at 1000rpm, 4 DEG C centrifugal 10 minutes, cell is resuspended with substratum, get 20 times of 20ul enchylema 1 × PBS dilutions, diluent adds the tongue of 1 times of volume to expect blue solution, after mixing, joins cell counting count board, under inverted microscope, observe counting, blue dyeing be dead cell, achromophil is viable cell, Cell viability reaches more than 98%.
Preferred cell phenotype detection method is: get tongue and expect 2 × 10 after blue dyeing counting
6cell, points two groups, first group is added to respectively and has 20 μ L FITC mark mouse-anti people's CD8 monoclonal antibodies and 20 μ L PE mark mouse-anti people NK1.1 monoclonal antibodies; Second group is homotype contrast, adds to respectively and has 20 μ L FITC mark mouse IgG1 and 20 μ L PE mark mouse IgG1 (BD company).Be placed in 4 DEG C of refrigerators dyeing 30 minutes, then use 1 × phosphate buffered saline buffer washing three times of 1mL, finally use the cell after the resuspended washing of 1 × PBS of 0.5mL, the cell after gained washing detects with FACSCalibur basic model flow cytometer.After testing, NK1.1
+/ CD8
+cell is 12.61%, NK1.1
+cell is 15.72%, CD8
+cell is 52.23%.Detected result shows that the cell obtaining by the method for the invention is that NKT cell has its typical phenotype of expression.
NKT cell detects interferon-gamma secretion level by dot enzyme-linked reaction kit (R & D company of the U.S.), NKT cell forms the enzyme connection spot of a lot of IFN-γ, show the ability of its energy secretion of gamma-IFN, thereby cause the immunne response of Th1 cell.
Brief description of the drawings
Fig. 1 represents the fluidic cell figure (FL1 path is FITC-CD8, and FL2 path is PE-NK1.1) of the NKT cell of Radix Aconiti Lateralis Preparata polysaccharide inducing culture acquisition of the present invention;
Fig. 2 represents that the NKT cellular enzymes connection immunodot test of Radix Aconiti Lateralis Preparata polysaccharide inducing culture of the present invention detects interferon-gamma secretion result figure.
Embodiment
The present invention finds by Aconitum carmichaeli polysaccharide associating interleukin-22, human peripheral, marrow or Cord blood to be induced through the mononuclearcell of purification, can breed easily and obtain a large amount of NKT cells, and the order of magnitude can be greater than 5 × 10
9if use 0.5 × 10 while treatment at every turn
9nKT cell, can be used for nearly more than 5 times.NKT cell can be expressed NK1.1 simultaneously
+and CD8
+, secretion of gamma-IFN, can produce the Th1 cellullar immunologic response of tumour cell is obtained to special NKT cell vaccine, can be used for nearly 13 above and 3 clinical applications more than course for the treatment of.
Preparation method to a kind of large scale culturing NKT cell vaccine the present invention relates to is specifically described.The present invention relates to adopt blood cell separator by gathering white corpuscle in human peripheral, obtain a large amount of mononuclearcells through in-vitro separation purifying, obtain a large amount of prematurity NKT cells at human granulocyte-macrophage colony stimulating factor and IL-4 inducing culture; After prematurity NKT cell loading tumour specific antigen, and under tumor necrosis factor alpha and lipopolysaccharide-induced culture condition, obtain the ripe NKT cell vaccine with antitumor action.
The present invention relates to the preparation method of Aconitum carmichaeli polysaccharide induced natural killer T cells propagation, it is characterized in that, comprise following operation: the mononuclearcell of human peripheral, marrow or Cord Blood-Derived, cultured continuously under Aconitum carmichaeli polysaccharide and people's recombinant interleukin 2 conditions, obtain a large amount of natural killer T cells, express NK1.1
+and CD8
+, and secrete interferon-gamma, and in the time of the 14th day, accounting for lymphocytic ratio more than 12.61%, the order of magnitude can reach 5 × 10
9above, interferon-gamma enzyme linked immunological spot detection is more than 30; There is effective antitumour and antiviral Th1 cellullar immunologic response function.
