CN103923879B - A kind of preparation method of NK cytokine mixture and application thereof - Google Patents
A kind of preparation method of NK cytokine mixture and application thereof Download PDFInfo
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Abstract
The invention belongs to tumor biotherapy field, the NK cell that the activation method relating to a kind of NK cell obtains and cytokine mixture and application thereof.The NK cell that the present invention obtains is the tumor-killing cell with cytotoxicity cultivated in vitro, activate and increase, to be fed back in patient body can direct killing tumour cell, improve immunizing power; NK cell culture supernatant of the present invention and give lyophilize and concentrate the freeze-dried powder preparation made and contain and a series ofly have bioactive natural cytokines mixture, the mixture of this natural cytokines has good anti-tumor activity in antineoplaston.
Description
Technical field
The invention belongs to tumor biotherapy field, relate to a kind of preparation method and application thereof of NK cytokine mixture.
Background technology
Natural killer cell (Naturalkillercells, NK) be one of inherent immune defence line member of first in human body, also be to inhibiting tumor cell or virus the most by force, the most effective immunological lymphocyte, with other antitumor immune cells as cytotoxic T cell (CytotoxicTcells, or dendritic cell (Dendriticcells CTL), DC) compare, NK cell tool broad spectrum, by histocompatibility antigen restriction and do not need the startup of sensitization can directly start the attack of cancer cells, be therefore called natural killer cell.NK cell plays a part immunosurveillance to multi-infection microorganism and tumour cell, have antitumor, anti-infective and immunoregulatory effect, its target cell mainly contains tumour cell, virus infected cell, some autologous tissue's cell (as hemocyte), parasite etc.
Clinical experiment shows: after obtaining the immunocyte of tumour patient, feed back in patient body after carrying out stimulated in vitro, amplification cultivation, can direct killing tumour cell, recovery or strengthen the effect that the immunity function of patient reaches Tumor suppression growth.
The NK cell of Nk Cell Activity its kill and wound medium and mainly contain pore-forming protein, NK cytotoxic factor and TNF etc.The NK cell of activation can synthesize and secrete cytokine profiles, play the effect of immunomodulatory and hemoposieis and direct killing target cell, these cytokines comprise interleukin (Interleukins, ILs), G CFS (ColonyStimulatingFactors, CSFs), tumour necrosis factor (TumorNecrosisFactors, TNFs), Interferon, rabbit (Interferons, IFNs) and pore-forming protein (Perforin) etc.IL-2 is the important cytokine participating in immunne response, participates in inflammatory reaction, antitumor reaction and graft-rejection, plays an important role in modulating T cell immunne response; CSFs has the cytotoxicity (ADCC) strengthening cell-mediated antibody-dependant, and have direct chemotaxis to granulocyte and scavenger cell, play an important role in antineoplastic immune, CFSs and IL-2 can also induce the growth etc. of Precursor T-cell together; TNFs can express the IL-2 acceptor of MHC-II quasi-molecule and high-affinity by inducing T cell, collaborative IL-2 promotes that T cell produces IFN-γ and strengthens cytotoxic activity, also can strengthening activity and kill capability, the immunne response ability of scavenger cell, also having promoter action to the activity of the neutrophil leucocyte of inflammation local with assembling; IFNs is the cytokine with broad-spectrum antiviral, antitumor and immunomodulatory isoreactivity found at first.Tumour cell function is affected, the activity of Promote immunity cell by number of mechanisms; The effect of pore-forming protein is on target cell membrane, form poly pore-forming protein tubular channel, makes granzyme enter target cell, causes target cell apoptosis.
At present, the recombinant cytokine utilizing genetic engineering technique to produce is treated tumour, hematopoietic disorders, infection etc. as biological response modifier (BRM) and has been received good curative effect.U.S. food Drug Administration (FDA) approved recombinant interleukin 12(rIL-12) as the clinical treatment medicine of hairy cell leukemia, Kaposi's sarcoma and chronic hepatitis, approval restructuring IFN-γ is used for the treatment of chronic granulomatous disease and the clinical application of approval rIL-2 in gastric cells cancer.
NK cell, in culturing process, produces a series of natural cytokines and secretes form natural cytokines mixture (nonrecombinantcytokinemixture, NCM) in culture supernatant.
