CN107064085A - Test Procedur of Splenic Natural Killer Cell Activity - Google Patents
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Abstract
A kind of Test Procedur of Splenic Natural Killer Cell Activity, " NK cell proportions " and " karyocyte motility rate " parameter is introduced when effector cell and target ration calculate, and carries out quality control in each link, reduces detection otherness.
Description
Technical field
The present invention relates to a kind of Test Procedur of Splenic Natural Killer Cell Activity, belong to field of biological detection.
Background technology
NK cells (Natural killer cells, NK cell, NK) are initially because without presensitization
Just autologous tumor cell can be killed, and is referred to as innate immunity lymph toxic cell.By for decades to its phenotype and function
Careful research, finding the principal biological function of NK cells mainly has secrete cytokines to participate in immunological regulation and to target cell
Direct killing effect [1,2].NK cell distributions Various Functions in different tissues, not only with antitumor, the various diseases of participation
Reason process, maintenance immune homeostasis, may additionally facilitate gestational period placenta vascularization process.Correspondingly, malignant tumour, various senses
Dye, autoimmune disease etc. all refer to NK cell disorders [3-6].
NK cell killing activities are the important indicators for evaluating immune state, available for immune shape during monitoring immunization therapy
In terms of the change of state.Existing document report, its also with Survival after pernicious Chemotherapy of Cervical Cancer curative effect, pancreatic cancer chemotherapy and Ah
Alzheimer's disease tempo correlation [7-9].In familial or acquired Hemophagocytic lymphohistocysis disease, inflammation
It is one of its feature that NK killing activities are impaired in disease, autoimmune disease and immunologic deficiency disease, but because the index is respectively tested
Room result can not compare, and lack normal reference value, and the confirmation frequently as other diagnosis indexs in relevant disease diagnosis refers to
Mark [10].The rigorous, determination method of standardization and corresponding normal reference value are badly in need of in the detection of NK killing activities, solve it
The bottleneck problem of application.
" the goldstandard method " of NK killing detection methods is 51Cr release experiments, but is limited the problems such as due to radioactive pollution
Its application [11,12] in clinical detection.Other methods for being used for the detection of NK cell killing activities also include low toxicity dyestuff
Calcein-AM release experiments, LDH release experiments, the detection method [13-17] based on flow cytometry or bioluminescence.Wherein
Detection method based on flow cytometry have it is easy to operate, stably, pollute less, baseline it is low and with 51Cr release experiment method phases
The advantages of closing property is good, existing clinic is more to carry out NK cell killing activities detection [14,18] using the method.
But the method for prior art is only calculated with number of nucleated cells and target cell number, may result in the inclined of testing result
Move, it is impossible to realize the rigorous assessment to NK cellkilling capacities.
The content of the invention
The present invention provides a kind of Test Procedur of Splenic Natural Killer Cell Activity, it is characterised in that comprise the following steps:
1) by target cell Secondary Culture;
2) NK ratio is analyzed in pairing effect cell;
3) by target cell with effector cell according to the effect target of setting than being cultivated;
4) detecting step 3) in target cell death rate to be measured;
5) by step 4) in target cell death rate bring into formula calculate natural killer cell activity:
Natural killer cell activity=(target cell death rate to be measured-control group target cell death rate)/(1- control group targets are thin
Born of the same parents' death rate) × 100%.
Further, step 3) in effect target ratio be more than 4.
Further, step 1) in enter the quality control of line density and/or the death rate to passage cell.
Further, step 2) in pairing effect cell carry out cell density, living cell rate and NK ratio extremely
One of few quality control.
Further, the method for quality control includes the target cell of Secondary Culture carrying out dye marker.
Further, including with antibody NK is marked.
Further, control target cell natural mortality rate is within 3%.
Further, the ratio between target cell and effector cell's FH1 passages MIF (average fluorescent strength) value are more than 80 times.
Further, step 4) in, give up the following detection number of degrees:Compared with only target cell control tube, effect target cell mixes
Close be incubated detection pipe intracellular targets concentration less than the former 85%.
