CN104611293A - Method for inducing amplification of NK(natural killer) cells by traditional Chinese medicine combination in vitro and application of method - Google Patents
Method for inducing amplification of NK(natural killer) cells by traditional Chinese medicine combination in vitro and application of method Download PDFInfo
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Abstract
The invention provides a method for inducing amplification of NK(natural killer) cells by a traditional Chinese medicine combination in vitro and the application of the method. The method comprises the following steps: adding cell factor IL(interleukin)-2, cell factor IL(interleukin)-15, astragalus polysaccharide and Chinese wolfberry polysaccharide to an NK cell culture fluid, wherein the concentration of the astragalus polysaccharide is 150-250 micogram/ml, and the concentration of the Chinese wolfberry polysaccharide is 100-200 micogram/ml. For the NK cells prepared by the cultural method, the amplification multiple, the cell activity and a lethal effect are greatly improved, stimulating factors used in the method are approved and can be used for clinic, besides, the method is simple and convenient to operate, is low in cost and is suitable for large-scale application.
Description
Technical field
The present invention relates to biomedicine field, specifically refer to a kind of method and application thereof of the external Chinese traditional medicine composition induced amplification NK cell in conjunction with Chinese medical extract astragalus polysaccharides and lycium barbarum polysaccharide and cytokine.
Background technology
Report that China's tumor incidence and mortality ratio raise year by year according to Chinese tumour annual report in 2013, tumour has become mankind's number one killer.Current China is clinically to the pattern that oncotherapy mainly takes operation, radiotherapy, chemotherapy to combine, and the body function of traditional therapy often major injury patient, makes patient's immunizing power sharply decline, cause quality of life generally poor.In recent years, along with the development of immunology, cytobiology, oncobiology, and to the further investigation of tumour Emergence and Development mechanism, start tumour cell treatment.Namely utilize and isolate mononuclearcell from autologous patient peripheral blood, feed back in patient body after Activation In Vitro, amplification, strengthen patient immune function or direct killing tumour cell and virus infected cell.Cell therapy can effective inhibition tumor cell metastasis and extension, capture that traditional therapy exists not thoroughly, the difficult problem such as easily transfer, side effect be large.
Natural killer cell (NK cell) treatment is wherein a kind of non-characteristic cell therapy, and clinical study shows that NK is antitumor and has certain curative effect.Research shows, amplification in vitro autologous NK cells can be used safely in clinical, does not have patient to occur serious toxic side effects, and the energy reduction of patient state of an illness, except autologous NK cells treatment, allosome NK cell therapy also has report.Current allosome NK cell therapy is mainly used in leukemia treating, and efficacy and saferry obtains clinical affirmative necessarily.Although NK cell is for clinical cancer therapy, NK cells in vitro separate complex, purification effect is poor, amplification times is low, cytoactive is weak, fragmentation effect is low, significantly limit it and applies clinically.For many years, investigator attempts developing a kind of method that preparation method is simple, amplification times is high, killing activity is strong always, although improve a lot in amplification times and purity, but still there are some problems.Present stage amplification in vitro NK cell mainly takes feeder layer cells Dual culture method and immunological magnetic bead sorting method, but feeder layer cells Dual culture adds to through improved tumor cell line by NK cell, the security that such transformation is applied clinically is not protected, and the operating process of immunological magnetic bead sorting method is loaded down with trivial details, cost is high, is not suitable for clinical large-scale application.
Summary of the invention:
In order to solve problems of the prior art, the invention provides a kind of method of the external Chinese traditional medicine composition induced amplification NK cell in conjunction with Chinese medical extract and cytokine, adopt the NK cell that the method obtains, amplification times, cytoactive and fragmentation effect are significantly increased, Chinese medical extract used, cytokine all go through to be used safely in clinical, the method is easy and simple to handle simultaneously, and cost is low, is applicable to large-scale application.
