CN107400659A - A kind of cultural method of NK cells - Google Patents
A kind of cultural method of NK cells Download PDFInfo
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Abstract
The invention belongs to medicine technology field, discloses a kind of cultural method of NK cells, comprises the following steps：Peripheral blood is separated, obtains PBMC cells；It is standby；The K562 cells of cellar culture are inoculated in culture medium；Then the DMEM nutrient solutions for changing serum-free synchronize, and are then resuspended using basal medium, add IL 2, IL 15, carry out cell culture, every fluid infusion in 3 days once, cultivate to K562 cells are added at the 6th~7,8 day, cultivate 16~18 days and obtain NK cells；It is inoculated with modeling.The present invention promotes the propagation of cell using autoserum, avoids the pollution of extrinsic protein and virus；The present invention only needs the IL 2 of relatively low-dose, greatly reduces production cost；It is higher that cultural method of the present invention turns out the NK cell purities come.
Description
Technical field
The invention belongs to medicine technology field, more particularly to a kind of cultural method of NK cells.
Background technology
Cell therapy is the disease treatment new technology risen in recent years, refers to utilize some cells with specific function
Characteristic, after being obtained using biological engineering method and/or being handled by amplification in vitro, specific culture etc., there are these cells and increase
Strong immune, the kill therapeutic efficiency such as pathogen and tumour cell, promotion organization neomorph and physical recovery, so as to reach treatment
The purpose of disease.Curative cell includes：NK, gamma delta T, CD3AK, DC-CIK etc., its curative effect, specificity, it is overall efficient,
The situation of side effect reaction etc. progressively improves.
NK cells (natural killer cell) are also referred to as NK, belong to lymphocyte linage cell, contain
The cellulotoxic lymphocyte of perforin and granzyme particle, identification to target cell without MHC (major histocompatibility complex,
Major histocompatibility complex) limitation is important immune cell.NK cell deriveds in marrow,
Belong to the large granular lymphocyte of lymphocyte linage, it accounts for the 5~10% of total number of peripheral blood, and no specificity is known
, be not required to by antigen presentation and can direct killing tumour cell, and can secrete cytokines adjust other immunocytes
Function, play the role of in oncotherapy, viral infection resisting and removing dissident's cell important.
NK cells achieve greater advance in terms of cancer treatment, mainly take with performing the operation, chemicotherapy is combined
Therapeutic modality, compared with the simple chemicotherapy of tradition, with reference to the treatment of NK cells, enhance the immunologic function or directly of patient
Kill tumour cell and virus infection cell, so aid in because chemicotherapy cause it is in poor health, have a low resistance and caused by two
It is secondary injury, easily infect it is sick etc..And cell therapy can effectively suppress the transfer diffusion of tumour cell, and make without secondary
With.
NK cells are first of natural defence lines that body is anti-infective, antitumor, are the important immunocytes of body.Activation
NK cells can synthesize and secrete cytokine profiles, play the work for adjusting immune and hemoposieis and direct killing target cell
With.At present, NK cells have been widely used in the clinical treatment for carrying out tumor patient, meanwhile, NK cell therapies are to virus or thin
Bacterium infection, immunological regulation relevant disease and anti-aging also have the effect of certain.
In summary, the problem of prior art is present be：At present, mainly using simple antigen presenting cell and NK cells
The method of co-incubation, to promote the propagation of NK cells.It is and this using simple Porcine HGF stimulation NK cell amplifications
The method NK cell purities that expand to obtain it is low, killing vigor is low；
At present, promote the method for NK cells to have much in vitro, there is the addition animal blood serum in cultivating system to be cultivated,
There is researcher by K562 cells by, by irradiation, then stimulating NK cells in vitro as feeder cells after genetic modification
Growth.Although the above method can stimulate NK cell high-efficients to expand, animal blood serum complicated component, it is possible to during materials
Bring mycoplasma, virus into, uncertain and insecurity is caused to NK cell culture；K562 is thin as a tumour in itself
Born of the same parents, very big query in terms of clinical safety be present, be not suitable for clinical NK cell therapies；The cell of culture is lacked correctly
Evaluation.
