CN109112103A - A kind of inducer and its rapid induction method of the extracellular trap baiting net of neutrophil leucocyte - Google Patents
A kind of inducer and its rapid induction method of the extracellular trap baiting net of neutrophil leucocyte Download PDFInfo
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Abstract
The present invention provides inducers and abductive approach that a kind of rapid induction neutrophil leucocyte in vitro generates extracellular trap baiting net, and wherein inducer includes following components: pretreatment component A, are selected from IgG2, IgG4；Inducing component B, the mixture selected from APD or APD and PMA composition；Lipopolysaccharides.Meanwhile the present invention also provides the abductive approach that a kind of rapid induction neutrophil leucocyte in vitro generates extracellular trap baiting net (NETs), comprising the following steps: separation peripheral blood neutrophil；Neutrophil leucocyte is induced using the above-mentioned inducer comprising component A, component B and lipopolysaccharides, to form NETs.Inducer and abductive approach of the present invention have a good application prospect in terms of the screening that mammal NETs is quickly detected and promotes or inhibit NETs to generate drug.
Description
Technical field
The present invention provides inducer and abductive approach that a kind of induction neutrophil leucocyte generates extracellular trap baiting net (NETs),
Specifically, providing a kind of neutrophil leucocyte of rapid induction in vitro for quickly detecting generates the compound of extracellular trap baiting net
Inducer and abductive approach, belong to biological detection and medical science.
Background technique
Neutrophil leucocyte is the highest leucocyte of abundance in effector cell and blood plasma important in natural immune system.
Neutrophil leucocyte has unique leaflet type nuclear structure, is usually in 3 ~ 5 leaves, therefore, also referred to as polymorphonuclear neutrophisls
(polymorphonuclear neutrophil, PMN).Under physiological conditions, neutrophil leucocyte accounts for the 50% of human peripheral leucocytes
~70%.It is for pathogen invasion generate response raises first to the cell of damage location, and be congenital immunity prevent
Imperial the first line of defence.When infection occurs, is raised arrive infection site at first, play the effect for removing pathogenic microorganism.It passes
Neutrophil leucocyte is considered to the effector cell of inflammatory response and acute immune on system, function is played by phagocytosis intracellular
Can, and using crack protein enzyme, active oxygen (ROS) and microprotein is killed for challenge infection agent.In the prior art indicate that
Property granulocyte can also by expression for other immunocytes, as Dendritic Cells, B cell and T cell transmit signal
Cell factor, chemotactic factor (CF), Fc receptor and complement component have immunoregulation capability (Mantovani, Cassatella etc.,
2011)。
Research in recent years is also shown that other than phagocytosis, neutrophil leucocyte can also be by discharging the extracellular trapping of neutrophil leucocyte
Pathogenic microorganism is sticked and removed to the mode of net (neutrophil extracellular trap, NETs), and can effectively limit
Pathogen is sent out to other tissue sites.
NETs is a kind of extracellular structure, causes neutrophil leucocyte to be activated because of bacterium infection, form, the dye of leaflet core
The distribution of chromaticness becomes unintelligible, and then nuclear membrane disappears, and cytoplasm, particulate component are mixed in chromatin Structure, and cell membrane is broken
It splits, at this point, the extracellular trap baiting net of the neutrophil leucocyte discharges reticular structure, captures bacterium, fungi, helminth, virus, play anti-
Bacterium effect.NETs is mainly by the enzymes such as histone, DNA and elastoser, cathepsin G and myeloperoxidase (MPO)
Composition.Specifically, NETs is to be discharged into a kind of extracellular reticular structure after being stimulated activation by neutrophil leucocyte, with DNA
For skeleton, it is inlaid with histone, myeloperoxidase (MPO), neutrophil elastase (NE), cathepsin therebetween
G, calprotectin, protease 3 etc. have the albumen sterilized and increase permeability effect, these albumen are embedded on DNA skeleton greatly
Its local concentration is increased greatly.NETs relies primarily on its unique tridimensional network capture pathogen, and by comprising it is big
Amount antibacterial protein kills pathogen, while NETs is to the fixed phagocytosis that other leucocytes can be enhanced of the capture of pathogen
Effect.NETs can catch and kill multiple pathogens, including gram-positive bacteria and Gram-negative bacteria, fungi, the soft spiral of Bai Shi packet
Body and certain helminths etc., or even can be stimulated by TLR-1 and TLR-8 including human immunodeficiency virus (HIV), HIV
Then NETs, which is generated, removes HIV by MPO and α-alexin.But certain pathogen such as streptococcus pneumonia, staphylococcus aureus
It is catched and killed etc. the DNA skeleton that can destroy NETs by generating DNase to escape.Due to generating NETs, cause neutrophil leucocyte dead
(NETosis) is died, if neutrophil leucocyte is stimulated by LPS, PMA, generates autophagy, meanwhile, generate active oxygen.Core is generated as a result,
Film rupture, chromatin decondensation contracting, histone are citrullinated, cause cell death.