The approximately 50mL human peripheral gathering carries out in-vitro separation purifying, remove red corpuscle, thrombocyte and granulocyte etc., obtain mononuclearcell, mononuclearcell is resuspended with RPMI1640 substratum, tongue is expected blue dyeing counting, using RPMI1640 perfect medium (containing 1ug/mL-1000ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 1IU-200IU/mL IL-2,10% (v/v) foetal calf serum) to adjust to cell concn is 1-5 × 10
6/ mL, is placed into 75cm by cell culture fluid
2in culturing bottle, every bottle of 30ml, in 37 DEG C, 5%CO
2in incubator, cultivate;
(contain 1ug/mL-1000ug/mL Radix Aconiti Lateralis Preparata polysaccharide in the 3rd, 6,9,11,13,15,17 days long-pending fresh RPMI1640 perfect mediums of supplementary monoploid, 1IU-200IU/mLIL-2,10% (v/v) foetal calf serum), cultivate the 14th, 16 and 18 days results NKT cells;
Preferably, the method for the invention is: the RPMI1640 perfect medium that inducing culture NKT cell uses is preferably and contains 10ug/mL-500ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 5IU-200IU/mL IL-2,10% (v/v) foetal calf serum.
Described NKT cell is carried out to Cell viability, cell phenotype and interferon-gamma enzyme linked immunological spot detection, and Cell viability reaches more than 98%; It expresses NK1.1
+and CD8
+molecule, wherein, NK1.1
+and CD8
+cell is for being greater than 12.61%, and interferon-gamma enzyme linked immunological spot detection is more than 30.
Cultivation all can be used for frozen to the NKT cell of 7-12 days results, be about to obtain NKT cell tongue and expect blue counting, 5 × 10
71 milliliter of individual NKT cell cryopreservation, cryopreservation tube is placed in program temperature reduction box, puts into-80 DEG C of Ultralow Temperature Freezers and spends the night, and within second day, goes to liquid nitrogen cryopreservation.NKT can recover when needed and carry out multiplication culture, still can keep same Cell viability, cell phenotype and secretion interferon-gamma, impels the function of Th1 cellullar immunologic response.
Preferably, the method for the invention is: the NKT cell of cultivating the 7th day results all can be used for frozen.
As the Tissue Culture Plate using in manufacture method of the present invention, Tissue Culture Dish, culturing bottle, cell culture bags, can exemplify, be 75cm
2tissue Culture Flask, 175cm
2the equipment (container) for cell cultures such as Tissue Culture Flask, 250mL cell culture bags, all can be used for the present invention, preferred cell culture bag.
In manufacture method of the present invention, NKT cell is carried out frozenly, to frozen storing liquid, without particular restriction, but preference is as being 50% calf serum, 40% cell culture fluid and 10% dimethyl sulfoxide (DMSO), wherein more preferably NKT cell culture fluid of cell culture fluid.
In substratum, serum can be added or blood plasma is cultivated.Their additions in substratum are not subject to particular restriction, as are greater than 0 capacity % to 20 capacity %, and can change according to different cultivation stages the consumption of serum or blood plasma, are preferably 5% (volume ratio).For example, can interim reduce serum or plasma concentration uses.In addition, as the source of serum or blood plasma, any that can be oneself in (meaning identical from institute's cultured cells source) or non-oneself (meaning with the source of institute cultured cells different), from the viewpoint of security, serum or the blood plasma of preferably oneself originating.In addition, also can add if human serum albumin and so on is through the serum composition of separation and purification.
The preparation of Aconitum carmichaeli polysaccharide induced NKT of the present invention cell proliferation is implemented with above-mentioned various compositions and substratum.The cultivation culture condition using in the present invention is also not particularly limited, and can use the condition using in common cell cultures.For example, can under the conditions such as 37 DEG C, 5%CO2, cultivate.The operation such as can also be implemented as follows: interval reasonable time adds fresh culture and carrys out diluting cells nutrient solution, or change substratum, or change cell cultures equipment etc.
The present invention also provides NKT cell antineoplaston of many courses for the treatment of repeatedly in clinical application, brings into play in vivo better anti-tumor immune response, reaches good result for the treatment of.In addition, above-mentioned NKT cell also tool has the following advantages, and repeatedly NKT cell therapy will cause Th1 cellullar immunologic response better many courses for the treatment of, produces efficiently, antineoplastic immune effect longer, is therefore beneficial to very much the raising of patient's curative effect and the prolongation of lifetime.
Below, the present invention is done more specifically and described in conjunction with the embodiments, but the invention is not restricted to this.