For reaching this goal of the invention, the technical solution used in the present invention is: a kind of method of external Chinese traditional medicine composition induced amplification NK cell, comprise and add cytokine IL-2 in NK cell culture fluid, cytokine IL-15, astragalus polysaccharides and lycium barbarum polysaccharide, described astragalus polysaccharides concentration is 150-250 μ g/ml, and lycium barbarum polysaccharide concentration is 100-200 μ g/ml.
More specifically, the method for external Chinese traditional medicine composition induced amplification NK cell of the present invention, comprises the steps:
(1) mononuclearcell is separated: venipuncture gathers peripheral blood in patients, is transferred in the anti-freezing bottle containing heparin sodium, mixes up and down.Peripheral blood sample is moved to centrifuge tube to carry out centrifugal, abandons upper plasma, normal saline dilution blood sample, and lymph parting liquid is separated mononuclearcell, the resuspended mononuclearcell of physiological saline, collected by centrifugation mononuclearcell;
(2) cell kind bottle: it is 5 × 10 that above-mentioned mononuclearcell serum-free medium is diluted to cell concn
5~ 2 × 10
6/ ml, adds cytokine N1 and N2, and the volume according to adding nutrient solution selects corresponding culturing bottle, and puts into 37 DEG C, 5%CO
2incubator is cultivated;
(3) cell amplification: be cultured to the 5th day, cytokine N2, N3, Chinese medical extract astragalus polysaccharides and lycium barbarum polysaccharide is added to nutrient solution, after this within 2-3 days, observation of cell growth conditions goes down to posterity, and adds same concentrations cytokine and Chinese medical extract simultaneously;
(4) cell suspension preparation: collect and be cultured to 21 days cells, centrifugally abandon supernatant liquor, physiological saline re-suspended cell, centrifuge washing, abandons supernatant, adds physiological saline, human serum albumin, abundant mixing, 70 μm of cell strainer filtration cells, and be transferred to the sealing of transfusion bags high-frequency thermocompressor.
Preferably, the microbiotic in the serum-free medium described in step (2) can be selected from penicillin, Streptomycin sulphate, gentamicin sulphate, is more preferably 80U gentamicin sulphate.
Preferably, in described step (2), initiator cell inoculum density is 1 × 10
6/ ml; Described serum-free medium is LONZA X-VIVO 15; Described cytokine N1 is CD3McAb, and concentration is 10ng/ml; Described cytokine N2 is IL-2, and concentration is 500U/ml.
Preferably, in described step (3), cytokine N2 is IL-2, and concentration is 500U/ml, and described cytokine N3 is IL-15, and concentration is 10ng/ml; The concentration of described astragalus polysaccharides is 150-250 μ g/ml, preferably 200 μ g/ml; The concentration of lycium barbarum polysaccharide is 100-200 μ g/ml, preferably 125 μ g/ml.
Preferably, in described step (4), human serum albumin concentration is 2%.
Another object of the present invention is also that the application of the natural killer cell obtained by cultural method of the present invention is included in the 4th day after cancer patients's the 1st end of chemotherapy and injects 3 NK cells in patient body, carried out the 2nd chemotherapy after 7 days, it is 10 that described average each cell feeds back dosage
9individual.
The present invention carries out the method that external efficient amplification natural killer cell is cultivated theoretical basis by adding astragalus polysaccharides and lycium barbarum polysaccharide in NK cell culture fluid is:
Astragalus polysaccharides and lycium barbarum polysaccharide are a kind of polysaccharide that can improve immune function of human body extracted from the Radix Astragali and matrimony vine, usually be used for clinical with immunostimulant form, research shows, astragalus polysaccharides APS and lycium barbarum polysaccharide LBP all has significant restraining effect to Several Kinds of Malignancy, as liver cancer, cancer of the stomach, colorectal carcinoma, lung cancer, mammary cancer etc., and can IL-2 be promoted, IL-6, IL-12, the cytokine-expressings such as TNF-a, strengthen NK cytophagy, the present inventor is by astragalus polysaccharides and lycium barbarum polysaccharide and cytokine combined induction NK cell, discovery can effectively improve NK cell proliferation and kill capability.