NCM therapy has been one methods for the treatment of safely and effectively by external confirmation, not only directly can play antitumor action, but also activates by number of ways and strengthen human immune system, remove the oncocyte having occurred to shift.Cytokine mixture Inter leukin Injection (the Multikine of Cel-Sci company of U.S. independent research
?) for antitumor application, by changing the cell cycle of tumour cell, thus improve tumour cell to the susceptibility of chemicotherapy.The mixture that natural IFN-α s injection (AlferonN) that Hemispherx biopharmaceutical company researches and develops is made up of twenties kinds of even more lymphokines, shows good therapeutic action in treatment pointed condyloma disease.
Need with PHA in tradition NK cell culture processes and/or tumor cell line (as K562, HFWT and K562-MB15-41BBL) makes stimulating factor and trophoderm cultivates NK cell, there is loaded down with trivial details cell culture step, use PHA and tumor cell line also to bring safety problem.
Summary of the invention
The present invention's technical problem first to be solved provides the NK cell culture processes of highly effective and safe more, and provides a kind of preparation method and application thereof of NK cytokine mixture.
For achieving the above object, the present invention adopts following technical scheme:
A kind of cultural method of NK cell, comprise the steps: the nutrient solution be placed in by the mononuclearcell of separator well containing OKT-3 antibody and IL-2 cytokine, cultivate after five days, within every 2 days, change a fresh NK cells substratum, and add IL-12 and IL-15, make its concentration in nutrient solution reach 10ng/ml and 50ng/ml respectively, be cultured to 21 days.
In tradition NK cell culture processes, PHA is as a kind of mitogen, be mainly used in activate immunity lymphocyte, due to its more difficult purification, and cost is high, all the time only in the lab as stimulating lymphopoietic reagent, being difficult to extensive popularization and application, and to immunocyte, there is the effect of overstimulation.The present invention only adopts IL-2, OKT-3, the efficient combination of IL-12 and IL-15, IL-12 and IL-15 added in the present invention can replace the effect of stimulating proliferation of the factor pair NK of cell strain secretion substantially, the Co stituation of these factors is conducive to the growth amplification of NK cell, can find that NK cell is in rapidly amplification state after within 5th day, adding IL-12 and IL-15, and the cell of other type starts to be in Developing restraint state gradually, the NK cell of higher degree can be obtained after 20th day, up to 64%, and obtain a large amount of NK cells, cell amplification efficiency is up to 510 times.
The present invention also provides the cell toxicant NK preparation method of cell preparation, specific as follows:
From cell cultures the 9th day every three days by part cell harvesting, supernatant is abandoned after centrifugal, cell precipitation Multiple electrolytes injection is centrifugal again after suspending, repeat once, abandon supernatant, suspended evenly by cell precipitation Multiple electrolytes injection, add human serum albumin, the preparation of toxicity NK cell preparation completes.
The present invention further provides a kind of preparation method of cytokine mixture, specific as follows:
From cell cultures 6th ~ 12 days centrifugal collecting cell supernatant liquors, by cell restored cells culturing bottle or cell culture bags; By supernatant liquor ultracentrifugation under low temperature, abandon precipitation, get supernatant ultrafiltration, micro-filtration, by for subsequent use for the filtrate 4 DEG C of short-term preservations obtained, or-20 DEG C save backup for a long time.
Under aseptic condition, be sub-packed in ampoule by above-mentioned filtrate equal-volume, and be placed in lyophilized plate ,-20 DEG C of pre-freeze supernatant liquors are to curdled appearance, vacuumize, sublimation drying simultaneously, remove ice crystal, fill in halogenated butyl rubber plug, add a cover aluminium-plastic cap sealing, preserve lyophilized powder or transport in 4 DEG C.
The NK cell that the inventive method employing itself has antineoplastic immune effect is cultivated, and can obtain the tumor-killing NK cell with cytotoxicity of activation after for some time.Simultaneously, can secrete in cell cultivation process the natural cytokines of multiple high-content in substratum (as table 1,2) in this specific period collecting cell culture supernatant, by ultracentrifugation, ultrafiltration micro-filtration, can obtain can for Clinical practice, containing multiple high-content natural cytokines mixture.Containing, for example pore-forming protein, granzyme and tumour necrosis factor etc. in obtained cytokine mixture, malignant cell can be caused to break or apoptosis, directly play antitumor action; Also containing other to have regulating effect as IL-10, TGF etc. the factor to body immune system, mutually can act synergistically in vivo, stimulate immune cell propagation, the maturation in body with tumor-killing effect, thus indirectly play antitumor action.Obtained cytokine mixture is natural cytokines, and compared with the recombinant cytokine of Clinical practice, have native conformation, activity is stronger; Various cytokine amount is immunocyte by optimum proportioning secretion, has reason to infer that the antitumor action that this proportioning may be powerful to NK cells play is relevant, also may be play a role in vivo most suitable.