Embodiment mono-
Human peripheral source mononuclearcell is cultivated and is obtained NKT cell
Patient with breast cancer, female, 24 years old, routine blood test detected result was 3.7 × 10
9individual white corpuscle/liter, patient's 50ml peripheral blood (signing Informed Consent Form with this patient) gathered; Centrifugal 10 minutes of the peripheral blood 1500rpm gathering, collects upper plasma, and deactivation is after 30 minutes in 56 DEG C of water-baths, and 3000rpm obtains autologous plasma in centrifugal 10 minutes, for subsequent use.After hemocyte solution is resuspended with the 1 × PBS (pH=7.4) of 1 times of volume, add the physiological saline of 1/4 0.6% hydroxyethylamyle, mix and leave standstill 30 minutes afterwards, draw white cellular layer as far as possible; White cellular layer, through Ficoll-Hypaque density gradient centrifugation, obtains mononuclearcell RPMI1640 substratum resuspended, and tongue expects that blue dyeing counting is 1.62 × 10
8.
It is 3 × 10 that peripheral blood mononuclear cell is adjusted to cell concn with RPMI1640 substratum
6, substratum contains 150ug/mL Radix Aconiti Lateralis Preparata polysaccharide, and 20IU/mL IL-2 and 10% (v/v) foetal calf serum, joins 75cm
2in Tissue Culture Flask (Corning Incorporated), at 37 DEG C, 5%CO
2in incubator, hatch; In the time cultivating by the 3rd day, supplement 1 times of fresh RPMI1640 substratum of volume, this fresh culture is containing 150ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 20IU/mL IL-2 and volume ratio 10% (v/v) foetal calf serum.
Cultured continuously to the 6 days, NKT cell culture fluid is transferred in 250mL cell culture bags, and the fresh RPMI1640 perfect medium that supplements 1 times of volume (contains 150ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 20IU/mLIL-2,10% (v/v) foetal calf serum), continue at 37 DEG C, 5%CO
2in incubator, cultivate.Supplemented the fresh RPMI1640 perfect medium of 1 times of volume in the 9th, 11,13,15,17 days (containing 150ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 20IU/mLIL-2,10% (v/v) foetal calf serum), cultivate the 14th, 16 and 18 days results NKT cells.
Cultivate and got wherein 1 bottle of NKT cell by the 7th day, results NKT cell, tongue is expected blue counting, quantity is 18.7 × 10
7, NKT cell can be frozen, and 50 × 10
61 milliliter of individual NKT cell cryopreservation, cryopreservation tube is placed in program temperature reduction box, puts into-80 DEG C of Ultralow Temperature Freezers and spends the night, and within second day, goes to liquid nitrogen cryopreservation, with for subsequent use.
Calculate the viable count of cultivating the 14th day.Get the nutrient solution that 1mL cultivates the last day, at 1000rpm, 4 DEG C centrifugal 10 minutes, cell is resuspended with RPMI1640 substratum, get 20 μ L, 20 times of 1 × PBS (pH7.4) dilutions for enchylema, diluent adds the tongue of 1 times of volume to expect blue solution, after mixing, joins cell counting count board, under inverted microscope, observe counting, blue dyeing be dead cell, achromophil is viable cell, Cell viability reaches more than 98%.
Get tongue and expect 2 × 10 after blue dyeing counting
6cell, points two groups, first group is added to respectively and has 20 μ LFITC mark mouse-anti people's CD8 monoclonal antibodies and 20 μ L PE mark mouse-anti people NK1.1 monoclonal antibodies; Second group is homotype contrast, adds to respectively and has 20 μ L FITC mark mouse IgG1 and 20 μ L PE mark mouse IgG1 (BD company).Be placed in 4 DEG C of refrigerators dyeing 30 minutes, then use 1 × phosphate buffered saline buffer washing three times of 1mL, finally use the cell after the resuspended washing of 1 × PBS of 0.5mL, the cell after gained washing detects with FACSCalibur basic model flow cytometer.Fluidic cell the results are shown in Figure one (FL1 path is FITC-CD8, and FL2 path is PE-NK1.1), NK1.1 after testing
+/ CD8
+cell is 12.61%, NK1.1
+cell is 15.72%, CD8
+cell is 52.23%.Detected result shows that the cell obtaining by the method for the invention is that NKT cell has its typical NK1.1 of expression
+phenotype.
