CN104894072B - A kind of preparation method and applications of autologous natural killer cells propagation - Google Patents
A kind of preparation method and applications of autologous natural killer cells propagation Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of autologous natural killer cells Multiplying culture, including following feature：Come from the mononuclearcell of cancer patient itself, using 3 phosphoinositide deopendent protein kinases 1 and CD122 is transfected and amplification activation acquisition natural killer cells under stablizing the K562 cells of expression as trophocyte and interleukin-22 effect, natural killer cells proliferation times reach more than 2500 times, CD16+/CD56+ phenotypes are more than 90%, and internal in vitro test is antitumor to have very strong killing toxicity；Present invention also offers a kind of kit containing the autologous natural killer cells Multiplying culture as obtained from the above method, there is efficient anti-tumor function.
Description
Technical field
The present invention relates to a kind of preparation method of autologous natural killer cells propagation, including following feature：Come from
The isolated mononuclearcell of cancer patient's derived from peripheral blood, is using 3- phosphoinositides deopendent protein kinase 1 and CD122
Transfect and stablize K562 cells and the lower amplification activation of interleukin-22 effect of expression, obtain natural killer cells；The present invention also provides
A kind of kit containing the autologous natural killer cells as obtained from the above method, available for all kinds of cancers includes solid tumor
With the immunization therapy of multiple courses for the treatment of including neoplastic hematologic disorder.
Background technology
Natural killer cells (Natural Killer cells, NK) quantity and dysfunction and tumour and persistent virus
The generation of infection is closely related.After NK cells add interleukin-22 (interleukin-2, IL-2) culture amplification in vitro at present, its
Antitumor action is 50-100 times stronger than LAK cell, and only needs a small amount of IL-2 to play a role, and because it is to certainly
Body tumour target cell has specificity, becomes the hot spot of domestic and international knubble biological research, and is used in clinical treatment.
NK cells mainly express CD16+、CD56+Phenotype, treat to melanoma, lung cancer, kidney, colon cancer, breast cancer,
The oncotherapies such as carcinoma of urinary bladder, liver cancer and leukaemia have obvious curative effects.NK cell quantities and killing ability are directly related with curative effect, control
Problem present in treatment is to obtain a large amount of NK cells finite sum incubation times long.NK cell expansion ex vivos are to 5 × 108It is average
Need 59 days, be expanded to 5 × 109Then averagely need 80 days, NK is selected in the culture medium containing IL-2 after cultivating after a while
Selecting property expands T lymphocytes, CD3+Account for more than 80%, CD4+And CD8+It is low to account for the amplification of more than 50%, NK cells, account for less than
10% or so.The low dose of interleukin-22 activation growth multiple that thus tradition uses is limited, and telomerase activation reduces, incubation time
It is long to cause NK cell killing hypofunctions.
The present invention provides the K562 for being transfected with 3- phosphoinositides deopendent protein kinase 1 and CD122 and stablize expression is thin
Born of the same parents are trophocyte, and a large amount of amplification NK cells, cultivate to 21 days cell Proliferation multiples and reach under the effect of Low dose IL -2
More than 2500 times, NK cells kill tumor activity and reached optimal at 21 days, greatly save the time for patient clinical treatment, and given play to
Best antitumor action, helps to improve clinical efficacy and extends patient survival, and can substantially reduce cell culture
Cost.
The content of the invention
The present invention is directed to the deficiency of prior art Multiplying culture NK cells, is particularly needed because NK cells are expanded to clinical treatment
The cell quantity wanted and incubation time is long, culture medium selective amplification T lymphocytes and NK cell occupation ratios containing IL-2
Example is very low, thus produce to kill tumor activity low.The present invention tests discovery by early-stage study and has transfected the dependence of 3- phosphoinositides
Property protein kinase 1 (3 '-phosphoinositide-dependent kinase 1, PDK1) and CD122 and stablize expression
K562 cells, as trophocyte and the lower culture NK cells of low dosage IL-2 effects, cultivate to 21 days cell Proliferation multiples and reach
To more than 2500 times, NK cell numbers reach 3 × 109The effect of clinical antineoplastic is sufficient for above, ensures that cancer patient is clinical
Treat curative effect.
