CN106932576A - A kind of detection method of the immune suppression function of people's regulatory T cells - Google Patents
A kind of detection method of the immune suppression function of people's regulatory T cells Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G01N15/149—
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/001—Assays involving biological materials from specific organisms or of a specific nature by chemical synthesis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4703—Regulators; Modulating activity
- G01N2333/4704—Inhibitors; Supressors
Abstract
The invention discloses a kind of detection method of the immune suppression function of people's regulatory T cells, the method is comprised the following steps：Collection peripheral blood extracts mononuclearcell；Cell surface antigen is marked using fluorescein-labeled anti-CD4 and anti-CD 25 antibody；Reuse fixed rupture of membranes agent and fix cell surface antibodies；Transcription factor Foxp3 and Helios in fluorescein-labeled anti-Foxp3 and anti-Helios antibody labeled cells core are used after rupture of membranes；Finally use flow cytomery CD4+CD25+FoxP3+Helios+Treg cell masses.The expression that the method passes through transcription factor Helios in quick detection children peripheral blood, the function of regulatory T cells in Direct Analysis peripheral blood.The method uses anti-Helios, anti-CD4, anti-CD25 and anti-FoxP3 streamings antibody, more rapidly can accurately mark the peripheral blood Treg cells with stronger immune suppression function.
Description
Technical field
The present invention relates to a kind of detection method of the immune suppression function of regulatory T cells in Fast Evaluation peripheral blood.Should
Method is used to monitor the inhibitory activity of regulatory T cells, belongs to biological technical field.
Background technology
The immune system of human body receives multiple fine adjustment, wherein regulatory T cells (regulatory T cell, Treg)
Important immune down regulation is played, is blocking immunity monitoring and the immune central immune cells of suppression effective antitumor.
Treg cells ratio shared in vivo seldom, only accounts for CD4 in normal human peripheral blood+5-10% of cell or so, and have and exempt from
The Treg cell proportions of epidemic disease rejection ability are lower.Treg cells express various kinds of cell surface molecular, such as CD4, CD25, FoxP3,
CTLA4, GITR and CD103 etc., it is that current cell is controlled that how recognition detection has the specific Treg of immune suppression function
Treat the emphasis of research.Early stage is thought that transcription factor FoxP3 is the most special label of Treg cells, but only expresses FoxP3
It is not enough to define Treg cells and determines the function and phenotype of Treg cells, current research finds, also other molecular markers
Have impact on the maturation and function of Treg cells.
The content of the invention
In order to solve the above technical problems, the present invention is on the basis of existing detection technique, optimizes a kind of vitro detection and adjust
The method of section property T cell immunologic function, by the use of Helios combinations CD4 CD25 FoxP3 as labelling functionality Treg cells
Method, the cell mass with practical adjustments function is found in overall adjustment T cell by finding target position, can be more effective
Detection possesses the cell colony of immune suppression function, and reaching scientific quantification has the purpose of regulatory T cells of inhibitory activity.
Step of the present invention is described in detail below：
1 collection：Take peripheral blood 5-10ml；
2 treatment samples：Take streaming pipe and be separately added into 100 μ l peripheral blood samples, often pipe adds 2ml erythrocyte cracked liquids, after mixing
Incubation at room temperature 15 minutes, 1500 revs/min of 20 DEG C of centrifugation 5min are washed 1 time, add 2mlPBS liquid the same terms to wash again 1 time,
Add 100 μ l PBS liquid resuspended into single cell suspension；
3. the mark of surface antibody：Each 5 μ l mark regulatory T cells surface molecular mark of antibody CD4, CD25 is added, room temperature is kept away
Light is incubated 20 minutes, and 20 DEG C of centrifugation 5min of PBS liquid 1500 rev/min are washed 1 time；
4. cell fixes rupture of membranes：1ml is added to fix rupture of membranes agent：10% formaldehyde/0.2%Triton-X/PBS re-suspended cells, room temperature
Lucifuge is incubated 1 hour, adds 2ml PBS liquid to wash 2 times；
5. core interior antibody dyeing：100 μ l PBS liquid re-suspended cells are added, each 5ul marks of core interior antibody Foxp3, Helios are added
Core interior antibody, room temperature lucifuge is incubated 1 hour, adds 2ml PBS liquid to wash 2 times；
6. flow cytometer detection：Add the PBS liquid of 400 μ l resuspended into single cell suspension, strainer filtering addition streaming orifice plate, fluidic cell
Machine testing CD4 on instrument+CD25+FoxP3+Helios+Cell accounts for CD4+Cell proportion；
7 mononuclearcells are separated：Peripheral blood whole blood sample is according to volume ratio 1：Peripheral blood is slowly added to Ficoll by 1 ratio
In lymphocyte separation medium, 2500 revs/min are centrifuged 25-30 minutes, collect the mononuclearcell of middle tunica albuginea layer, add 15ml
PBS liquid, is centrifuged 10 minutes by 1800 revs/min, washs 2 times；
8 sortings：Magnetic bead sorting CD4+CD25+Treg cells, wherein negative sorting CD4+Cell, positive sorting CD25+Cell,
Different groups of CD4 of CFSE experiment detections+CD25+Treg cells are to CD4+CD25-The immune suppression function of Th cells；
9 detections：Helios siRNA are built, Helios siRNA are transfected into the ALL CD4 after sorting+CD25+Treg cells
In, CFSE experiment detection Treg Cellular immunity suppression functions, untransfected Treg cells are as a control group.