Embodiment bis-
Enzyme linked immunological Spot Jest (Elispot) detects NKT emiocytosis IFN-γ level
With the coated Elispot96-orifice plate of IFN-gamma antibody:
Anti-human IFN-gamma antibodies R4-6A2 (Pharmingen company-18181D) 25ul that adds 1mg/mL in 5ml PBS, makes concentration reach 5ug/ml, and every hole adds 50ul, adds a cover 4 DEG C and spends the night.Discard coated antibody, use containing the perfect medium (RPMI-10) of 10% foetal calf serum and wash once, every hole adds 200ul perfect medium, adds and is placed on room temperature sealing 2 hours, discards substratum.
NKT cell-stimulating:
(1) get and cultivate to the 14th day NKT cell, the blue counting of placenta, uses R-5 without 3 extent of dilution (1 × 10 of phenol red medium dilution
6, 3.3 × 10
5with 1 × 10
5).Join respectively in the coated Elispot96 orifice plate of IFN-gamma antibodies, making every pore volume is 100ul.
(2) the every hole of experimental group adds 100ul to contain the R1640-10 of restricted 9 peptides of MHC-1 (final concentration is that 1ug/ml stores concentration 1ug/ul, should be dilution in 1: 500).
(3) the every hole of control group adds 100ul substratum.
(4) negative control adds substratum.
Mix in 37 DEG C 5%CO
2in incubator, cultivate 24 hours.
The operation of ELISPOT test kit:
(1), with aseptic washing plate 2 times, with 1 × lavation buffer solution or PBST washing 6 times, while washing, embathe 1～2min at every turn.
(2) antibody (vitamin H coupling mouse-anti people IFN-gamma antibodies) is added in the dilution buffer liquid 1 of 12ml (10 groups of amounts), final concentration is 2ug/ml.
(3) every hole adds the upper step of 50ul mixed solution, and 4 DEG C are spent the night or room temperature 2 hours.
(4) wash 3 times with 1 × lavation buffer solution or PBST.
(5) Streptavidin-horseradish peroxidase concentrated solution A (BD company) being added to dilution buffer liquor ratio value is 1: 100, and every hole adds 50ul mixed solution.
(6) 4 DEG C are spent the night, with PBST washing 4 times, with PBS washing 2 times.
(7) every hole adds 50ul Elispot staining fluid (AEC substrate, the chromogen (chronogen) of article No.: 551951,20ul joins in the AEC substrate solution of 1mL).
(8) lucifuge room temperature is placed 5-60 minute, discards staining fluid, uses distilled water wash
(9) dry 2 hours or dried overnight of air at room temperature, save data.
96 hole Elispot plates join spot image automatic analyser by enzyme and detect spot number, and in Fig. 2, for enzyme connection spot image automatic analyser detects figure and spot count results, left side is corresponding dilution control group, produces without IFN-γ Elispot spot; Right side is 3 dilution test group, and IFN-γ Elispot spot is respectively 37,12 and 8; Show that NKT cell prepared by the present invention can secrete the IFN-gamma cells factor, 10
6the order of magnitude be that IFN-γ Elispot spot can reach more than 30.
Embodiment tri-
The cultivation of peripheral blood in patients source NKT cell
Patient one: patient with breast cancer, and female, 34 years old, routine blood test detected result was 6.1 × 10
9individual white corpuscle/liter.
Patient one: liver cancer patient, and man, 36 years old, routine blood test detected result was 3.6 × 10
9individual white corpuscle/liter.
Patient three: melanotic tumor patient, and man, 56 years old, routine blood test detected result was 7.0 × 10
9individual white corpuscle/liter.
Patient four: ovarian cancer patients, and female, 45 years old, routine blood test detected result was 6.6 × 10
9individual white corpuscle/liter.
Patient five: patients with lung cancer, and man, 49 years old, routine blood test detected result was 4.7 × 10
9individual white corpuscle/liter.
All sign Informed Consent Form with patient.
Gather patient 50mL peripheral blood through Ficoll-Hypaque density gradient centrifugation, obtain mononuclearcell.Patient one, patient two and patient three cultivate NKT cell according to the method Aconitum carmichaeli polysaccharide induced of embodiment mono-.
The mononuclearcell application prior art of patient four and patient's five derived from peripheral bloods is carried out inducing culture, use containing the RPMI1640 substratum (Gibco company of the U.S.) of 100ng/mL α-semi-lactosi schwann's sheath amine alcohol (α-Galcer), 50IU/mL interleukin-22 and 10% (v/v) foetal calf serum and carry out cultured continuously, use 75cm
2culturing bottle expands bottle cultured continuously to the 18 days.