The K562 cell transfectings expression vector of 3- phosphoinositides deopendent protein kinase 1 and CD122, can be steady on cell membrane
Surely the polypeptide of PDK1 and CD122 is expressed.PDK1 is the master regulators of AGC protein kinases family, and research finds that PDK1 is NK thin
Born of the same parents' early development key molecule, have activated interleukin 15 expression rise, has also activated the raising of CD122 developed by molecule so that NK is thin
Born of the same parents can be abnormal in spontaneous kill body in the case of from extraneous " sensitization " cell.Thus relied on using 3- phosphoinositides
Property protein kinase 1 and CD122 transfect and stablize expression K562 cells can be used as artificial antigen presenting cell, NK can be promoted
Cell development and a large amount of propagation, suppress the propagation of T lymphocytes and generation in NK cells, extend NK Cell Telomerase Activities, can
Produce the killing activity optimal to tumour cell.
The present invention relates to a kind of preparation method of autologous natural killer cells propagation, it is characterised in that using PDK1 and
CD122 is transfected and is stablized the K562 cells and interleukin-22 of expression, and amplification activation natural killer cells, obtains natural killer cells
Largely to breed.
K562 cell line transfections stabilization expression 3- phosphoinositides deopendent protein kinase 1 and CD122 of the present invention
Expression vector, contains viral promotors and selectable marker gene in carrier, can obtain PDK1 on K562 cell membranes and
CD122 polypeptides, K562 cells still stablize expression 3- phosphoinositides deopendent protein kinase 1 and CD122 after culture is bred.Through training
There is expression 3- phosphoinositides deopendent protein kinase 1 and the cell of CD122, by strong acid, by force on K562 cell membranes after supporting
Alkali and/or irradiation and pass through high speed centrifugation and collect K562 cells and carry out purified pool.
Mononuclearcell is thin through lymph from the peripheral blood of cancer patient's collection or marrow etc. in the method for the invention
Born of the same parents' separating liquid, gradient of continuous density centrifugation obtain.Preferably, after cancer patient's operation one month, chemicotherapy one
The fresh peripheral blood or marrow gathered after month.
The K562 cells of stable expression 3- phosphoinositides deopendent protein kinase 1 and CD122 of the present invention with killing naturally
Hinder cell to co-culture first 5 days, the usage ratio of the K562 cells and mononuclearcell is K562 cell numbers: mononuclearcell
Number=1: 0.1-1: 10, it is highly preferred that being K562 cell numbers: mononuclearcell number=1: 1-1: 5.
After co-culturing 5 days, natural killer cells is suctioned out into new blake bottle with 5mL pipettes, and it is new to supplement 10-20mL
Fresh cell culture medium (active unit containing 50-500/mL interleukin-22s), in 37 DEG C, 5%CO2Continue culture 2-3 days in incubator；
Continue to supplement 20-40mL Fresh cell culture mediums (active unit containing 50-500/mL interleukin-22s), in 37 DEG C, 5%CO2Incubator
In continue culture 2-3 days；Then the continuous Multiplying culture of natural killer cells was by 18-21 days so that cell number reaches 3 × 109With
On.
Source 30ml peripheral blood mononuclear cells Fiber differentiation obtains natural killer cells, propagation in the method for the invention
Multiple is cultivated to 21 days natural killer cells numbers up to 3 × 10 up to more than 2500 times9More than.
Culture obtains natural killer cells, height expression its specific markers CD16+/CD56+, sun in the method for the invention
Property rate be higher than more than 90%, and CD3+ positive rates be less than less than 7%.
Culture obtains natural killer cells in the method for the invention, and internal in vitro test has very powerful antitumor killing poison
Property.
The cell culture medium that the present invention uses adds 50- for lymphocyte serum or 1640 culture mediums of RPMI
2000 active units/mL interleukin-22s and 10% (volume ratio) autoserum or hyclone, it is preferable that cell culture medium is leaching
Bar cell non-serum culture medium, such as CellGro SCGM, AIM-V, X-VIVO and GT-T551, contain 1000 active units/mL
Interleukin-22.