The present invention is from scale operation operability, and clinical application security and validity angle are set out, the preferred plan of optimization Combined with Chinese Herbal extract and cytokine induction NK cell, and compared with additive method, the present invention has following advantage:
1. the present invention adds cytokine IL-2 and can directly activate NK cell, promotes NK cell proliferation, and strengthens NK emiocytosis IFN-γ, IL-4; Utilize IL-15 can raise NK cell surface NKG2D expression of receptor, strengthen the release of cytotoxic molecule TRAIL and pore-forming protein, improve it to tumor cell lysis activity.These two kinds of combination of cytokines are utilized can greatly to improve NK cell proliferation and killing activity.
2. the present invention add Chinese medical extract astragalus polysaccharides and lycium barbarum polysaccharide, the oral liquid of these two kinds of polysaccharide is used for protective foods by state approval, its security is protected, and its validity demonstrated in enhancing human immunity activity has obtained clinical confirmation.Research proves, astragalus polysaccharides and lycium barbarum polysaccharide can raise the cytokine-expressings such as IL-2, TNF-a, IL-4, impel DC cell maturation, stimulate T cell propagation, improve the phagocytic function of scavenger cell, NK cell, strengthen the effect of the aspects such as NK cell killing activity, can obviously grow and propagation by Tumor suppression.
3. utilize the NK cell that the present invention obtains, all be significantly increased at cell quantity, purity, killing activity, levels of cytokine secretion, utilize present method to cultivate, cell quantity improves 678.46 times, cell killing activity reaches to 79.21%, NK cell purity and reaches 75.61%.Found by clinical application, after present method acquisition NK cell is used for the treatment of tumour patient, there is not serious adverse reaction in all patients, patient's muscle power and appetite have clear improvement.
Accompanying drawing explanation
Fig. 1 is the comparison diagram of the NK cells expanded added before and after astragalus polysaccharides and lycium barbarum polysaccharide;
Fig. 2 is the comparison diagram of the NK cell purity added before and after astragalus polysaccharides and lycium barbarum polysaccharide;
Fig. 3 is the comparison diagram of the T cell purity of adding before and after astragalus polysaccharides and lycium barbarum polysaccharide;
Fig. 4 is the comparison diagram adding Treg cell purity before and after astragalus polysaccharides and lycium barbarum polysaccharide;
Fig. 5 adds before and after astragalus polysaccharides and lycium barbarum polysaccharide the K562 cell killing activity comparison diagram of the 14th day;
Fig. 6 adds before and after astragalus polysaccharides and lycium barbarum polysaccharide the K562 cell killing activity comparison diagram of the 21st day;
Fig. 7 is the comparison diagram adding cytokine secretion IFN-γ before and after astragalus polysaccharides and lycium barbarum polysaccharide;
Fig. 8 is the comparison diagram adding cytokine secretion IL-4 before and after astragalus polysaccharides and lycium barbarum polysaccharide;
Fig. 9 be to the cellkilling capacity detection experiment of amplified production NK cell often group establish the schematic diagram of three parallel groupings.
Embodiment
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
(1) peripheral blood mononuclear cell is separated: sign informed consent postscript patient, patient 50ml peripheral blood and the mixing of 10ml sodium heparin anticoagulant is gathered under aseptic condition, it is on average assigned in 2 pipe 50ml centrifuge tubes, often pipe 30ml blood sample sample, the centrifugal 10min of 800g, after centrifugal end, abandon upper plasma, in centrifuge tube, volume is about 15ml ± 3ml, adds equal-volume physiological saline to 30ml, fully mix in the blood sample being separated blood plasma.Then manage in new 50ml centrifuge tube to 2 and respectively add 15ml lymph parting liquid, the volume ratio of lymph parting liquid and dilution blood sample is 1:2, and pipettor is drawn dilution blood sample and slowly added on Ficoll along tube wall at distance liquid level 1cm place, the then centrifugal 20min of 700g.After centrifugal, 2 pipe tunica albuginea layers absorption managed in new 50ml centrifuge tube to 1, is settled to 50ml with 0.9% physiological saline, the centrifugal 10min of 800g, abandons supernatant, resuspended to 50ml with 0.9% physiological saline, get 300 μ l samples for counting, carry out the 2nd centrifugal 300g × 10min afterwards.