In sum, the purpose of the present invention is directed to imperfection at present to Treg cellular elements mark, there is provided Yi Zhongxin
With stronger immune suppression function human peripheral Treg cellular immunities mark, so as to detect the immune suppression function of T cell.
Brief description of the drawings
Fig. 1：ALL and CD4 in healthy children peripheral blood+CD25+FoxP3+Helios+The ratio of Treg cells；
Fig. 2：The difference of ALL and healthy children peripheral blood T reg immune suppression functions；
Fig. 3：Helios expresses the influence to Treg immune suppression functions；
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited to that：
Embodiment 1
Treg cell detections
The present embodiment material therefor is mainly as follows：
（1）Specimen origin：The example uses the peripheral blood of parents of hospitalized children agreement mandate of being had a medical check-up through initial ALL infants and normal health
As the source of mononuclearcell, 10 parts of ALL peripheral bloods and 10 parts of normal control peripheral bloods are gathered altogether, add anticoagulant heparin, often
Part 3-5ml；
（2）Main agents：Anti-human CD4-FITC, FoxP3-APC antibody of mouse is purchased from ebiosicence companies of the U.S., and mouse is anti-human
CD25-PE-Cy5, Helios-PE antibody are purchased from Biolegend companies of the U.S. respectively, and FoxP3 Fix/Perm buffer are purchased from
Ebioscience, erythrocyte cracked liquid is purchased from BD Biosciences companies of the U.S., and cell counter is Beck-man
Coulter (U.S.), GuavaeasyCyte 6HT flow cytometer are produced for EMD Millipore companies of the U.S.；
（3）CD4+CD25+FoxP3+Helios+Cell accounts for CD4+The flow cytometer detection of T cell ratio：Take streaming pipe and be separately added into 100
μ l peripheral blood samples, often pipe add 2ml erythrocyte cracked liquids, after mixing room temperature lucifuge be incubated 15 minutes, 1500 revs/min 20 DEG C from
Heart 5min is washed 1 time, adds 2ml PBS the same terms to wash again 1 time, adds 100 μ l PBS resuspended into single cell suspension.To
Each 5 μ l of CD4-FITC and CD25-PE-Cy5 antibody are wherein added, is mixed, room temperature lucifuge is incubated 20min, after PBS washing centrifugations
It is separately added into 1ml Fixation/Permeabilization and fixes rupture of membranes agent, room temperature lucifuge is incubated 30-60min, with 1 ×
After Perm buffer wash 2 times, FoxP3-APC (Isotype control pipe adds 5 μ l mouse IgG r1-APC) and Helios- is added
PE (Isotype control pipe adds 5 μ lArmenian Hamster IgG) each 5 μ l, lucifuge is incubated 30-60min, through 1 × Perm
Buffer is washed 2 times, up flow type machine testing CD4 resuspended with 400 μ l PBS+ CD25+FoxP3+ Helios+ Treg accounts for whole blood
CD4+The percentage of cell（Fig. 1）.Experimental result is shown：In pre-B ALL groups, CD4+CD25+ FoxP3+ Helios+Treg is thin
Born of the same parents account for CD4+T cell ratio is（6.16±0.79）%, hence it is evident that higher than control group（2.62±0.34）%, difference has conspicuousness
Statistical significance（p=0.005）.