Present invention also offers a kind of kit for preparing autologous natural killer cells, it is characterised in that the kit
Including：
(1) 3- phosphoinositides deopendent protein kinase 1 and CD122 are expressed through strong acid, highly basic and/or stablizing for radiation treatment
K562 cells；
(2) interleukin-22；
(3) lymphocytes culture medium；
(4) tissue digestion liquid；
(5) lymphocyte separation medium；
(6) physiological saline；
(7) operation instructions；
Wherein, the operation instructions include the method described in claim 1-6.
The K562 cells of the stable expression 3- phosphoinositides deopendent protein kinase 1 and CD122, it is preferable that further include
Nourished after strong acid, highly basic and/or radiation treatment containing the K562 for stablizing expression 3- phosphoinositides deopendent protein kinase 1 and CD122
The culture plate of confluent monolayer cells, such as 24 porocyte culture plates, 12 porocyte culture plates and 6 porocyte culture plates.
Present invention also offers the above method and its kit to prepare NK cells, available for all kinds of cancers include solid tumor and
The immunization therapy of multiple courses for the treatment of including neoplastic hematologic disorder.
Brief description of the drawings
Fig. 1 shows the advanced breast cancer patient for two in vitro culture of embodiment and normal people source NK cell number change curves
Figure；
Fig. 2 is expressed as the advanced breast cancer patient of two in vitro culture of embodiment and normal people source NK cell Proliferations multiple becomes
Change curve map；
Fig. 3 is expressed as the advanced breast cancer patient NK cell streaming cell detection results figures of the preparation of embodiment two；
Fig. 4 is expressed as normal person's NK cell streaming cell detection results figures of the preparation of embodiment two；
Fig. 5 is expressed as the killing of the advanced breast cancer patient and normal people source NK cells of the preparation of embodiment two to MCF-7
Toxicity profile figure；
Fig. 6 is expressed as the killing of the advanced breast cancer patient and normal people source NK cells of the preparation of embodiment two to HepG2
Toxicity profile figure；
Fig. 7 is expressed as the complete tumor size afterwards of NK cell therapies of the preparation of lotus breast cancer MCF-7 mouse models embodiment two
Change curve.
Embodiment
Present invention discover that the K562 for being transfected by 3- phosphoinositides deopendent protein kinase 1 and CD122 and stablizing expression is thin
Born of the same parents can be used as artificial antigen presenting cell, and NK cells can be promoted largely to breed, and suppress the propagation of T lymphocytes in NK cells
And generation, extend NK Cell Telomerase Activities, produce the killing activity optimal to tumour cell.NK cell culture was to 21 days cells
Proliferation times reach more than 2500 times, and NK cell numbers reach 3 × 109Meet clinical treatment needs above, available for repeatedly anti-swollen
The clinical practice of knurl.
A kind of preparation method of autologous natural killer cells propagation of the present invention is specifically described.The present invention relates to
And transfected using PDK1 and CD122 and stablize the K562 cells and interleukin-22 of expression, amplification activation natural killer cells so that
NK cells largely expand, and obtain the NK cells with antitumor action.
The present invention relates to a kind of preparation method of autologous natural killer cells propagation, it is characterised in that using PDK1 and
CD122 is transfected and is stablized the K562 cells and interleukin-22 of expression, and amplification activation natural killer cells, obtains natural killer cells
Largely to breed.
K562 cell line transfections stabilization expression 3- phosphoinositides deopendent protein kinase 1 and CD122 of the present invention
Expression vector, contains viral promotors and selectable marker gene in carrier, can obtain PDK1 on K562 cell membranes and
CD122 polypeptides, K562 cells still stablize expression 3- phosphoinositides deopendent protein kinase 1 and CD122 after culture is bred.Through training
There is expression 3- phosphoinositides deopendent protein kinase 1 and the cell of CD122, by strong acid, by force on K562 cell membranes after supporting
Alkali and/or irradiation and pass through high speed centrifugation and collect K562 cells and carry out purified pool.
Mononuclearcell is thin through lymph from the peripheral blood of cancer patient's collection or marrow etc. in the method for the invention
Born of the same parents' separating liquid, gradient of continuous density centrifugation obtain.Preferably, after cancer patient's operation one month, chemicotherapy one
The fresh peripheral blood or marrow gathered after month.