In addition, the prior art reports the formation with NETs, neutrophil leucocyte secretes a large amount of protein by degranulation,
Especially aniline blue particles, also secrete relevant to eucaryotic cell structure protein etc. (Q Remijsen etc., 2011, Cell
Death and Differentiation, 18:581-588).These protein are also referred to as NETs structural protein.
NETs original effect is to capture external microorganism and be limited in part, is sterilized.But further study table
It is bright, as infection disease etc. switchs to chronic, the formation of NETs is observed in the case where external microorganism is not present.Such as
The systemic loupus erythematosus (SLE) of one of autoimmune disease form for itself DNA, related protein itself is anti-
Body.SLE is shown in damage location, and there are a large amount of neutrophil leucocytes.The neutrophil leucocyte of SLE patient is thinner than the neutral grain of Healthy People
Born of the same parents are easier to cause NETosis.In view of the improvement of the state of an illness, generally require to inhibit the phenomenon that NETs formation.
The substance for inhibiting NETs to be formed, including the substance that antibody, the inhibition superoxides for histone generate, such as two
(the Tobias such as phenylene nicotine chloride (Diphenylene Iodonium Chloride, DPI), catalase
A.Fuchs etc., 2007, J Cell Biol, 176:231-241).Myeloperoxidase (Myeloperoxidase, MPO) activity
Also NETosis (K.Akong-Moore etc., 2012, PLOS ONE, 7:e42984) are influenced.
In addition, the prior art also discloses a series of substances for inhibiting NETs, such as following patent.
Japanese Unexamined Patent Publication JP2008-189637 discloses the alkaline protein group from cream and is divided into effective component inhibition NETs,
And prevent the autoimmune disease of type-1 diabetes mellitus, rheumatoid arthritis.
WO2014/168253 disclose treatment because NETs formation caused by the medicament of disease specifically provide one
The novel medicament that kind inhibits the extracellular trap baiting net of leucocyte to be formed.The extracellular trapping of its leucocyte for containing lactoferrin
Net forms inhibitor and the extracellular trap baiting net for treating the leucocyte containing lactoferrin forms the combination of relevant disease
Object.
Patent WO2016/056665 discloses a kind of extracellular trapping net form of leucocyte comprising lactoferrin fragment
It is used to treat the group for forming relevant disease to the extracellular trap baiting net of leucocyte at inhibitor and comprising lactoferrin
Object is closed, it includes contain the amino acid sequence indicated by X1-K-C-X2-X3-X4-Q-X5-X6-X7-X8-X9 as effective component
The peptide of column.
In NETs detection and its drug screening of inhibitor, it is required to induce neutral grain thin in vitro using inducer
Born of the same parents.