(2) cell kind bottle: after centrifugal, removes supernatant liquor, with the serum-free medium X-VIVO15 containing gentamicin sulphate, cell concn is adjusted to 1.0 × 10
6/ ml, the volume according to adding nutrient solution selects corresponding culturing bottle, adds nutrient solution volume and is designated as V, V=number concentration × 50ml ÷ 1 × 10
6/ ml, if V≤7ml, cell is inoculated in T25 culturing bottle; If 7ml≤V≤30ml, cell is inoculated in T75 culturing bottle; If V>=30ml, be inoculated in T175 culturing bottle.Add the cytokine of N1=10ng/mlCD3 monoclonal antibody and N2=500U/ml IL-2 subsequently, and put into 37 DEG C, 5%CO2 incubator cultivates, cultivate and be designated as the 1st day the same day.
(3) cell amplification: be cultured to the 5th day, sampling counting, with X-VIVO15 nutrient solution by cell dilution to 1.0 × 10
6/ ml, cell is on average assigned to culturing bottle, the cytokine, the N3 that add N2=500U/ml IL-2 are 10ng/ml IL-15, Chinese medical extract astragalus polysaccharides concentration be 150-250 μ g/ml and lycium barbarum polysaccharide concentration is 100-200 μ g/ml, after this within 2-3 days, observation of cell growth conditions goes down to posterity, add same concentrations cytokine and Chinese medical extract simultaneously, cultivate 21 days collecting cells.
(4) cell suspension preparation: collect and be cultured to 21 days cells, the centrifugal 10min of 800g, abandon supernatant liquor, physiological saline re-suspended cell, the centrifugal 10min of 800g, abandons supernatant, add physiological saline 10ml, 2% human serum albumin 4ml, abundant mixing, 70 μm of cell strainer filtration cells, and be transferred to the sealing of 200ml transfusion bags high-frequency thermocompressor.
Embodiment 2 (control group)
Except in (3) cell amplification, do not add Chinese medical extract astragalus polysaccharides and other concrete operation steps of lycium barbarum polysaccharide as embodiment 1.
Embodiment 3: the detection of NK cells expanded is carried out to amplified production
500 μ l cells are collected respectively at the 1st, 7,14,21 day, blow and beat into individual cells, or centrifugal 400g × 5min, abandon supernatant, add 500 μ lPBS, use Counterstar automatic blood cell count miriam, the cell count before being cultivated divided by the 1st day by counting cells sum on the same day according to Trypan Blue principle is cells expanded.This method is utilized to calculate cells expanded.According in culturing process, cell state is observed, find astragalus polysaccharides concentration to be 200 μ g/ml and lycium barbarum polysaccharide concentration be that the experimental group cell state of 125 μ g/ml is best, afterwards related experiment all with astragalus polysaccharides 200 μ g/ml and lycium barbarum polysaccharide 125 μ g/ml for experimental group.
Embodiment 4: the detection of NK cell surface marker is carried out to amplified production
The cell suspension collecting 5ml at the 1st, 7,14,21 day respectively carries out phenotype analytical.Counting after collecting, PBS damping fluid diluting cells concentration to 1 × 10
6the centrifugal 10min of/ml, 400g, abandons supernatant, and add 5mlPBS damping fluid re-suspended cell, centrifugal condition is the same, abandons supernatant after centrifugal, and it is resuspended to add 500 μ lPBS.The corresponding antibodies adding to respectively FACS test tube (Falcon) according to following table and cell suspension, vibration mixing, lucifuge hatches 15min, add 500 μ lPBS damping fluids, the centrifugal 10min of 400g, remove non-binding antibody, in Falcon pipe, add 500 μ lPBS resuspended, machine testing in preparation.Wherein Treg cell phenotype detects needs fixing agent and rupture of membranes agent process, concrete steps are as follows: add 10 μ l cell suspensions, 5 μ lCD4,5 μ l CD25 at FACS test tube (Falcon), PBS washed cell, what add 1ml after vortex concussion re-suspended cell fixes/rupture of membranes working fluid, and vortex mixing again, lucifuge hatches 30-60min.Add 2ml rupture of membranes agent centrifuge washing cell and abandoning supernatant.Add the normal rabbit serum 100 μ l of 2% (2 μ l), 15min is hatched lucifuge 4 DEG C, directly add the fluorescent mark FOXP3 antibody 20 μ l diluted, lucifuge 4 DEG C hatches at least 30min, adds 2ml rupture of membranes agent centrifuge washing cell and abandoning supernatant.With the PBS re-suspended cell of appropriate volume, and upper machine testing analyzing.