The K562 cells of stable expression 3- phosphoinositides deopendent protein kinase 1 and CD122 of the present invention with killing naturally
Hinder cell to co-culture first 5 days, the usage ratio of the K562 cells and mononuclearcell is K562 cell numbers: mononuclearcell
Number=1: 0.1-1: 10, it is highly preferred that being K562 cell numbers: mononuclearcell number=1: 1-1: 5.
After co-culturing 5 days, natural killer cells is suctioned out into new blake bottle with 5mL pipettes, and it is new to supplement 10-20mL
Fresh cell culture medium (active unit containing 50-500/mL interleukin-22s), in 37 DEG C, 5%CO2Continue culture 2-3 days in incubator；
Continue to supplement 20-40mL Fresh cell culture mediums (active unit containing 50-500/mL interleukin-22s), in 37 DEG C, 5%CO2Incubator
In continue culture 2-3 days；Then the continuous Multiplying culture of natural killer cells was by 18-21 days.
Source 30ml peripheral blood mononuclear cells Fiber differentiation obtains natural killer cells, propagation in the method for the invention
Multiple is cultivated to 21 days natural killer cells numbers up to 3 × 10 up to more than 2500 times9More than.
Fiber differentiation obtains natural killer cells, height expression its specific markers CD16+/CD56+, and positive rate is higher than 90%
More than, and CD3+ positive rates are less than less than 7%；Internal in vitro test has very powerful antitumor killing toxicity.
The cell culture medium that the present invention uses adds 50- for lymphocyte serum or 1640 culture mediums of RPMI
2000 active units/mL interleukin-22s and 10% (volume ratio) autoserum or hyclone, it is preferable that cell culture medium is leaching
Bar cell non-serum culture medium, such as CellGro SCGM, AIM-V, X-VIVO and GT-T551, contain 1000 active units/mL
Interleukin-22.
Present invention also offers a kind of kit for preparing autologous natural killer cells, it is characterised in that the kit
Including：
(1) 3- phosphoinositides deopendent protein kinase 1 and CD122 are expressed through strong acid, highly basic and/or stablizing for radiation treatment
K562 cells；
(2) interleukin-22；
(3) lymphocytes culture medium；
(4) tissue digestion liquid；
(5) lymphocyte separation medium；
(6) physiological saline；
(7) operation instructions；
Wherein, the operation instructions include the method described in claim 1-6.
The K562 cells of the stable expression 3- phosphoinositides deopendent protein kinase 1 and CD122, it is preferable that further include
Nourished after strong acid, highly basic and/or radiation treatment containing the K562 for stablizing expression 3- phosphoinositides deopendent protein kinase 1 and CD122
The culture plate of confluent monolayer cells, such as 24 porocyte culture plates, 12 porocyte culture plates and 6 porocyte culture plates.
As used in NK cells amplification cultivation of the present invention Tissue Culture Plate, Tissue Culture Dish, blake bottle, cell culture
Bag, can enumerate, be 75cm2Tissue Culture Flask, 175cm2The cell culture equipment such as Tissue Culture Flask, 250mL cell culture bags
(container), is used equally for the present invention, preferred cell culture bag.
NK cells are frozen in the manufacturing method of the present invention, to frozen stock solution without specifically limited, but are preferably, for example,
50% calf serum, 40% cell culture fluid and 10% dimethyl sulfoxide (DMSO), wherein cell culture fluid are more preferably NK cell culture
Liquid.
Serum can be added in the medium or blood plasma is cultivated.Their additive amounts in the medium are from special limit
System, such as larger than 0 capacity % is to 20 capacity %, and can change the dosage of serum or blood plasma according to different cultivation stages, excellent
Elect 5% (volume ratio) as.For example, interim serum or plasma concentration can be reduced to use.In addition, as serum or blood plasma
Source, can be oneself (meaning identical with the cell derived cultivated) or oneself non-(cell for meaning and being cultivated
Any of source is different), set out from a security point, preferably the serum or blood plasma in oneself source.Alternatively, it is also possible to add
Add such as serum composition of the human serum albumins etc through isolating and purifying.
The preparation of the autologous NK cells propagation of the present invention is implemented using above-mentioned various composition and culture medium.Make in the present invention
Culture condition of culture is also not particularly limited, and can use the condition used in common cell culture.For example, can be 37
DEG C, 5%CO2Cultivated Deng under the conditions of.It can also implement such as inferior operation：Interval reasonable time adds fresh culture to dilute
Cell culture fluid, or culture medium is replaced, or replace cell culture equipment etc..