Compared to inhibitor, for the inducer of NETs, then type is more, in addition to microorganism, lipopolysaccharides (LPS), (12-) ten
During tetracid phorbol exters (- 13-) acetate (PMA, also known as phorbol ester), blood platelet of activation etc. can stimulate to a certain extent
Property granulocyte generate NETs.In addition, certain cell factors such as tumor necrosis factor-alpha (TNF-α), interleukin 8 (IL-8),
Interferon (INF) etc. can also stimulate to a certain extent and promote the generation of NETs.The prior art is it has been reported that using highly concentrated
PMA is spent as inducer, and sytox green is added, and detects NETs formational situation using Laser Scanning Confocal Microscope.Due to PMA
It induces neutrophil leucocyte to generate NETs, ROS probe is added, can also be distributed by confocal detection green fluorescence.
But existing NETs is external evoked, in detection method, used above-mentioned inducer otherwise it is expensive, obtain
It takes and is not easy (such as interleukins, tumor necrosis factor etc.) or there is strong carcinogenicity (such as PMA), or need highly concentrated
Degree just has good inducibility (such as LPS).
Importantly, defect existing for above-mentioned inducer is in addition to other than needing high concentration, existing inducer exists
Pass through host's pattern recognition receptors and there is low efficiency in terms of inducing NETs releasability, induction stimulation time generally requires
It is more than up to several hours；Can not rapid induction NETs release, seriously affected induced efficiency.
For example, generalling use inducer of the PMA as NETs in prior art experiment in vitro.But lower PMA
It is very poor using concentration (such as less than 40 nM) inducing effect or even do not have inductive effect, the effect of this inducing effect difference with
Induction time length of time is almost unrelated, that is to say, that inducing effect can hardly be improved by extending induction time；What is worse,
Even if using higher induced concentration (such as higher than 50 nM), it is also desirable to longer stimulation time, it usually needs four or five hours
Significant NETs could be generated above.
In addition, using the existing direct inducing cell of other inducers including PMA including further disadvantage is that, even if use
High concentration (such as PMA of 100 nM or more) induction, will not form NETs in a short time, only lure in high concentration and enough
The NETs that conspicuousness could be generated in the case that the two has both is led the time and (be greater than 2-3 h), can be used to detect.
Therefore, although the inducers such as PMA are commonly used because the NETs of neutrophil leucocyte is induced to form effect obviously in vitro for it
Make inducer, but there are many restrictions for its abductive approach.
Meanwhile NETs rapid detection method is established for improving detection efficiency, improving testing result accuracy and exploitation
Application of NETs detection kit etc. is of great significance.
Up to the present, there is no open quickly for inducing the extracellular trap baiting net of neutrophil leucocyte to generate in the prior art
In the method for detection, hospital, colleges and universities, scientific research institutions or third party inspection mechanism is caused to carry out the extracellular trapping of neutrophil leucocyte
Difficulty is larger when the detection of net generative capacity, needs voluntarily to carry out experiment condition to grope；Reality used when different institutions are detected
Proved recipe method is different, and then causes the testing result otherness of same sample very big, brings biggish puzzlement to result judgement.Cause
This, it is necessary to providing one kind can overcome above-mentioned prior art defect, the stable easy extracellular trap baiting net of neutrophil leucocyte to lure
Lead object and abductive approach.
To sum up, although detecting the generative capacity of cell NETs under physiology or pathological conditions, to promotion or inhibit NETs related
The screening of drug or immunotherapy targeted autoantibody drug are particularly significant, also have significant significance in field of biological medicine, but
It is the current shortage quickly and effectively external evoked generation NETs especially method of people NETs.
Summary of the invention
In order to overcome many defects present in the external evoked generation NETs method of the prior art as indicated above, seek
A kind of stabilization, quickly and effectively method generate NETs to induce, and efficiently, the inducer of low concentration, present inventor has performed
In-depth study, after having paid a large amount of creative work, so as to complete the present invention.
Specifically, it is an object of that present invention to provide a kind of rapid induction neutrophil leucocytes in vitro to generate extracellular trap baiting net
Inducer and abductive approach, in particular, a kind of luring of significantly forming of the neutrophil leucocyte NETs for providing stable rapid induction people
Composition and abductive approach are led, for every NETs detection including kit.
More specifically, the present invention provides following scheme.