Embodiment
1, the vitro culture of NK cell, amplification and activation
1) under aseptic condition from the peripheral blood of patient's venous puncture 20ml in 50ml centrifuge tube, add 300IU sodium heparin anticoagulant;
2) physiological saline or PBS dilution is added by 1:1;
3) in the 15ml centrifuge tube of sky, add 5ml lymphocyte separation medium, get 10ml dilute blood and slowly add in 15ml centrifuge tube, 1500rpm, centrifugal 20min, gets tunica albuginea confluent monolayer cells and uses brine, 1200rpm, after centrifugal 10min, PBS is resuspended, repeats 1 time, abandons supernatant;
4) with the resuspended above-mentioned cell of NK cell culture medium, and anti-cd 3 antibodies (OrthocloneOKT-3 is added; OrthoBiotech, Raritan, NJ), antibody final concentration is adjusted to 10ng/ml;
| NK cell culture medium | Manufacturer |
| Serum-free stem cell growth substratum SCGM | CellGenix, Germany |
| 5% human serum | Biowhittaker-Cambrex, the U.S. |
| 500U/ml rhIL-2 | Proleukin, markon's welfare is sub- |
5) after being cultured to the 5th day, within every 2 days, change a fresh NK cells substratum (not containing OKT-3), and add IL-12 that final concentration is 10ng/ml and final concentration be the IL-15 of 50ng/ml until 21 days, within the 0th, 5,10,15,20 day, add up cell count with Trypan Blue what cultivate respectively;
6) in order to avoid the intercellular contact inhibition growth of NK, NK cell is transferred in large Tissue Culture Flask or culture bag.
Result: Trypan Blue cell counting (total cell count)
| Number of days | 0 | 5 | 10 | 15 | 20 |
| Cell count | 2×10 6 | 12×10 6 | 138×10 6 | 426×10 6 | 1026×10 6 |
2, with flow cytometer, sorting is carried out to cultured cells:
For the phenotype CD3 that NK cell, T cell and NK sample T cell are corresponding
-cD56
+, CD3
+cD56
-and CD3
+cD56
+carry out sorting with streaming antibody, and add up its ratio.
Result: selected by flow cytometry apoptosis cell type ratio (often kind of cell/total cell)
3, enzyme linked immunological kit (ELISA) detects supernatant cytokine content
Get the cell culture supernatant of the 0th, 3,6,9,12,15,18 and 21 day respectively, operate according to the standard operating procedure of ELISA kit (R & DSystems, Minneapolis, MN).
Result: ELISA detects supernatant cytokine content (note: in form, the concentration of IL-2, IL-12 and IL-15 is deduct the concentration being added into the factor in substratum)
The each composition of table 1 natural cytokines mixture and concentration (unit: pg/ml)
| IL-2 | IFNs | TNF-α | GM-CSF | |
| 0d | 2.4±0.3 | 4.6±0.7 | 2.1±1.3 | 7.8±0.9 |
| 3d | 443.8±63.7 | 13.6±2.0 | 10.8±1.8 | 23.1±4.6 |
| 6d | 1135.7±139.6 | 384.1±71.9 | 346.6±4.7 | 96.8±9.5 |
| 9d | 3692.2±954.8 | 1233.2±108.9 | 789.3±86.0 | 209.2±10.4 |
| 12d | 2454.3±665.9 | 960.3±112.5 | 249.9±20.5 | 392.3±43.0 |
| 15d | 1018.8±285.0 | 353.2±41.3 | 120.5±20.9 | 203.2±39.8 |
| 18d | 415.2±85.8 | 182.0±32.0 | 97.3±29.3 | 150.3±24.2 |
| 21d | 172.9±35.9 | 78.4±19.0 | 18.2±11.0 | 37.3±11.0 |
The each composition of table 2 natural cytokines mixture and concentration (unit: pg/ml)
| Number of days | IL-6 | IL-12 | IL-15 |
| 0d | 3.1±0.4 | 3.9±0.2 | 1.7±0.3 |
| 3d | 14.3±2.5 | 11.6±1.8 | 6.7±0.5 |
| 6d | 492.4±43.6 | 56.7±22.6 | 97.3±21.5 |
| 9d | 904.5±372.1 | 732.6±110.4 | 687.6±102.8 |
| 12d | 1130.6±142.1 | 798.5±98.0 | 365.8±119.4 |
| 15d | 223.4±198.3 | 286.0±42.9 | 87.3±28.3 |
| 18d | 64.5±110.1 | 79.6±15.4 | 39.0±10.5 |
| 21d | 0.28±0.0 | 18.3±7.1 | 8.3±1.4 |
4, the preparation of cell toxicant NK cell preparation
By a series of preliminary experiment, obtain the preparation technology of following optimal N K cell preparation:
1) the 9th day, part cell and supernatant liquor thereof is collected from cell cultures every three days;
2) be placed in 50mL centrifuge tube, 1200rpm, centrifugal 10min, abandons supernatant;
3) cell precipitation Multiple electrolytes injection suspends, 1000rpm, centrifugal 10min, repeats once, to abandon supernatant;
4) suspended evenly by cell precipitation 95mL Multiple electrolytes injection, transfer in 100mL vial, add 5mL20% human serum albumin, the preparation of toxicity NK cell preparation completes.
5, the preparation of cytokine mixture and lyophilized powder thereof
Homogeneous for ensureing the steady quality of NK cell culture supernatant, adopting Freeze Drying Technique to be prepared into freeze-dried powder preparation, in-20 DEG C of preservations, laying the foundation for making a search to its biological activity further.
1) from 6th ~ 12 days centrifugal collecting cell supernatant liquors, by cell restored cells culturing bottle or cell culture bags;
2) at 4 DEG C by supernatant by 50000rpm ultracentrifugation 2h, abandon precipitation; Get supernatant ultrafiltration, then use 0.11 μm of millipore filtration micro-filtration;
3) under aseptic condition, be sub-packed in by 1ml filtrate in the ampoule of 3ml capacity, and be placed in lyophilized plate ,-20 DEG C of pre-freeze supernatant liquors to curdled appearance,
4) supernatant liquor solidified is vacuumized, sublimation drying, remove ice crystal, fill in halogenated butyl rubber plug, add a cover aluminium-plastic cap sealing, preserve lyophilized powder or transport in 4 DEG C.
6, cytokine mixture anti-tumor activity detects
1) animal model is set up
Buy the BALB/c mouse of body weight 18-22 gram in 18 6 week ages (purchased from pharmaceutical college of Zhongshan University Experimental Animal Center), every mouse web portion injection inoculation Human Prostate Cancer PC-3 Cell Line 1 × 10
6individual, all see after inoculation that tumor mass grows, and is then divided into 3 groups at random, often organize each 6 mouse.
2) administering mode and dosage
3 groups of all totally 5 intravenous injections in the 5th day, the 7th day, the 9th day, the 11st day, the 13rd day after inoculation Human Prostate Cancer PC-3 Cell Line, dosage is as follows:
NK cell culture supernatant preparation: the dosage of 0.2ml/ day is injected to mouse peritoneal, every day timed drug administrations;
The freeze-dried powder preparation of NK cell culture supernatant: specification is 1ml supernatant stoste/bottle, every bottle of physiological saline with 0.2ml redissolves, and the medicament obtained is equivalent to 5 times of concentrated supernatants, then injects to mouse peritoneal with the dosage of 0.2ml/ day, every day timed drug administrations
3) record also statistical experiment result
After treatment, within the 42nd day, dissect that each mouse gets knurl block, precision electronic balance claims knurl weight, calculate tumor control rate respectively
Table three NK cell conditioned medium preparation is to the restraining effect of mouse prostate growth of cancer cells
| Group | Dosage ml/d | Tumor mass g | Tumor control rate % |
| Saline control group | 0.2 | 3.58±0.96 | |
| Supernatant preparations | 0.2 | 2.01±0.57* | 43.85 |
| Freeze-dried powder preparation | 0.2 | 1.26±0.36* | 64.80 |
* P<0.01, has significant difference
Experimental result shows: NK culture supernatant and freeze-dried powder preparation, compared with physiological saline group, reach 43.85% and 64.80% respectively to the inhibiting rate of Prostate Carcinoma of Mice, shows that cytokine mixture can play certain anti-tumor activity really in vivo.