The content of the invention
To solve the problems, such as that prior art is present, the present invention provides a kind of cultural method of NK cells.
The present invention is achieved in that a kind of cultural method of NK cells, and the cultural method of the NK cells is including following
Step：
Peripheral blood is separated, obtains PBMC cells；It is standby；
By the K562 cells of cellar culture be inoculated in containing in 10%~15%DMEM culture mediums at 36 DEG C~37 DEG C, 6%~
12%CO2At least 15h~20h is incubated in incubator, it is standby after being allowed to completely adherent 80%；First washed with anteserum-less substrate standby
K562 cells；
Then the DMEM nutrient solutions for changing serum-free synchronize 8h~16h, then add 2 μm of ol/L~2.5 μm ol/L
PBMC, 36 DEG C~37 DEG C are placed in, 60h~72h is incubated in 10%~15%DMEM medium culture casees, is taken out from incubator,
Culture supernatant is abandoned, is then washed 3 times~6 times with 0.01mol/L~0.05mol/L PBS solutions,
Then it is resuspended using basal medium, adds IL-2, IL-15, carries out cell culture, every fluid infusion in 3 days once, training
Support to K562 cells are added at the 6th~8 day, cultivate 16~18 days and obtain NK cells；
It is inoculated with modeling：K562 rat model groups are inoculated in cultured NK cells；By NK cell liquid 0.05ml~0.1ml
Vaccinization 60 days；Analyzing rat appetite, fur, body weight, activity performance.
Further, also need to carry out RNA microarrayed genes detections after being inoculated with modeling：After 60 days, by the K562 rats after inoculation
Model group rats are put to death, and marrow, which is promptly released, is placed in cryopreservation tube, is directly placed into liquid nitrogen and freezes, and whole behaviour is completed in the short time
Make；Total serum IgE is collected to specifications with MirVana microRNA isolation kit；Appropriate RNA solution is taken, through ultraviolet point
Light photometric quantification determines RNA sample OD260 and OD280 value.
Further, then calculate RNA concentration, 1% agarose electrophoresis observation total serum IgE, take the μ g Microcon of 3 μ g~6
The micro- centrifugal filtration post filterings of centrifugal filter, obtain the tiny RNA that fragment is less than 300；Will using Poly polymerases
3 ' ends of tiny RNA add poly (A) tail, reconnect an oligonucleotide mark, for follow-up fluorescence labeling, complete μ
ParafloTM microRNA microarrayed genes
Further, also carry out expressing experiment and analysis after the detection of RNA microarrayed genes：Hybridize instrument by micro circulation pump to exist
On μ ParafloTM micro-fluid chips overnight, hybridized using the buffer solutions of SSPE containing ammonium formate, rinsing dries, and uses laser scanning
Instrument gathers hybridization image, and Array--Pro softwares are digitally converted to hybridization image, data processing and analysis, calculates two groups
The p value that the ratio of signal and t are examined is detected, with p<There were significant differences property that 0.01 is defined as signal, analyze the position of differential expression
Point, rat micro-array chip include 454 microRNA, include microRNA and Partial mature ripe in database
MicroRNA complementary strand；Detection probe on chip carries out 27 repetitions, and experiment is carried out 6 times.
Further, also need to carry out after expression experiment and analysis：
Real-time fluorescence quantitative PCR detects：Collected to specifications always with MirVana microRNA isolation kit
RNA, calculate RNA concentration；Special reverse transcriptase primer obtains from Taqman MicroRNA Assays, and uses Taqman
RNA reverse transcriptions are that cDNA carries out real-time fluorescence quantitative PCR detection by MicroRNA ReverseTranscription Kit, instead
ABI Prism7900 software analysis results are used after should terminating, PCR experiment is carried out 6 times, and number is analyzed with threshold cycle method is compared
According to obtaining Ct values, i.e., fluorescence reaches the PCR cycle number needed for threshold value, and analyzes gene relative expression quantity.