Further, tumour selection effect target ratio whether was suffered from according to detection object.
The present invention establishes a kind of brand-new NK cell killing vigor testing methods, in effector cell and target ration meter
" NK cell proportions " and " karyocyte motility rate " parameter is introduced during calculation, the preciseness of the detection is improved, at the same with by NK cells
Carry out detection after sorting to compare, in the absence of NK cell magnetic bead sortings are with high costs and mechanical damage of selected by flow cytometry apoptosis etc. is asked
Topic.
This method improves NK cell killing vitality assessments between separate sources sample compared with conventional traditional detection method
Comparativity, with broader practice prospect.
Present invention firstly provides carry out quality control to each link of the detection, it is therefore an objective to reduces the otherness detected between room,
It is expected to solve the present situation that each laboratory detection result can not be referred to mutually.
The present invention assesses the human peripheral N K cell killing vigor normal reference value models for being available for different experiments room to refer to first
Enclose.
Brief description of the drawings
The NK cell proportion regularities of distribution in Fig. 1 sample karyocytes to be checked
Fig. 2 tumor patients group organizes peripheral blood NK cell killing activity with healthy volunteer
Embodiment
1 data and method
1.1 research objects are chosen to go to a doctor in affiliated hospital of Military Medical Science Institute and had a medical check-up in June, 2014 in October, 2016
Healthy volunteer and tumor patient totally 108, collects age, sex and NK killing activities detection correlated results.Separately collect other
NK killing activities detect the karyocyte NK percent informations of sample 158.Reference value group inclusion criteria:1. healthy population;2. blood
As normal;3. informed consent form is signed.Reference value exclusion standard:1. immune correlated disease person;2. there are long-term steroid and immune tune
Save agent application history person;3. there is clear and definite the infected in 3 weeks;4. the person that has cardiovascular and cerebrovascular disease;5. it is doubtful or clarify a diagnosis for tumour suffer from
Person;6. other influences immune function disease or medical history.
1.2 reagents and instrument PI fluorescent dyes, the analysis of NK cells of human beings subgroup monoclonal antibody (CD45-APC, CD56-
PE, CD3-PerCP) and its Isotype control be purchased from U.S. company BD, CSFE fluorescent dyes are purchased from Japanese colleague company, and people's lymph is thin
Born of the same parents' separating liquid is purchased from the foreign China Tech bio tech ltd of Tianjin Hao, and cell culture is purchased from 1640 culture mediums and hyclone
Thremo Fisher companies.Detection BD Accuri C6 flow cytometers.
1.3 experimental method
1.3.1 the Secondary Culture of target cell:K562 cells press 1-2 × 105Cells/ml density, is inoculated in containing 20ml's
In 1640 complete medium T75 blake bottles, passage in every three days once, maintains its good vegetative state.
1.3.2 the dye marker of target cell:K562 suspension cultures are collected in 15ml centrifuge tubes, horizontal centrifugal is placed in
1000rpm is centrifuged 10 minutes in machine.With brine twice after, unicellular is resuspended in 500ul serum-frees 1640 and trained
Support in base, sampled after fully mixing, count stand-by using C6.It is another to take the serum free medium of certain volume 1640 to dilute CSFE dyestuffs
Working solution is simultaneously fully mixed, and is rapidly added in stand-by K562 cell suspensions and mixed, and it is 5 μM to make CSFE dye concentrations,
Cell suspension density is 1 × 107cells/ml.It is placed in 37 DEG C of 5%CO2In incubator, shake once within every 5 minutes.It is incubated 10 points
The hyclone of isometric 4 DEG C of precoolings is added after clock, is placed in 4 DEG C of refrigerators and terminates staining reaction 10 minutes.Supply physiological saline
1000rpm is centrifuged 10 minutes after mixing, is abandoned supernatant and is resuspended in 1640 complete mediums, is placed in incubator and is incubated 1 hour.Receive
Collect cell, once, cell is resuspended in 1640 complete mediums to brine, and sampling counts stand-by with C6.