Employing tongue is expected NKT cell count and the Cell viability that the blue calculating of dyeing is cultivated 14 days, and NKT cell passes through streaming antibody staining and flow cytometer detects, analysis of cells phenotype; Get 10
6individual NKT cell detects NKT emiocytosis IFN-γ level by Elispot.
Table one
Claims (5)
1. Aconitum carmichaeli polysaccharide induced natural killer T cells (Nature Killer T cell, NKT) preparation method of propagation, it is characterized in that the mononuclearcell of human peripheral, marrow or Cord Blood-Derived, cultured continuously under Aconitum carmichaeli polysaccharide and people's recombinant interleukin 2 conditions, obtain a large amount of natural killer T cells, express NK1.1
+and CD8
+, and secrete interferon-gamma, and in the time of the 14th day, accounting for lymphocytic ratio more than 12.15%, the order of magnitude can reach 5 × 10
9above.
2. preparation method according to claim 1, it is characterized in that human peripheral, marrow or Cord blood are through in-vitro separation purifying, remove red corpuscle, thrombocyte and granulocyte etc., obtain mononuclearcell, mononuclearcell is resuspended with RPMI1640 substratum, and tongue is expected blue dyeing counting, uses RPMI1640 perfect medium (containing 1ug/mL-1000ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 1IU-400IU/mL IL-2,10% (v/v) foetal calf serum) to adjust to cell concn be 1-5 × 10
6/ mL, is placed into 75cm by cell culture fluid
2in culturing bottle, every bottle of 30ml, in 37 DEG C, 5%CO
2in incubator, cultivate;
(contain 1ug/mL-1000ug/mL Radix Aconiti Lateralis Preparata polysaccharide in the 3rd, 6,9,11,13,15,17 days long-pending fresh RPMI1640 perfect mediums of supplementary monoploid, IIU-400IU/mLIL-2,10% (v/v) foetal calf serum), cultivate the 14th, 16 and 18 days results NKT cells.
3. preparation method according to claim 2, it is characterized in that, the RPMI1640 perfect medium that inducing culture NKT cell uses is preferably and contains 10ug/mL-500ug/mL Radix Aconiti Lateralis Preparata polysaccharide, 5IU-200IU/mL IL-2,10% (v/v) foetal calf serum.
4. preparation method according to claim 1, is characterized in that, described NKT cell is carried out to cell proliferation and cell phenotype detection, and tongue expects that blue counting cells propagation reaches 5 × 10
9above; Its high expression level NKT cell phenotype NK1.1
+and CD8
+, and secrete interferon-gamma (IFN-γ), wherein, in the 14th day NKT cell, contain NK1.1
+and CD8
+two positive cells are more than 12.61%, and enzyme linked immunological spot detection interferon-gamma is more than 30.
5. described in claim 1-4, prepare NKT and meet the application in clinical antitumor, antiviral therapy.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017854A (en) * | 2014-06-15 | 2014-09-03 | 浙江大学 | Method for evaluating killing capacity of NKT cells to liver parenchymal cells |
| CN104593326A (en) * | 2014-12-30 | 2015-05-06 | 杭州特马赛生物技术有限公司 | Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines |
| CN105219712A (en) * | 2015-11-16 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of NKT cell culture medium and NKT cell culture processes |
| CN110501483A (en) * | 2019-07-25 | 2019-11-26 | 同济大学 | A kind of active detection method of Chinese herbal medicine |
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2014
- 2014-03-12 CN CN201410087827.0A patent/CN103834614A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017854A (en) * | 2014-06-15 | 2014-09-03 | 浙江大学 | Method for evaluating killing capacity of NKT cells to liver parenchymal cells |
| CN104017854B (en) * | 2014-06-15 | 2016-03-30 | 浙江大学 | A kind ofly assess the method for NKT cell to hepatic parenchymal cells kill capability |
| CN104593326A (en) * | 2014-12-30 | 2015-05-06 | 杭州特马赛生物技术有限公司 | Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines |
| CN105219712A (en) * | 2015-11-16 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of NKT cell culture medium and NKT cell culture processes |
| CN110501483A (en) * | 2019-07-25 | 2019-11-26 | 同济大学 | A kind of active detection method of Chinese herbal medicine |
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