Further, need to carry out Bioinformatics Prediction after real-time fluorescence quantitative PCR detection：Using miRanda,
TargetScan and PicTar3 bioinformatics microRNA target prediction software, to select in microRNA chip results
MicroRNA target gene is made prediction.
Further, need to carry out statistical procedures after Bioinformatics Prediction：It is special according to the property of data, distribution and design
Point, data are analyzed using SPSS13.0 statistical softwares.
Further, included with laser scanner collection hybridization image method：
Probe used in ultrasonography is carried out to K562 rat models to pop one's head in for L15-7io superficials, and marrow abnormal echo is remembered
Record, and gather image after measuring size；
Carry out Micro CT enhancing scannings to the K562 rat models of structure, the contrast agent that Micro CT are used for
Fenestra LC, contrast agent dosage are 0.4ml/20g, scan the enhancement method taken every time：Enter within 4 hours after injection contrast agent
Row scanning；
The cross-section position of magnetic resonance marrow and Coronal scanning, scanning sequence are carried out to the K562 rat models of structure：T2WI is horizontal
Section and Coronal.
NK cell culture mediums security provided by the invention is preferable, and the NK cells cultivated under the culture medium of said components are fast
The stable propagation of speed, and there is preferably killing vigor to tumour cell.Test result indicates that：NK cell culture provided by the invention
For base culture NK cells after two weeks, proliferation times have nearly turned over 50 times；To K562 killing activity 40:Reach during 1 effect target ratio
92%.
The present invention promotes the propagation of cell using autoserum, avoids the pollution of extrinsic protein and virus；This hair
The bright IL-2 for needing relatively low-dose, greatly reduces production cost；Cultural method of the present invention turn out come NK cell purities compared with
It is high.
The present invention discloses virus on molecular level by grouping experiment and infects essential part scientific meaning.Demonstrate
Complex relationship between the macrophenotypic and microbiological molecule of time.
The construction method of the present invention has the characteristics of simple to operate, cost is low, and cell model stability is high, autophagy activity
Height, and there is chemotherapeutics tolerance caused by high autophagy activity, help to analyze in vitro；Can be it is demonstrated experimentally that passing through this hair
The cell model of bright foundation has the repellence for stablizing more than 96 hours.Model group occur mobility significantly reduce, chaeta it is in disorder；
Weight loss.
Animal model provided by the invention, different infection biological characteristics are analyzed first, find height not
There is significant difference with biological characteristics index expression between cancer strain and the different Nodules of height；First to high and low different turns
The image for moving the cancer strain of characteristic is analyzed；A variety of PET are imaged first and carry out best of breed, infection is fully reflected and gives birth to
Thing characteristic, new method is provided for early diagnosis.
The present invention has high flux property, compared with high specific, outstanding advantages of detection quick and precisely, has to diagnosis important
Clinical meaning.
Brief description of the drawings
Fig. 1 is the cultural method flow chart of NK cells provided in an embodiment of the present invention.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The application principle of the present invention is described in detail below in conjunction with the accompanying drawings.
As shown in figure 1, the cultural method of NK cells provided in an embodiment of the present invention, comprises the following steps：
S101:Peripheral blood is separated, obtains PBMC cells；It is standby；
S102:By the K562 cells of cellar culture be inoculated in containing in 10%~15%DMEM culture mediums at 36 DEG C~37 DEG C,
6%~12%CO2At least 15h~20h is incubated in incubator, it is standby after being allowed to completely adherent 80%；First washed with anteserum-less substrate
Wash standby K562 cells；
S103:Then the DMEM nutrient solutions for changing serum-free synchronize 8h~16h, then add the μ of 2 μm of ol/L~2.5
Mol/L PBMC, 36 DEG C~37 DEG C are placed in, 60h~72h is incubated in 10%~15%DMEM medium culture casees, from incubator
Take out, abandon culture supernatant, then washed 3 times~6 times with 0.01mol/L~0.05mol/L PBS solutions；
S104:Then it is resuspended using basal medium, adds IL-2, IL-15, cell culture is carried out, every fluid infusion one in 3 days
It is secondary, cultivate to K562 cells are added at the 6th~8 day, cultivate 16~18 days and obtain NK cells；
S105:It is inoculated with modeling：K562 rat model groups are inoculated in cultured NK cells；By NK cell liquid 0.05ml~
0.1ml vaccinizations 60 days；Analyzing rat appetite, fur, body weight, activity performance.