First aspect, the present invention provide the induction that a kind of rapid induction neutrophil leucocyte in vitro generates extracellular trap baiting net
Object, it includes following components:
Pretreatment component A, selected from one or both of IgG2, IgG4；
Inducing component B, the mixture selected from APD (12-O- acetyl phorbol -13- caprinoyl ester) or APD and PMA composition；
Lipopolysaccharides.
The second aspect, based on the component in first aspect of the present invention, the present invention provides a kind of rapid induction in vitro
Neutrophil leucocyte generates the abductive approach of extracellular trap baiting net NETs, comprising the following steps:
(1) peripheral blood neutrophil is separated；
(2) neutrophil leucocyte is induced using the inducer comprising component A, component B and lipopolysaccharides, to form NETs.
Further, including post-processing step: NETs identification and quantitative analysis.
In the method for the present invention, peripheral blood neutrophil described in step (1) comes from mammal.
Preferably, the peripheral blood neutrophil comes from people.
In the method for the present invention, the induction of neutrophil leucocyte is to induce stage by stage, including pre-process, is fast in the step (2)
Speed induction extends induction three phases.
In the method for the present invention, the final concentration of 1-20nM of IgG in the component A, it is preferable that the final concentration of 10- of IgG
20nM。
In the method for the present invention, the final concentration of 5-20nM of APD in the component B, it is preferable that the final concentration of 10- of APD
20nM。
In the method for the present invention, when in the component B contain PMA when, used PMA concentration be not higher than 20nM, such as
It can be 1-10nM, preferably 5nM-10nM.
In the method for the present invention, used lipopolysaccharide concentration is 10-50nM, preferably 10-30nM.
In the method for the present invention, the NETs induction of conspicuousness is induction stimulation stage by stage, including pretreatment, rapid induction stage
The extension induction period extended with enhancing core；It is directly induced in addition, above-mentioned co-induction object can also be used.
In the method for the present invention, induction time is that 30-60min is to reach last core to extend the stage, to be formed significant
NETs structure.
Specifically, in the method for the present invention, detailed process is as follows for step (1):
(a) anticoagulant peripheric venous blood 5-10ml is taken, with PBS according to the dilution proportion of 1:1；
(b) in centrifuge tube be added 10-20 ml separating liquid, then by diluted anticoagulation slowly along tube wall blow slowly drip be added
To separating liquid upper layer；
(c) 700-900 g horizontal centrifugal 30-40min at room temperature；Different layerings is presented after centrifugation, is stretched with capillary
Into leukocytic cream, whole leucocytes are sucked out to a new EP pipe along tube wall；
(d) leucocyte is washed with 1-2ml PBS, 1500-2500rpm is centrifuged 10-15min, leaves and takes precipitating, repeats 1-2 times；
(e) cell, 3-4g/L Trypan Blue is resuspended with the RPMI culture medium containing 5% FBS, tally differential counting is simultaneously identified
Cell purity；When purity is lower, repeat step (d).
In the method for the present invention, detailed process is as follows by external evoked neutrophil leucocyte formation NETs in step (2):
(1) neutrophil leucocyte of extraction is diluted with the above-mentioned RPMI culture medium for containing 5% FBS, adjust to cell density be 1 ×
106A/ml；
(2) cell is pressed 5 × 105The number in a/hole is seeded in 24 orifice plates for being covered with slide, every 0.5 ml of hole；Except real in plate
It is outer to test group, is additionally provided with control group, experimental group adds inducer, and isometric PBS buffer is added in control group, and every group of sample is each
If 3 multiple holes；
(3) neutrophil leucocyte is induced stage by stage using the inducer comprising component A, component B and lipopolysaccharides, detailed process
Are as follows:
S1: pretreatment cell: pretreatment component A is added into hole, in 37 DEG C, 5 %CO2Pretreatment cell is stood in incubator
15-30min；Wherein, the final concentration of 10-20nM of pretreatment component A；
S2: rapid induction: in above-mentioned solution after pretreatment be added component B, while make temperature from 37 DEG C be rapidly heated to
39-42 DEG C, 5-15min is kept the temperature, is then cooled to 37-38 DEG C again with 0.5-1 DEG C per minute of speed, heat preservation induction 15-
30min；Wherein, the final concentration of 10-20nM of APD in component B, PMA final concentration are not higher than 10nM；
S3: extend induction: the lipopolysaccharides (LPS) of final concentration of 10-30nM is added in the solution after above-mentioned rapid induction, keeps
37-39 DEG C of temperature, continue to extend induction 30-60min, to enhance core extension, to form the NETs of conspicuousness.