The present invention also provides NK cells antineoplaston multiple in clinical practice, preferably plays in vivo anti-
Tumor immune response, reaches good therapeutic effect.In addition, above-mentioned NK cells also have the following advantages that, multiple NK cell therapies
It will preferably trigger NK cells to kill tumor activity, and suppress Autoimmune disease immunosuppressive activity, generation is efficient, resists longer
Tumour immunity effect, therefore it is especially advantageous for the raising of patient's curative effect and the extension of life cycle.
Hereinafter, the present invention is done in conjunction with the embodiments and more specifically described, however, the present invention is not limited thereto.
Embodiment one
The preparation of the K562 cell lines of expression PDK1 and CD122 is stablized in transfection
The expression vector for stablizing expression PDK1 and CD122 is transcribed with K562 cell lines, in carrier containing viral promotors and
Selectable marker gene；After heterogenous expression carrier enters host cell, promoter is activated, cultivates K562 cells for a period of time, i.e.,
It can obtain 3- phosphoinositides deopendent protein kinase 1 and CD122 polypeptides on cell membrane；K562 cell membranes after culture
The upper cell with expression PDK1 and CD122, by strong acid, highly basic and/or irradiation and passes through high speed centrifugation and collects K562 cells
Carry out purified pool.
Embodiment two
The preparation of autologous natural killer cells
Advanced breast cancer patient and normal healthy people 30ml (signing informed consent form with it) are gathered, adds 1 × PBS of equivalent
Dilution, is gently superimposed on lymphocyte separation medium along centrifugation tube wall, forms sharp interface, 2000rpm is centrifuged at room temperature
20min, draws the PBMC of boundary layer, is washed twice with 1 × PBS (pH value 7.4).After the blue counting of tongue disk, CellGro is used
SCGM adjustment mononuclearcell concentration is 1 × 107/ml。
Stablize the K562 cells for expressing 3- phosphoinositides deopendent protein kinase 1 and CD122 and natural killer cells first
Co-culture 5 days, the usage ratio of the K562 cells and lymphocyte is K562 cell numbers: mononuclearcell number=1: 5.
After co-culturing 5 days, natural killer cells is suctioned out into new blake bottle with 5mL pipettes, and it is fresh to supplement 20mL
Cell culture medium (contains 1000 active units/mL interleukin-22s), in 37 DEG C, 5%CO2Continue culture 2-3 days in incubator；Continue
40mL Fresh cell culture mediums (containing 1000 active units/mL interleukin-22s) are supplemented, in 37 DEG C, 5%CO2Continue to train in incubator
Support 2-3 days；Then the continuous Multiplying culture of natural killer cells was by 21 days so that cell number reaches 1 × 109More than.
Using viable count instrument (Millipore companies of the U.S.) respectively at the 0th day, 6 days, 9 days, 12 days, 15 days, 18 days
Counted with 21 days, analyze NK Cell viabilities and proliferation times.
With the increase of in vitro culture number of days in Fig. 1, NK cell numbers are continuously improved, to patient with breast cancer's NK cells at the 21st day
Number reaches 3.34 × 109It is a, normal artificial 4.06 × 109It is a；Fig. 2 is with the increase of in vitro culture number of days, NK cells expandeds
It is continuously improved, is that patient with breast cancer's NK cells expandeds are 2594.75 times, normal person's amplification times is 2629.59 times；Both
The equal no significant difference in NK cell counts and proliferation times, shows that there is cancer patient source NK cells same propagation to imitate
Fruit.BC-NK is patient with breast cancer source NK cells, and NB-NK is normal health people source NK cells.