Also need to carry out RNA microarrayed genes detections after inoculation modeling：It is after 60 days, the K562 rat model groups after inoculation is big
Mouse is put to death, and marrow, which is promptly released, is placed in cryopreservation tube, is directly placed into liquid nitrogen and freezes, and whole operation is completed in the short time；With
MirVana microRNA isolation kit collect total serum IgE to specifications；Appropriate RNA solution is taken, through ultraviolet spectrometry light
Degree meter quantitative determination RNA sample OD260 and OD280 value.
Calculate RNA concentration again, 1% agarose electrophoresis observation total serum IgE, take the μ g Microcon centrifugal of 3 μ g~6
The micro- centrifugal filtration post filterings of filter, obtain the tiny RNA that fragment is less than 300；3 ' ends of tiny RNA are added using Poly polymerases
Upper poly (A) tail, an oligonucleotide mark is reconnected, for follow-up fluorescence labeling, completes μ ParafloTM
MicroRNA microarrayed genes
Also carry out expressing experiment and analysis after the detection of RNA microarrayed genes：Instrument is hybridized in μ by micro circulation pump
On ParafloTM micro-fluid chips overnight, hybridized using the buffer solutions of SSPE containing ammonium formate, rinsing dries, and uses laser scanning
Instrument gathers hybridization image, and Array--Pro softwares are digitally converted to hybridization image, data processing and analysis, calculates two groups
The p value that the ratio of signal and t are examined is detected, with p<There were significant differences property that 0.01 is defined as signal, analyze the position of differential expression
Point, rat micro-array chip include 454 microRNA, include microRNA and Partial mature ripe in database
MicroRNA complementary strand；Detection probe on chip carries out 27 repetitions, and experiment is carried out 6 times.
Also need to carry out after expression experiment and analysis：
Real-time fluorescence quantitative PCR detects：Collected to specifications always with MirVana microRNA isolation kit
RNA, calculate RNA concentration；Special reverse transcriptase primer obtains from Taqman MicroRNA Assays, and uses Taqman
RNA reverse transcriptions are that cDNA carries out real-time fluorescence quantitative PCR detection by MicroRNA ReverseTranscription Kit, instead
ABI Prism7900 software analysis results are used after should terminating, PCR experiment is carried out 6 times, and number is analyzed with threshold cycle method is compared
According to obtaining Ct values, i.e., fluorescence reaches the PCR cycle number needed for threshold value, and analyzes gene relative expression quantity.
Need to carry out Bioinformatics Prediction after real-time fluorescence quantitative PCR detection：Using miRanda, TargetScan and
3 bioinformatics microRNA target prediction softwares of PicTar, to the target of select microRNA in microRNA chip results
Gene is made prediction.
Need to carry out statistical procedures after Bioinformatics Prediction：According to the property of data, distribution and design feature, application
SPSS13.0 statistical softwares are analyzed data.
Included with laser scanner collection hybridization image method：
Probe used in ultrasonography is carried out to K562 rat models to pop one's head in for L15-7io superficials, and marrow abnormal echo is remembered
Record, and gather image after measuring size；
Carry out Micro CT enhancing scannings to the K562 rat models of structure, the contrast agent that Micro CT are used for
Fenestra LC, contrast agent dosage are 0.4ml/20g, scan the enhancement method taken every time：Enter within 4 hours after injection contrast agent
Row scanning；
The cross-section position of magnetic resonance marrow and Coronal scanning, scanning sequence are carried out to the K562 rat models of structure：T2WI is horizontal
Section and Coronal.