Embodiment 5: the cellkilling capacity of amplified production NK cell is detected
K562 clone is taken out as target cell from liquid nitrogen, recover in 37 DEG C of water-baths, 5ml substratum RPMI1640 re-suspended cell is added in super clean bench, the centrifugal 5min of 400g, abandon supernatant, add 5ml nutrient solution resuspended, put into 37 DEG C, the incubator quiescent culture of 5%,CO2 24 hours countings, with PBS damping fluid, target cell concentration is adjusted to 1.0 × 10
5/ ml, by the cultivation NK cell action effect cell of 14,21 days, imitates target ratio according to 1:1,1:10,1:20,1:50, and adjustment NK cell concn is 1.0 × 10
5/ ml, 1.0 × 10
6/ ml, 2.0 × 10
6/ ml, 5.0 × 10
6/ ml, and mix with K562 target cell, join in 96 orifice plates, simultaneously laying effect cell hole action effect control group, Target cell wells is as target cell group, and often group establishes 3 parallel group, as shown in Figure 9.Experimental group: effector cell and target cell respectively add 100 μ l, effect group: effector cell and each 100 μ l of RPMI1640 substratum, target cell group: target cell and each 100 μ l of RPMI1640 substratum, every hole final volume is made to be 200 μ l, three groups of cells put into 37 DEG C simultaneously, 5%CO2 incubator hatches 24 hours, every hole adds 10 μ lMTT solution, is placed in 37 DEG C, 5%CO
2incubator hatches 4 hours, hatches OD value during end microplate reader mensuration 570nm, calculation formula: kill rate=[1-(effect target mixing OD value-target cell group OD value)/effect OD value] × 100%.
Embodiment 6:ELISA detects IL-4 and IFN-gamma cells factor content.
Supernatant liquor 500 μ l is collected respectively at the 1st, 7,14,21 day.Operate according to Zheng Bai bio tech ltd, Beijing four test kit specification sheets.Establish blank well, standard orifice, testing sample hole respectively, blank well adds sample diluting liquid 100 μ l, and standard orifice adds 100 μ l standard substance, and testing sample hole adds 100 μ l liquid to be measured, and mixing, adds enzyme plate lid, 37 DEG C of reaction 120min; Every orifice plate adds 100 μ l biotin labelled antibodies working fluids afterwards, and 37 DEG C of reaction 60min, discard reacted liquid in hole, dry, wash plate 3 times, soak 1-2min at every turn, the 350 every holes of μ l/, dry; Every hole adds substrate solution 90 μ l successively, 37 DEG C of lucifuge colour developings; Every hole adds stop bath 50 μ l, termination reaction successively; The OD value in each hole is measured successively with microplate reader 450nm.
Claims (10)
1. a method for external Chinese traditional medicine composition induced amplification NK cell, is characterized in that comprising and in NK cell culture fluid, adds cytokine IL-2, cytokine IL-15 and Chinese medical extract.
2. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 1, it is characterized in that described Chinese medical extract is astragalus polysaccharides and lycium barbarum polysaccharide, described astragalus polysaccharides concentration is 150-250 μ g/ml, and lycium barbarum polysaccharide concentration is 100-200 μ g/ml; The concentration of described cytokine IL-2 is 500U/ml, and the concentration of described cytokine IL-15 is 10ng/ml.
3. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 1 and 2, is characterized in that being made up of following steps:
(1) mononuclearcell is separated: venipuncture gathers peripheral blood in patients, is transferred in the anti-freezing bottle containing sodium heparin anticoagulant, mixes up and down; Peripheral blood sample is moved to centrifuge tube to carry out centrifugal, abandons upper plasma, normal saline dilution blood sample, and lymph parting liquid is separated mononuclearcell, the resuspended mononuclearcell of physiological saline, collected by centrifugation mononuclearcell.
(2) cell kind bottle: above-mentioned mononuclearcell is diluted to 5 × 10 with containing antibiotic serum-free medium
5~ 2 × 10
6/ ml, adds cytokine N1 and N2, and the volume according to adding nutrient solution selects corresponding culturing bottle, and puts into 37 DEG C, 5%CO
2incubator is cultivated.
(3) cell amplification: be cultured to the 5th day, basis of microscopic observation cell state, add cytokine N2, N3, astragalus polysaccharides and lycium barbarum polysaccharide to nutrient solution, within after this 2-3 days, observation of cell growth conditions goes down to posterity, and adds same concentrations cytokine and Chinese medical extract simultaneously.
(4) cell suspension preparation: collect and be cultured to the cell of 21 days, centrifugally abandons supernatant liquor, physiological saline re-suspended cell, centrifuge washing, abandon supernatant, add physiological saline, human serum albumin, abundant mixing, 70 μm of cell strainer filtration cells, and be transferred to the sealing of transfusion bags high-frequency thermocompressor.
4. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 3, is characterized in that the serum-free medium described in step (2) is LONZA X-VIVO 15.
5. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 3, is characterized in that the microbiotic in the serum-free medium described in step (2) can be selected from penicillin, Streptomycin sulphate, the large element of sulfuric acid celebrating.
6. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 5, it is characterized in that, described microbiotic is 80U gentamicin sulphate.
7. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 3, is characterized in that, described cytokine N1 is 10ng/ml CD3McAb, N2 be 500U/ml IL-2, N3 is 10ng/ml IL-15; Described astragalus polysaccharides concentration is 150-250 μ g/ml, and preferred concentration is 200 μ g/ml, and lycium barbarum polysaccharide concentration is 100-200 μ g/ml, and preferred concentration is 125 μ g/ml.
8. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 3, is characterized in that described in step (2), cell implantation concentrations is 1 × 10
6/ ml.
9. the method for external Chinese traditional medicine composition induced amplification NK cell according to claim 3, is characterized in that described in step (4), human serum albumin concentration is 2%.
10. the application of the natural killer cell obtained for the method for the arbitrary described external Chinese traditional medicine composition induced amplification NK cell of claim 1 to 9, it is characterized in that in patient body, injecting 3 natural killer cell cells in the 4th day after being included in cancer patients's the 1st end of chemotherapy, carried out the 2nd chemotherapy after 7 days, it is 10 that described average each cell feeds back dosage
9individual.
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| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
| CN114874984A (en) * | 2022-06-16 | 2022-08-09 | 杭州中赢生物医疗科技有限公司 | Method for in vitro induction amplification of NK cells by adopting plant extract |
| CN114874984B (en) * | 2022-06-16 | 2024-05-03 | 杭州中赢生物医疗科技有限公司 | Method for in-vitro induction amplification of NK cells by adopting plant extracts |
| CN114891743A (en) * | 2022-06-23 | 2022-08-12 | 杭州中赢生物医疗科技有限公司 | High-purity NK cell in-vitro amplification culture method |
| CN117487750A (en) * | 2023-11-08 | 2024-02-02 | 青岛西凯生物技术有限公司 | Use of NK cells in the treatment of immune related disorders |
| CN117487750B (en) * | 2023-11-08 | 2024-06-21 | 青岛西凯生物技术有限公司 | Use of NK cells in the treatment of immune related disorders |
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