Embodiment three
Autologous NK cells phenotype FCM analysis
The 1 × 10 of patient with breast cancer and normal health people source culture acquisition in the 14th day prepared by Example two6NK is thin
Born of the same parents, carry out the dyeing detection and analysis of streaming antibody.One group of NK cells are respectively that the anti-human CD16-FITC of 20 μ L mouse, 20 μ L mouse are anti-human
The CD56-PE and 20 anti-human CD8-PerCP of μ L mouse；Another group is respectively the anti-human CD3-FITC of 20 μ L mouse, the 20 anti-human CD25-PE of μ L mouse
With the 20 anti-human CD4-PerCP of μ L mouse；Isotype control group is 20 μ L mIgG1-FITC, 20 μ L mIgG1-PE and 20 μ L mIgG1-
PerCP；Place after 4 DEG C of refrigerators dye 30 minutes and washed 3 times with PBS, then in FACS Calibur instruments (U.S. company BD)
Upper machine testing and analysis.
Patient with breast cancer's NK cell FCM analysis prepared by Fig. 3 embodiments two.Patient with breast cancer NK cells CD56 and
CD16 phenotypes, CD16CD56 positive rates are 94.4%；The T lymphocytes contained are few, and CD3 positive rates are only 5.9%；Fig. 4 is real
Apply normal person's NK cell FCM analysis of the preparation of example two.Normal NK cells of human beings CD56 and CD16 phenotype, CD16/CD56 are positive
Rate is 93.4%；3 positive rate of T lymphocyte CDs is only 6.4%, and the cell for showing both in vitro cultures is mainly NK cells, containing T
Lymphocyte quantity is very low.
Example IV
NK cells in vitro kills knurl experiment
Respectively using breast cancer cell line MCF-7 and liver cancer cell lines HepG2 as target cell, with the NK prepared in embodiment two
Cell is separately added into 96 well culture plates than 40: 1,20: 1,10: 1,5: 1 and 2.5: 1 by effect target, then put as effector cell
4h is cultivated under the conditions of 37 DEG C, 5%CO2, is all provided with 3 multiple holes.Using LDH cellulotoxic experiments method measure NK activity：Draw supernatant
50 μ L of liquid, are placed in 96 orifice plates, add LDH base fluids 50 μ L, room temperature lucifuge 30min, add 50 μ L terminate liquids and terminate reaction,
Absorbance (OD) value is surveyed at 490nm wavelength.Calculate the killing rate of MCF-7 and HepG2 cells respectively by following equation: killing rate=
(experimental group OD values-effector cell Spontaneous release group OD values-target cell Spontaneous release group OD values)/(target cell maximum release group OD
Value-target cell Spontaneous release group OD values) × 100%.
Fig. 5 is the killing activity of advanced breast cancer patient and normal people source NK cells to MCF-7, as effect target ratio carries
Height, cellular cytoxicity activity are also continuously improved, and the anti-MCF-7 killings toxicity of advanced breast cancer patient NK cells is slightly above normal person,
But both no significant differences.Fig. 6 is the killing activity of advanced breast cancer patient and normal people source NK cells to HepG2, normally
NK cells of human beings anti-MCF-7 killings toxicity is slightly above advanced breast cancer patient, but tumor killing effect also no significant difference between the two,
The NK cells in vitro antitumor actions in patient with breast cancer source are disclosed in the similar of normal person.
Embodiment five
The effect in knurl experiment is killed in NK cell bodies
18 8 weeks SCID mice bellies flanks are subcutaneously injected 5 × 106Breast cancer cell line MCF-7, after raising 7 days, at random
It is divided into 3 groups of A, B and C, every group 6：A groups are patient with breast cancer source NK cell therapy groups prepared by embodiment two；B groups are real
Apply the normal people source NK cell therapy groups of the preparation of example two；C groups are normal saline placebo group.A groups are in patient with breast cancer source
NK cell culture was to the 14th day and the 16th day tail vein injection 2 × 104Cell/gram, B groups are trained in Normal human patients source NK cells
Support to the 14th day and the 16th day tail vein injection 2 × 104Cell/gram, C groups and A and B treatment groups same time, tail vein injection are same
The physiological saline of volume, each 1mL.Draw neck to put to death respectively at 7d, 14d, 21d, 28d and 35d after treatment, take lotus MCF-7 mouse moulds
Type tumor tissues, calculate mouse model lotus knurl size.
Tumor size change curve after Fig. 7 lotus breast cancer MCF-7 mouse model NK cell therapies are complete, physiological saline group C
With BC-NK treatment groups A compared with NB-NK treatment groups B, treatment group A is reduced significantly with breast cancer tumour size in treatment group B, but
The killing activity indifference of both NK cells, it is same with normal NK cells of human beings to show that patient with breast cancer source NK cells have triggered
Kill tumor activity in vivo.