NK cell culture mediums security provided by the invention is preferable, and the NK cells cultivated under the culture medium of said components are fast
The stable propagation of speed, and there is preferably killing vigor to tumour cell.Test result indicates that：NK cell culture provided by the invention
For base culture NK cells after two weeks, proliferation times have nearly turned over 50 times；To K562 killing activity 40:Reach during 1 effect target ratio
92%.
The present invention promotes the propagation of cell using autoserum, avoids the pollution of extrinsic protein and virus；This hair
The bright IL-2 for needing relatively low-dose, greatly reduces production cost；Cultural method of the present invention turn out come NK cell purities compared with
It is high.
The present invention discloses virus on molecular level by grouping experiment and infects essential part scientific meaning.Demonstrate
Complex relationship between the macrophenotypic and microbiological molecule of time.
The construction method of the present invention has the characteristics of simple to operate, cost is low, and cell model stability is high, autophagy activity
Height, and there is chemotherapeutics tolerance caused by high autophagy activity, help to analyze in vitro；Can be it is demonstrated experimentally that passing through this hair
The cell model of bright foundation has the repellence for stablizing more than 96 hours.Model group occur mobility significantly reduce, chaeta it is in disorder；
Weight loss.
Animal model provided by the invention, different infection biological characteristics are analyzed first, find height not
There is significant difference with biological characteristics index expression between cancer strain and the different Nodules of height；First to high and low different turns
The image for moving the cancer strain of characteristic is analyzed；A variety of PET are imaged first and carry out best of breed, infection is fully reflected and gives birth to
Thing characteristic, new method is provided for early diagnosis.
The present invention has high flux property, compared with high specific, outstanding advantages of detection quick and precisely, has to diagnosis important
Clinical meaning.
The application principle of the present invention is further described with reference to specific embodiment.
The amplification cultivation of embodiment 1NK cells
1st, the separation of PMNC
40~100mL peripheral blood is gathered, by it by volume 1 with physiological saline：1 ratio mixes, after mixing
Blood mixed liquor and lymphocyte separation medium by volume 2:1 ratio slowly adds to lymphocyte separation medium upper strata,
3000rpm centrifuges 20min；
Centrifuge tube is seen from top to bottom after centrifugation, respectively blood plasma, tunica albuginea layer (i.e. mononuclearcell layer, PBMC), lymph
Cell separating liquid, red blood cell layer, PBMC layers are extracted, after the PBMC being collected into is resuspended with brine, 1500rpm centrifugations
10min, repeated washing twice, are collected into PBMC；
2nd, the amplification cultivation of NK cells
By the PBMC being collected into and the antigen presenting cell of above-mentioned preparation by 1:1 ratio mixed culture, by 1 × 106
Density is inoculated in blake bottle, using 1640 medium cultures, adds Porcine HGF IL-2 and IL-15, final concentration difference
For 200U/mL, 20U/mL；The autoserum of cumulative volume 10% is added again, and adds pseudo-ginseng saponin, final concentration of 10mg/mL；Put
In 37 DEG C, concentration is 5% CO2Cultivated in incubator.Every 3 days supplement fresh cultures and add IL-2, IL-15 and pseudo-ginseng
Saponin(e, final concentration are respectively 200U/mL, 20U/mL and 10mg/mL.Cell density is not more than 3 × 106/mL, fluid infusion before fluid infusion
Cell density is in 0.5~1.0 × 106/mL afterwards.Antigen presenting cell is added when cultivating by the 5th day again and carries out secondary stimulus,
Until the 14th day collects cell.