1.3.3 the separation of effector cell:If sample is whole blood, then PMNC (Peripheral is carried out
Blood mononuclear cell, PBMC) separation, i.e., by whole blood sample 1800rpm centrifuge 10 minutes, go after blood plasma wait body
Product physiological saline gravity treatment cell, is gently added in after being centrifuged on lymphocyte separation medium, carefully siphons away tunica albuginea layer.Washed with physiology salt
Wash twice, cell is resuspended in 1640 complete mediums, sampling is diluted in white blood cell count(WBC) liquid, count stand-by with C6.If sample is to exempt from
Epidemic disease cell culture product, then only with brine twice.Sampling is diluted in 1640 complete mediums, is counted and treated with C6
With.
1.3.4 the mark of effector cell NK cell subsets:Take containing about 5 × 105The 100ul effects of individual mononuclearcell (PBMC)
Cell suspension is answered, CD3-CD56+NK cell subsets antibody labelings is carried out by flow cytometer detection antibody specification, uses BD FACS
Calibur flow cytometers are detected, analyze NK cell proportions in karyocyte.PBMC then carries out erythrocyte splitting step in this way
Suddenly, in this way immunocyte cultured products then without erythrocyte splitting step.
1.3.5 the detection of effector cell's living cell rate:Such as sample is immunocyte cultured products, then needing detection, it has core thin
Born of the same parents' living cell rate.100ul cell suspensions are taken, PI dyestuffs is added to final concentration 3.75ug/ml, balances 5-10 minutes, fluidic cell
Instrument detects effector cell's death rate.
1.3.6 effector cell is mixed with target cell:Setting effect target ratio (NK cells and mark CSFE target cell it
Than) it is 0: 1,1: 1,2: 1,4: 1 and 6: 1, according in effector cell's karyocyte density, effector cell's motility rate and its karyocyte
Effector cell's volume, melange effect cell and target cell, are complemented to 1640 complete mediums needed for NK cell proportions are calculated
200ul co-culture systems.The multiple holes of each group at least three, mix and are incubated 2.5 hours altogether.
1.3.7 target cell death rate is detected:The mixed culture cell of 1.3.6 steps is resuspended, PI dyestuffs are added to final concentration
3.75ug/ml, is balanced 5-10 minutes, target cell death rate positive each pipe CSFE of flow cytomery.
1.3.8NK the calculating of cell killing vigor:Bring each pipe target cell death rate into formula (experimental group target cell death
Rate-control group target cell death rate)/(1- control group target cell deaths rate) × 100%, calculate each effect target and lived than NK cell killing
Mild-natured average.
1.4 quality control
1.4.1 the quality control of target cell observes growth conditions and speed by constant density inoculation passage K562 cells,
The indexs such as cell cycle are detected if necessary.Control balance durations of the K562 in 1640 complete mediums after CSFE marks, it is desirable to
K562 cell lines natural mortality rate after mark is less than 3%, target cell and effector cell's FH1 passage average fluorescent strengths
The ratio between (Median Fluorescence Intensity, MIF) value is more than 80 times, ensures that the target used of detection every time is thin with this
The uniformity of born of the same parents.
1.4.2 the quality control EDTA anticoagulant tubes collection periphery blood specimen of effector cell, in vitro to be sent to reality in 3 hours afterwards
Room is tested, room temperature placement is placed in, is resuspended in when effector cell is standby in 1640 complete mediums.As non-fresh blood sample or it is long when
Between be positioned over other conditions, karyocyte motility rate need to be detected, with the cell density of karyocyte, living cell rate and NK ratios calculate
Each effect target is than required PBMC suspension volumes.
1.4.3 the quality control of flow cytometer is calibrated by instrument fixed maintenance, with FH3 Air conduct measurement PI fluorescent dyes
Signal, the manual measuring effect cell of cell absolute number function replacement or target cell suspension density are measured with Accuri C6.
1.4.4 the same technical staff of the quality control of data analysis carries out reading under same standard, if any data
Accept or reject and analysis is then completed jointly by fixing three technical staff.Compared with only target cell control tube, effect target cell mixing is incubated
Educate detection pipe intracellular targets concentration less than the former 85% when, it is believed that be completely severed after the target cell death of part, cause testing result
Less than actual target cell death rate, then the testing result is insincere.