Claims (9)
1. a kind of preparation method of autologous natural killer cells propagation, it is characterised in that come from the single of cancer patient itself
Nucleus, the K562 cells expressed are transfected and stablize as nourishing using 3- phosphoinositides deopendent protein kinase 1 and CD122
Confluent monolayer cells and the lower amplification activation of interleukin-22 effect obtain natural killer cells, natural killer cells is largely bred；Culture
Reach more than 2500 times to 21 days cell Proliferation multiples, NK cell numbers reach 3 × 109More than；CD16+/CD56+ phenotypes are 90%
More than；The natural killer cells has antitumor killing toxicity；
The K562 cells of the stable expression 3- phosphoinositides deopendent protein kinase 1 and CD122 are with deriving from outside autologous patient
The mononuclearcell of all blood co-cultures 5 days first, and the usage ratio of the K562 cells and lymphocyte is K562 cell numbers: leaching
Bar cell number=1: 0.1-1: 10；After co-culturing 5 days, natural killer cells is suctioned out into new blake bottle with 5mL pipettes, and
10-20mL Fresh cell culture mediums are supplemented, in 37 DEG C, 5%CO2Continue culture 2-3 days in incubator；Continue to supplement 20-40mL
Fresh cell culture medium, in 37 DEG C, 5%CO2Continue culture 2-3 days in incubator；Then natural killer cells continuously breeds training
Support 18-21 days so that NK cells are largely bred；The Fresh cell culture medium active unit containing 50-2000/mL interleukin-22s.
2. according to the method described in claim 1, it is characterized in that, expression 3- is stablized with K562 cell line transfections in the method
The expression vector of phosphoinositide deopendent protein kinase 1 and CD122, contains viral promotors and selectable marker gene in carrier,
3- phosphoinositides deopendent protein kinase 1 and CD122 polypeptides on K562 cell membranes can be obtained, K562 cells increase through culture
Still stablize expression 3- phosphoinositides deopendent protein kinase 1 and CD122 after growing；There is expression on K562 cell membranes after culture
The cell of 3- phosphoinositides deopendent protein kinase 1 and CD122, by strong acid, highly basic and/or irradiation, and by high speed from
The heart collects K562 cells and carries out purified pool.
3. method according to any one of claim 1 to 2, it is characterised in that 30ml peripheral bloods in source in the method
Mononuclearcell Fiber differentiation obtains natural killer cells, and proliferation times are cultivated thin to 21 days natural kill up to more than 2500 times
Born of the same parents' number is up to 3 × 109More than.
4. method according to any one of claim 1 to 2, it is characterised in that culture obtains nature and kills in the method
Hinder cell, height expression its specific markers CD16+/CD56+, positive rate is higher than 90%, and CD3+ positive rates are less than 7%.
5. method according to any one of claim 1 to 2, it is characterised in that culture obtains nature and kills in the method
Hinder cell, internal in vitro test has very powerful antitumor killing toxicity.
6. a kind of kit for preparing autologous natural killer cells, it is characterised in that the kit includes：
(1) through stablizing expression 3- phosphoinositides any one of the claim 1-5 of strong acid, highly basic and/or radiation treatment
The K562 cells of deopendent protein kinase 1 and CD122；
(2) interleukin-22；
(3) lymphocytes culture medium；
(4) tissue digestion liquid；
(5) lymphocyte separation medium；
(6) physiological saline；
(7) operation instructions；
Wherein, the operation instructions include the method any one of claim 1-5.
7. kit according to claim 6, wherein, stable 1 He of expression 3- phosphoinositides deopendent protein kinase
The K562 cells of CD122, it is preferable that further include after strong acid, highly basic and/or radiation treatment containing stablize expression 3- phosphoinositides according to
Rely property protein kinase 1 and the culture plate of the K562 trophocyte of CD122, the culture plate is selected from 24 porocyte culture plates, 12
Porocyte culture plates or 6 porocyte culture plates.