Embodiment 2
The amplification cultivation of NK cells
1st, the separation of PMNC
40~100mL peripheral blood is gathered, by it by volume 1 with physiological saline：1 ratio mixes, after mixing
Blood mixed liquor and lymphocyte separation medium by volume 2:1 ratio slowly adds to lymphocyte separation medium upper strata,
3000rpm centrifuges 20min；
Centrifuge tube is seen from top to bottom after centrifugation, respectively blood plasma, tunica albuginea layer (i.e. mononuclearcell layer, PBMC), lymph
Cell separating liquid, red blood cell layer, PBMC layers are extracted, after the PBMC being collected into is resuspended with brine, 1500rpm centrifugations
10min, repeated washing twice, are collected into PBMC；
2nd, the amplification cultivation of NK cells
By the PBMC being collected into and the antigen presenting cell of above-mentioned preparation by 1:3 ratio mixed culture, by 1 × 106
Density is inoculated in blake bottle, using 1640 medium cultures, adds Porcine HGF IL-2 and IL-15, final concentration difference
For 500U/mL, 100U/mL；The autoserum of cumulative volume 10% is added again, and adds pseudo-ginseng saponin, final concentration of 50mg/mL；
37 DEG C are placed in, concentration is 5% CO2Cultivated in incubator.Every 3 days supplement fresh cultures and add IL-2, IL-15 and field
Seven saponin(es, final concentration are respectively 500U/mL, 100U/mL and 50mg/mL.Cell density is not more than 3 × 106/mL before fluid infusion,
Cell density is in 0.5~1.0 × 106/mL after fluid infusion.It is secondary that antigen presenting cell progress is added when cultivating by the 5th day again
Stimulate, until the 14th day collects cell.
The amplification cultivation of embodiment 3NK cells
1st, the separation of PMNC
40~100mL peripheral blood is gathered, by it by volume 1 with physiological saline：1 ratio mixes, after mixing
Blood mixed liquor and lymphocyte separation medium by volume 2:1 ratio slowly adds to lymphocyte separation medium upper strata,
3000rpm centrifuges 20min；
Centrifuge tube is seen from top to bottom after centrifugation, respectively blood plasma, tunica albuginea layer (i.e. mononuclearcell layer, PBMC), lymph
Cell separating liquid, red blood cell layer, PBMC layers are extracted, after the PBMC being collected into is resuspended with brine, 1500rpm centrifugations
10min, repeated washing twice, are collected into PBMC；
2nd, the amplification cultivation of NK cells
By the PBMC being collected into and the antigen presenting cell of above-mentioned preparation by 1:2 ratio mixed culture, by 1 × 106
Density is inoculated in blake bottle, using 1640 medium cultures, adds Porcine HGF IL-2 and IL-15, final concentration difference
For 300U/mL, 50U/mL；The autoserum of cumulative volume 10% is added again, and adds pseudo-ginseng saponin, final concentration of 30mg/mL；Put
In 37 DEG C, concentration is to be cultivated in 5% CO2 incubators.Every 3 days supplement fresh cultures and add IL-2, IL-15 and pseudo-ginseng
Saponin(e, final concentration are respectively 300U/mL, 50U/mL and 30mg/mL.Cell density is not more than 3 × 106/mL, fluid infusion before fluid infusion
Cell density is in 0.5~1.0 × 106/mL afterwards.Antigen presenting cell is added when cultivating by the 5th day again and carries out secondary stimulus,
Until the 14th day collects cell.
Embodiment 4NK cell Indexs measures
The NK cells for detecting existing method and the method culture of the embodiment of the present invention 1~3 carry out indices detection contrast, examination
It is as follows to test group setting：
Experimental group 1：Embodiment 1 cultivates the NK cells of 14 days；
Experimental group 2：Embodiment 2 cultivates the NK cells of 14 days；
Experimental group 3：Embodiment 3 cultivates the NK cells of 14 days；
Control group：In addition to antigen presenting cell and pseudo-ginseng saponin is not added, other operations are identical with the inventive method.
1st, the survival rate test of cell
After NK cells after culture are carried out into Trypan Blue, cell count is carried out with cell counting count board, calculates cell
Motility rate.And the Cell viability of experimental group is all higher than control group.