1.5 statistical methods carry out statistical analysis using SPSS21.0 softwares, and each index is represented with X ± s, and factor is related
Property analysis use between multiple linear regression analysis, two groups mean to compare to use independent samples t test.P < 0.05 are that difference has system
Meter learns meaning.
2 results
2.1 include research object this basic condition
Normal reference value group includes healthy volunteer 47, women 22 [- 68 years old 28 years old, average (47.5 ± 9.1) year],
Male 25 [- 69 years old 27 years old, average (47.3 ± 11.2) year].Tumor group includes all kinds of tumor patient 61 [liver cancer 23, pancreas
Gland cancer 8, lung cancer and each 5 of colon cancer, the carcinoma of the rectum, stomach cancer, breast cancer and each 3 of oophoroma, thyroid cancer 2, kidney, the moon
Road cancer, hypopharyngeal cancer, cancer of the esophagus, hepatobiliary cancer and each 1 of cholangiocarcinoma], women 18 [- 84 years old 36 years old, average (60.2 ± 11.9)
Year], male 43 [- 85 years old 17 years old, average (53.9 ± 13.5) year] refers to table 1.
The ordinary circumstance table (example) of table 1
2.2NK proportional difference
NK cells occupy nucleus ratio distribution situation (see Fig. 1) in each source sample in analysis NK killing activity detections,
As a result the parameter distribution situation is 10.8% ± 7.87% (3%-42%) in prompting tumor patient group, and the degree of bias is 1.714, kurtosis
For 3.609;The parameter distribution situation is 12.31% ± 5.25% (4%-23%) in healthy volunteer person's group, and the degree of bias is
0.576, kurtosis is -0.860;The parameter distribution situation is 13.48% ± 12.30% (0.11%- in other source groups
75.19%), the degree of bias is 3.095, and kurtosis is 11.283, it can be seen that in each source NK killing activities sample karyocyte to be checked
The NK cell proportion regularities of distribution are different, do not consider the greatest differences of the parameter when calculating effector cell with target ration such as,
Only calculated with number of nucleated cells and target cell number, may result in the skew of testing result, it is impossible to realized to NK cell killing energy
The rigorous assessment of power.
2.3NK cell killing activity Study on Relative Factors
It will be grouped by less than 44 years old, -59 years old 45 years old, more than 60 years old at age, analyze age, sex and whether suffered from
It is related between tumour and NK killing activities.As a result point out age, sex and whether suffered from tumour three when imitating target ratio and being 1 and 2
Non-correlation (P > 0.05) between person and NK killing activities.When effect target ratio is 4 and 6, sex, age property associated therewith are without statistics
Meaning (P > 0.05) is learned, suffered from tumour property whether associated therewith statistically significant (P < 0.05).Based on this data
Analysis, age and Effect of gender peripheral blood NK cell killing ability are not found, and whether suffering from tumour will produce to the index
Influence, and effect target ratio should be set in 4 or more than 6.
2.4NK cell killing activity normal reference values
It is that age and sex are unrelated with NK cell killing activities to consider this research healthy control group Data Representation, therefore true
When determining reference range, no longer it is grouped according to age or sex.The determination of term of reference uses method of percentiles, sets NK
The reference range of cell killing activity bilateral 90%, the results are shown in Table 2.
The peripheral blood NK cell killing activity normal reference value (%) of table 2
The NK killing activity features of 2.5 tumor patients
Comparison of tumor patient organizes organizes peripheral blood NK cell killing activity difference with healthy volunteer, as a result the target score of prompting effect
Not Wei 4 and 6 when, two group differences are statistically significant (P > 0.05).When it is 1 and 2 to imitate target ratio, two group differences are without statistics
Learn meaning (P < 0.05).Point out the tumor patient index to be less than health donors, and the detection project be considered as set imitate target ratio as
4 and 6.