Embodiment 2
The separation and the sorting of Treg cells of mononuclearcell
（1）Specimen origin：The example uses the peripheral blood of parents of hospitalized children agreement mandate of being had a medical check-up through initial ALL infants and normal health
As the source of mononuclearcell, 5 parts of ALL peripheral bloods and 5 parts of normal control peripheral bloods are gathered altogether, add anticoagulant heparin, every part
10ml；
（2）Main agents：Lymphocyte separation medium is purchased from Sigma companies, CD4+CD25+It is Treg cell sortings kit, immune
Magnetic bead sorting instrument (autoMACS Pro Separator), MACS buffer, LD and MS MACS splitters are purchased from Germany
Miltenyi Biotec companies；
（3）Mononuclearcell is separated：Extract anticoagulant heparin peripheral blood 10ml, peripheral blood adds the dilution of equivalent PBS liquid, take 50ml from
Heart pipe, often pipe be carefully added into lymphocyte separation medium 20ml, blood is slowly positioned at lymphocyte point along tube wall after 20ml is diluted
On chaotropic aspect, 2500 revs/min of 20 DEG C of centrifugation 30min collect the lymphocyte in buffy coat, add 15ml PBS,
1800 revs/min × 10 minutes centrifuge washings 2 times, cell count about 1 × 107-2×107Mononuclearcell, every 1 × 107Carefully
Born of the same parents add 90ulPBS resuspended；
（4）The MACS sortings of Treg cells：Take the mononuclearcell after separating, every 107Individual cell adds Biotin-
Antibody10ul is mixed, and is placed in 2-8 DEG C of incubation 5min, adds 20 μ l Anti-Biotin Microbeads/ every 107It is individual thin
Born of the same parents, are mixed, 2-8 DEG C of incubation 10min, and cell is adjusted to 500 μ l volumes, cross LD posts, are collected the cell flow down after post and are hanged
Liquid is CD4+T cell (the moon choosing), after cell is washed into centrifugation with PBS, every 107Individual cell adds the μ l of PBS 90 resuspended, and every 107It is individual
Cell adds CD25MicroBeads20ul, mixes, on 2-8 DEG C of incubation 15min, addition 1ml PBS washs centrifugation, incline
Clearly, it is resuspended to 500 μ l, LS posts are crossed, separator is removed, 1ml PBS are placed on LS posts, cell is laid rapidly, collect
That flow down is CD4+CD25+T cell (sun choosing), adds Treg medium cultures base to continue to cultivate.A part of cell is left and taken to be flowed
The detection of formula cytology, flow cytomery CD4+CD25+Positive cell sorting index is up to more than 90%.
Embodiment 3
The detection of Treg Cellular immunity suppression functions
（1）Main agents：Co-culture culture medium：10% hyclone, 100U/ml penicillin+chain are added in 1640 culture mediums
Mycin, 50 μM/L 2 mercapto ethanols and 2mM/L Glus, 200IU/mL rhIL-2.CD3/CD28
Dynbeads is purchased from Invitrogen companies of the U.S., and CFSE is purchased from Japanese colleague's chemistry institute；
（2）The CD4 that will be sub-elected in embodiment 2+CD25-Cell is washed, and adds PBS to be adjusted to 1 × 107Concentration, according to
5 μM of CFSE of CFSE specifications mark, 37 DEG C are incubated 15-30 minutes, and PBS is washed 2 times, takes 1 × 106By cell number 2：1 ratio
6 orifice plates are added to co-culture with experimental group 1, experimental group 2, control group Treg cells respectively, culture medium is trained using culture medium is co-cultured
Another additional proportion is 1 in supporting base：1 CD3/CD28 dynbeads, culture carries out cell after 72 hours using flow cytometer
Propagation detection, using Modfit FLT software analysis results, such as Fig. 2.Result is shown:Experimental group CD4+CD25+T cell rejection ability
Compared with the rising that control group has obvious statistical significance（11.40 ± 0.64 vs 14.53 ± 0.20,p=0.009）.
Embodiment 4
Helios expresses the influence to Treg cell functions
To detect the influence of Helios gene pairs Treg cell functions, Helios-siRNAs is transfected in leukaemia Treg cells
Helios expression is lowered, Helios-siRNAs is synthesized by lucky horse biology Co., Ltd.Experiment is divided into two groups：Helios-
SiRNA transfection groups and control group, are operated according to the explanation of Lipofectamine 2000 and are transfected into two groups of siRNA respectively
In Treg cells, transfection carries out quantitative PCR detection Helios gene expressions decline after 48 hours, and such as Fig. 3, CFSE co-cultures suppression
The immune suppression function of experimental result display siRNA-Helios group Treg cells processed averagely declines 34% compared with control group（24.00±
1.73 vs 16.33 ± 0.88,p=0.001）.