2nd, the detection of the amplification quantity of cell
After NK cells after culture are carried out into Trypan Blue, cell count is carried out with cell counting count board.
It can be seen that the cell proliferating number amount after each group culture, and the propagation quantity of experimental group is above control group.
3rd, the killing viability examination after cell culture
NK cells after culture are carried out to the detection of killing activity, using lactic dehydrogenase (LDH) method, using K562 cells as
Target cell, the killing vigor of each group NK cells is detected respectively.Imitate target ratio respectively 40:1、20:1、10:1、5:1 cell number ratio
Effector cell and target cell are added 96 orifice plates by example, and target cell number is 104/hole, and every kind of effect target ratio sets 4 parallel holes, per hole
200μL.It is resuspended using RPMI1640 culture mediums.In 37 DEG C, 5%CO24h is incubated in incubator, then uses LDH-
Cytotoxicity Colorimetric Assay Kit II (U.S., BioVision, article No.：K313-500) kit enters
Row detection, using light absorption value (A) of the ELIASA Detection wavelength in 490nm.The calculating process of killing vigor is as follows：
The light absorption value of all experimental ports, the spontaneous LDH release apertures of K562 and the spontaneous LDH release apertures of NK cells is subtracted into culture medium
The average of background light absorption value；The light absorption value of K562 maximums LDH release controls is subtracted to the average of volume correction control light absorption value；
Cell killing vigor %=(spontaneous-ODK562 of OD experiment-ODNK cells is spontaneous)/(ODK562 maximums-ODK562 is certainly
Hair) × 100.
It can be seen that killing of each group NK cells to K562 cells it is living with effect target than increase and strengthen, the NK of each experimental group
Cell killing vigor no significant difference, the result of experimental group 3 is optimal, and the NK cells of control group kill in different effect target ratios
Vigor is below each experimental group.
4th, immunophenotype (purity of NK cells) detects after cell culture
The NK cells collected after culture are resuspended with PBS, density is adjusted to 106/mL, add FITC marks CD16 and
The CD56 antibody of PE marks, after room temperature lucifuge is incubated 30min, with flow cytomery, detection has corresponding immunophenotype
Cell proportion testing result.
Conclusion：
From the point of view of above-mentioned each laboratory test results, the result of experimental group is better than control group, illustrates the culture side of the present invention
Method is better than conventional cultural method.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. a kind of cultural method of NK cells, it is characterised in that the cultural method of the NK cells comprises the following steps：
Peripheral blood is separated, obtains PBMC cells；It is standby；
By the K562 cells of cellar culture be inoculated in containing in 10%~15%DMEM culture mediums at 36 DEG C~37 DEG C, 6%~12%
CO2At least 15h~20h is incubated in incubator, it is standby after being allowed to completely adherent 80%；First washed with anteserum-less substrate standby
K562 cells；
Then the DMEM nutrient solutions for changing serum-free synchronize 8h~16h, then add 2 μm of ol/L~2.5 μm ol/L PBMC,
36 DEG C~37 DEG C are placed in, 60h~72h is incubated in 10%~15%DMEM medium culture casees, is taken out from incubator, abandon culture
Supernatant, then washed 3 times~6 times with 0.01mol/L~0.05mol/L PBS solutions,
Then it is resuspended using basal medium, adds IL-2, IL-15, carry out cell culture, every fluid infusion in 3 days once, culture is extremely
K562 cells are added at the 6th~8 day, cultivates 16~18 days and obtains NK cells；
It is inoculated with modeling：K562 rat model groups are inoculated in cultured NK cells；NK cell liquid 0.05ml~0.1ml is continuous
Inoculation 60 days；Analyzing rat appetite, fur, body weight, activity performance.