A kind of 8. preparation method of autologous natural killer cells propagation, it is characterised in that
Step 1: the preparation of the K562 cell lines of expression PDK1 and CD122 is stablized in transfection
The expression vector for stablizing expression PDK1 and CD122 is transcribed with K562 cell lines, contains viral promotors and selection in carrier
Marker gene；After heterogenous expression carrier enters host cell, promoter is activated, culture K562 cells for a period of time, that is, obtain
3- phosphoinositides deopendent protein kinase 1 and CD122 polypeptides on cell membrane；There is table on K562 cell membranes after culture
It is pure by strong acid, highly basic and/or irradiation, and by high speed centrifugation collection K562 cell progress up to the cell of PDK1 and CD122
Change and collect；
Step 2: the preparation of autologous natural killer cells
Advanced breast cancer patient and normal healthy people 30ml peripheral bloods are gathered, adds 1 × PBS of equivalent dilutions, along centrifugation tube wall
Gently it is superimposed on lymphocyte separation medium, forms sharp interface, 2000rpm centrifuges 20min at room temperature, draws boundary layer
PBMC, is washed twice with 1 × PBS；It is 1 × 10 with CellGro SCGM adjustment mononuclearcell concentration after the blue counting of tongue disk7/
ml；
Take the K562 cells and natural kill for stablizing expression 3- phosphoinositides deopendent protein kinase 1 and CD122 described in step 1
Cell co-cultures 5 days first, and the usage ratio of the K562 cells and lymphocyte is K562 cell numbers: mononuclearcell number=
1∶5；
After co-culturing 5 days, natural killer cells is suctioned out into new blake bottle with 5mL pipettes, and supplement 20mL fresh cells
Culture medium, in 37 DEG C, 5%CO2Continue culture 2-3 days in incubator；Continue supplement 40mL Fresh cell culture mediums, 37 DEG C,
5%CO2Continue culture 2-3 days in incubator；Then the continuous Multiplying culture of natural killer cells was by 21 days so that cell number reaches
3×109More than；Wherein, the Fresh cell culture medium contains 1000 active units/mL interleukin-22s；
Counted respectively the 0th day, 6 days, 9 days, 12 days, 15 days, 18 days and 21 days using viable count instrument, analysis NK is thin
Born of the same parents' motility rate and proliferation times；The results show that with the increase of in vitro culture number of days, NK cell numbers are continuously improved, to breast at the 21st day
Adenocarcinoma patients' NK cell numbers reach 3.34 × 109It is a, normal artificial 4.06 × 109It is a；As the increase of in vitro culture number of days, NK are thin
Born of the same parents' amplification times are also continuously improved, and are that patient with breast cancer's NK cells expandeds are 2594.75 times, normal person's amplification times are
2629.59 again；Both equal no significant differences in NK cell counts and proliferation times, show that cancer patient source NK cells have
Same cultivation effect；
Step 3: autologous NK cells phenotype FCM analysis
Take 1 × 10 that patient with breast cancer and normal health people source culture in the 14th day prepared by step 2 obtains6NK cells, carry out
The dyeing detection and analysis of streaming antibody；One group of NK cells be respectively the anti-human CD16-FITC of 20 μ L mouse, the anti-human CD56-PE of 20 μ L mouse and
The 20 anti-human CD8-PerCP of μ L mouse；Another group is respectively the anti-human CD3-FITC of 20 μ L mouse, the anti-human CD25-PE of 20 μ L mouse and 20 μ L mouse
Anti-human CD4-PerCP；Isotype control group is 20 μ LmIgG1-FITC, 20 μ LmIgG1-PE and 20 μ LmIgG1-PerCP；Place 4
DEG C refrigerator is washed 3 times after dyeing 30 minutes with PBS, then machine testing and analysis on FACS Calibur instruments；The results show that
Patient with breast cancer NK cell CD56 and CD16 positive rate are 94.4%；Normally NK cells of human beings CD56 and CD16 positive rate is
93.4%；Patient with breast cancer's CD3 positive rates are only 5.9%；Normal person's CD3 positive rates are only 6.4%, show both in vitro cultures
Cell be mainly NK cells, lymphocyte quantity containing T is very low.
9. according to the method described in claim 1, it is characterized in that, the K562 cell numbers: lymphocyte number=1: 1-1: 5.
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