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Claims (10)
1. a kind of Test Procedur of Splenic Natural Killer Cell Activity, it is characterised in that comprise the following steps:
1) by target cell Secondary Culture;
2) NK ratio is analyzed in pairing effect cell;
3) by target cell with effector cell according to the effect target of setting than being cultivated;
4) detecting step 3) in target cell death rate to be measured;
5) by step 4) in target cell death rate bring into formula calculate natural killer cell activity:
Natural killer cell activity=(target cell death rate to be measured-control group target cell death rate)/(1- control group target cells are dead
Die rate) × 100%.
2. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 1, it is characterised in that step 3) in effect target ratio be
More than 4.
3. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 1, it is characterised in that step 1) in passage cell
Enter the quality control of line density and/or the death rate.
4. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 1, it is characterised in that step 2) in pairing effect cell
Carry out the quality control of at least one cell density, living cell rate and NK ratio.
5. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 3, it is characterised in that the method for quality control bag
Include and the target cell of Secondary Culture is subjected to dye marker.
6. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 4, it is characterised in that including with antibody to killing naturally
Hinder cell to be marked.
7. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 3, it is characterised in that control target cell natural death
Rate is within 3%.
8. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 3, it is characterised in that target cell and effector cell
The ratio between FH1 passages MIF (average fluorescent strength) value is more than 80 times.
9. the Test Procedur of Splenic Natural Killer Cell Activity as described in one of claim 1-8, it is characterised in that step 4) in, house
Abandon the following detection number of degrees:Compared with only target cell control tube, effect target cell mixing is incubated detection pipe intracellular targets concentration and is less than
The former 85%.
10. Test Procedur of Splenic Natural Killer Cell Activity as claimed in claim 1, it is characterised in that whether foundation detection object
Suffered from tumour selection effect target ratio.
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CN110231275A (en) * | 2019-05-20 | 2019-09-13 | 西安交通大学 | A method of based on Flow cytometry NK cell killing activity |
CN112285081A (en) * | 2020-10-28 | 2021-01-29 | 上海睿钰生物科技有限公司 | Method for detecting cell killing efficacy and application thereof |
CN112285083A (en) * | 2020-10-28 | 2021-01-29 | 上海睿钰生物科技有限公司 | Method for evaluating cell killing efficacy |
CN112304851A (en) * | 2020-10-28 | 2021-02-02 | 上海睿钰生物科技有限公司 | Evaluation method of in vitro natural killer cell immunocompetence and application thereof |
CN113899725A (en) * | 2021-10-12 | 2022-01-07 | 江苏省人民医院(南京医科大学第一附属医院) | Method for real-time quantitative detection of degranulation and killing capacity of NK effector cells |
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Cited By (8)
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CN110231275A (en) * | 2019-05-20 | 2019-09-13 | 西安交通大学 | A method of based on Flow cytometry NK cell killing activity |
CN110231275B (en) * | 2019-05-20 | 2021-05-28 | 西安交通大学 | Method for detecting killing activity of NK (natural killer) cells based on flow cytometry |
CN112285081A (en) * | 2020-10-28 | 2021-01-29 | 上海睿钰生物科技有限公司 | Method for detecting cell killing efficacy and application thereof |
CN112285083A (en) * | 2020-10-28 | 2021-01-29 | 上海睿钰生物科技有限公司 | Method for evaluating cell killing efficacy |
CN112304851A (en) * | 2020-10-28 | 2021-02-02 | 上海睿钰生物科技有限公司 | Evaluation method of in vitro natural killer cell immunocompetence and application thereof |
CN112285081B (en) * | 2020-10-28 | 2021-11-19 | 上海睿钰生物科技有限公司 | Method for detecting cell killing efficacy and application thereof |
CN113899725A (en) * | 2021-10-12 | 2022-01-07 | 江苏省人民医院(南京医科大学第一附属医院) | Method for real-time quantitative detection of degranulation and killing capacity of NK effector cells |
CN113899725B (en) * | 2021-10-12 | 2022-04-29 | 江苏省人民医院(南京医科大学第一附属医院) | Method for real-time quantitative detection of degranulation and killing capacity of NK effector cells |
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