2. the cultural method of NK cells as claimed in claim 1, it is characterised in that also need to carry out the micro- battle arrays of RNA after inoculation modeling
Row genetic test：After 60 days, the K562 rat model groups rat after inoculation to be put to death, marrow, which is promptly released, is placed in cryopreservation tube,
It is directly placed into liquid nitrogen and freezes, whole operation is completed in the short time；With MirVana microRNA isolation kit according to
Specification collects total serum IgE；Appropriate RNA solution is taken, RNA sample OD260 and OD280 value is quantitative determined through ultraviolet specrophotometer.
3. the cultural method of NK cells as claimed in claim 2, it is characterised in that calculate RNA concentration, 1% agarose electricity again
Swimming observation total serum IgE, takes the μ g of 3 μ g~6 to be filtered with the micro- centrifugal filtration posts of Microcon centrifugal filter, obtains fragment
Tiny RNA less than 300；3 ' ends of tiny RNA are added into poly (A) tail using Poly polymerases, reconnect an oligomerization core
Thuja acid marks, and for follow-up fluorescence labeling, completes μ ParafloTM microRNA microarrayed genes.
4. the cultural method of NK cells as claimed in claim 2, it is characterised in that also carried out after the detection of RNA microarrayed genes
Expression experiment and analysis：Stayed overnight by micro circulation pump hybridization instrument on μ ParafloTM micro-fluid chips, using containing ammonium formate
SSPE buffer solutions are hybridized, and rinsing dries, and gather hybridization image with laser scanner, Array--Pro softwares are to hybridization image
It is digitally converted, data processing and analysis, the p value that two groups of ratios for detecting signal and t are examined is calculated, with p<0.01 is fixed
Justice is signal there were significant differences property, analyzes the site of differential expression, and rat micro-array chip includes 454 microRNA, comprising
Ripe microRNA and Partial mature microRNA complementary strand in database；Detection probe on chip carries out 27 weights
Multiple, experiment is carried out 6 times.
5. the cultural method of NK cells as claimed in claim 4, it is characterised in that also need to carry out after expression experiment and analysis：
Real-time fluorescence quantitative PCR detects：Total serum IgE is collected to specifications with MirVana microRNA isolation kit,
Calculate RNA concentration；Special reverse transcriptase primer obtains from Taqman MicroRNA Assays, and with Taqman MicroRNA
RNA reverse transcriptions are that cDNA carries out real-time fluorescence quantitative PCR detection by ReverseTranscription Kit, and reaction is used after terminating
ABI Prism7900 software analysis results, PCR experiment are carried out 6 times, with threshold cycle method analyze data is compared, obtain Ct values,
I.e. fluorescence reaches the PCR cycle number needed for threshold value, and analyzes gene relative expression quantity.
6. the cultural method of NK cells as claimed in claim 5, it is characterised in that real-time fluorescence quantitative PCR detection after need into
Row Bioinformatics Prediction：Using 3 bioinformatics microRNA target prediction softwares of miRanda, TargetScan and PicTar,
The target gene of select microRNA in microRNA chip results is made prediction.
7. the cultural method of NK cells as claimed in claim 6, it is characterised in that need to be counted after Bioinformatics Prediction
Handle：According to the property of data, distribution and design feature, data are analyzed using SPSS13.0 statistical softwares.
8. the cultural method of NK cells as claimed in claim 4, it is characterised in that gather hybridization image side with laser scanner
Method includes：
Probe used in ultrasonography is carried out to K562 rat models to pop one's head in for L15-7io superficials, and marrow abnormal echo is recorded, and
Image is gathered after measurement size；
Micro CT enhancing scannings are carried out to the K562 rat models of structure, the contrast agent that Micro CT are used is Fenestra
LC, contrast agent dosage are 0.4ml/20g, scan the enhancement method taken every time：It is scanned within 4 hours after injection contrast agent；
The cross-section position of magnetic resonance marrow and Coronal scanning, scanning sequence are carried out to the K562 rat models of structure：T2WI cross sections
And Coronal.
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| CN106635987A (en) * | 2016-12-27 | 2017-05-10 | 宁波枫林生物科技有限公司 | Method for carrying out in-vitro efficient amplification on natural killer (NK) cells, and application of method |
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