4) safety evaluation
The mouse growth of two kinds of preparation groups of NK cell culture supernatant is in good condition, and hair is more smooth, and feed is normal, and body weight increases and tumor growth is slower.And control group mice growth conditions is poor, hair is not smooth, and body weight has no obvious weightening finish, and tumor growth is rapidly simultaneously with hemorrhage and phenomenon of festering.As can be seen here, this preparation also has the little advantage of toxic side effect while having the growth of remarkable Tumor suppression in vivo, be a kind of comparatively safe effective medicine, the present invention has absolutely proved that the cytokine mixture in NK culture supernatant is preparing the applied research prospect in antitumor drug.
Claims (4)
1. a cultural method for NK cell, is characterized in that:
The mononuclearcell of separator well is placed in the nutrient solution containing OKT-3 antibody and IL-2 cytokine, cultivate after five days, within every 2 days, change a fresh NK cells substratum, and add IL-12 and IL-15, make its concentration in nutrient solution reach 10ng/ml and 50ng/ml respectively, be cultured to 21 days.
2. a preparation method for cell toxicant NK cell preparation, is characterized in that:
The mononuclearcell of separator well is placed in the nutrient solution containing OKT-3 antibody and IL-2 cytokine, cultivate after five days, within every 2 days, change a fresh NK cells substratum, and add IL-12 and IL-15, make its concentration in nutrient solution reach 10ng/ml and 50ng/ml respectively, be cultured to 21 days;
From cell cultures the 9th day every three days by part cell harvesting, be placed in 50mL centrifuge tube, 1200rpm, centrifugal 10min, abandons supernatant, and cell precipitation Multiple electrolytes injection suspends, 1000rpm, centrifugal 10min, repeats once, to abandon supernatant, cell precipitation 95mL Multiple electrolytes injection is suspended evenly, transfer in 100mL vial, add 5mL20% human serum albumin, the preparation of toxicity NK cell preparation completes.
3. a preparation method for cytokine mixture, comprises the steps:
The mononuclearcell of separator well is placed in the nutrient solution containing OKT-3 antibody and IL-2 cytokine, cultivate after five days, within every 2 days, change a fresh NK cells substratum, and add IL-12 and IL-15, make its concentration in nutrient solution reach 10ng/ml and 50ng/ml respectively, be cultured to 21 days;
From cell cultures 6th ~ 12 days centrifugal collecting cell supernatant liquors, by cell restored cells culturing bottle or cell culture bags; At 4 DEG C, supernatant is passed through 50000rpm ultracentrifugation 2h, abandon precipitation, get supernatant ultrafiltration, then use 0.11 μm of millipore filtration micro-filtration, filtrate is added in cell preparation, or 4 DEG C of short-term preservations are for subsequent use, or-20 DEG C save backup for a long time.
4. the cytokine mixture prepared by claim 3.
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| EP3725296A4 (en) * | 2017-12-14 | 2021-09-29 | Green Cross WellBeing Corporation | Cosmetic composition and pharmaceutical composition for alleviating atopic dermatitis, hair loss, and wounds or reducing skin wrinkles |
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| WO2016122014A1 (en) * | 2015-01-27 | 2016-08-04 | 한국생명공학연구원 | Method for mass-producing natural killer cells and use of natural killer cells obtained by the method as anticancer agent |
| CN104928241B (en) * | 2015-03-27 | 2018-04-13 | 北京康爱瑞浩生物科技股份有限公司 | A kind of activation method and cell preparation method of enhanced NK cells |
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| CN108220236B (en) * | 2017-12-20 | 2020-06-30 | 中国科学院遗传与发育生物学研究所 | Human NK cell line |
| CN109294985B (en) | 2018-10-25 | 2022-02-18 | 江苏普瑞康生物医药科技有限公司 | Culture medium system for NK cell in-vitro amplification and NK cell in-vitro amplification method |
| CN112251402A (en) * | 2020-10-20 | 2021-01-22 | 埃尔利生物技术(上海)有限公司 | Protein peptide, freeze-dried powder, oral liquid and preparation method thereof |
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