Claims (1)
1. a kind of detection method of the immune suppression function of people's regulatory T cells, it is characterised in that specific detecting step is as follows：
1）Collection：Take peripheral blood 5-10ml；
2）Treatment sample：Take streaming pipe and be separately added into 100 μ l peripheral blood samples, often pipe adds 2ml erythrocyte cracked liquids, mix
It is incubated at room temperature afterwards 15 minutes, 1500 revs/min of 20 DEG C of centrifugation 5min are washed 1 time, add 2mlPBS liquid the same terms to wash 1 again
Time, add 100 μ l PBS liquid resuspended into single cell suspension；
3）The mark of surface antibody：Each 5 μ l mark regulatory T cells surface molecular mark of antibody CD4, CD25 is added, room temperature is kept away
Light is incubated 20 minutes, and 20 DEG C of centrifugation 5min of PBS liquid 1500 rev/min are washed 1 time；
4）Cell fixes rupture of membranes：1ml is added to fix rupture of membranes agent：10% formaldehyde/0.2%Triton-X/PBS re-suspended cells, room temperature
Lucifuge is incubated 1 hour, adds 2ml PBS liquid to wash 2 times；
5）Core interior antibody is dyeed：100 μ l PBS liquid re-suspended cells are added, each 5ul marks of core interior antibody Foxp3, Helios are added
Core interior antibody, room temperature lucifuge is incubated 1 hour, adds 2ml PBS liquid to wash 2 times；
6）Flow cytometer detection：Add the PBS liquid of 400 μ l resuspended into single cell suspension, strainer filtering addition streaming orifice plate, fluidic cell
Machine testing CD4 on instrument+CD25+FoxP3+Helios+Cell accounts for CD4+Cell proportion；
7）Mononuclearcell is separated：Peripheral blood whole blood sample is according to volume ratio 1：Peripheral blood is slowly added to Ficoll by 1 ratio
In lymphocyte separation medium, 2500 revs/min are centrifuged 25-30 minutes, collect the mononuclearcell of middle tunica albuginea layer, add 15ml
PBS liquid, is centrifuged 10 minutes by 1800 revs/min, washs 2 times；
8）Sorting：Magnetic bead sorting CD4+CD25+Treg cells, wherein negative sorting CD4+Cell, positive sorting CD25+Cell,
Different groups of CD4 of CFSE experiment detections+CD25+Treg cells are to CD4+CD25-The immune suppression function of Th cells；
9）Detection：Helios siRNA are built, Helios siRNA are transfected into the ALL CD4 after sorting+CD25+Treg cells
In, CFSE experiment detection Treg Cellular immunity suppression functions.
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| CN108508196A (en) * | 2018-04-13 | 2018-09-07 | 沈阳汇敏源生物科技有限责任公司 | A kind of kit and detection method of detection people's regulatory T cells hypotype |
| US10414755B2 (en) | 2017-08-23 | 2019-09-17 | Novartis Ag | 3-(1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
| CN110672500A (en) * | 2019-09-29 | 2020-01-10 | 杭州联科生物技术股份有限公司 | Th detection method of non-heparin anticoagulant fluid sample |
| CN110823847A (en) * | 2018-08-08 | 2020-02-21 | 澳门大学 | Method for quantitatively analyzing content of transcription factors in cell nucleus based on flow cytometry |
| CN111235209A (en) * | 2020-03-09 | 2020-06-05 | 山东大学齐鲁医院 | Method for evaluating immune regulation function of stem cells |
| CN112147340A (en) * | 2020-09-30 | 2020-12-29 | 北京银丰鼎诚生物工程技术有限公司 | Method for detecting immunoregulation function of neural stem cells |
| US11185537B2 (en) | 2018-07-10 | 2021-11-30 | Novartis Ag | 3-(5-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
| US11192877B2 (en) | 2018-07-10 | 2021-12-07 | Novartis Ag | 3-(5-hydroxy-1-oxoisoindolin-2-yl)piperidine-2,6-dione derivatives and uses thereof |
| CN114112868A (en) * | 2020-08-26 | 2022-03-01 | 上海睿昂基因科技股份有限公司 | Flow cytometry kit for monitoring tumor-related immune microenvironment and monitoring method |
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