The inventors discovered that the fixed temperature that compares directly induces, can be in short-term when using abductive approach so
The interior NETs for obtaining formation rate higher (being higher than 40%), conspicuousness appearance form, and when change induced processes parameter or induction
Group timesharing, then can not obtain the NETs of the conspicuousness form of effect same in the same time.
NETs quantitative analysis and authentication step in the method for the present invention, in post-processing step are as follows:
(1) after cell induction, solution supernatant is collected, 4 DEG C, 100-200 × g centrifugation 5-10min remove cell precipitation,
Final proof is taken, quantitative analysis NETs content is detected using dissociative DNA；
(2) 4% paraformaldehyde, 0.5 ml is added into the hole after absorption supernatant, is taken from 24 orifice plates after fixed cell climbing sheet 10min
Cell climbing sheet out successively embathes 3 times with 0.01 mol/L PBS, every time 5 min；Later with containing 0.5% Triton X-100
The penetrating 1-2 min of PBS, 0.5 mg/L DAPI dye liquor is protected from light dyeing 5-10 min, and 0.01 mol/L PBS embathes 2 times, seals
Piece liquid (is configured) mounting, confocal microscopy by isometric glycerol and water.
Wherein, neutrophil leucocyte separation and NETs analysis or identification can also be used it is as known in the art other often
Rule method.
For example, passing through the expression quantity of immuno-fluorescence assay cell surface DNA/ histone, the generation of NETs is determined
Property analysis.
The DNA specific dye that acellular permeability can also be used dyes the post-stimulatory neutrophil leucocyte of induction,
The expression quantity for passing through Flow cytometry cell surface DNA again carries out quantitative analysis to the generation of NETs.
Other NETs Rapid Quantifications are also well known to those skilled in the art, for example, using green DNA fluorescence
Dyestuff Picogreen is dyed, and after specifically binding dsDNA, it is strong that fluorescence is detected at 485 nm of fluorescence microplate reader wavelength
Degree.
Since NETs is mainly that dissociative DNA exists in conjunction with neutrophil leucocyte protein, identification and analysis method is removed can
It takes dissociative DNA to detect except quantitative analysis NETs content, Protein Assay analysis also can be used, including but not limited to:
Enzyme linked immunosorbent assay (ELISA) (ELISA), mass spectrography, chromatography, electrophoresis, radioimmunoassay, flow cytometry, fluorescence are living
Change cell sorting (FACS) or western trace determines.
The analysis or identification method selection of separation and NETs for neutrophil leucocyte, those skilled in the art can basis
Actual conditions are suitably selected and are determined, this is no longer going to repeat them.
In terms of third, the present invention provides the kit that can be used for the extracellular trap baiting net detection of neutrophil leucocyte comprising this
The invention inducer, or including abductive approach of the present invention.
In the technical effect, it using method of the invention, can not only be formed with rapid induction NETs, compare the prior art
It is substantially shorter induction time, with conspicuousness NETs can be induced to be formed.By shorter induction time, contaminated using nucleic acid
Color or confocal scanning are that can observe intuitive, conspicuousness NETs image.
Method of the invention is directed to the process that NETs is formed, and takes and is targetedly segmented induction mode, is lured with lower
It leads object concentration and shorter induction time has reached unexpected technical effect, and overcome direct one-step method in the prior art
Induce the defect of high concentration inducer required for (such as PMA is directly induced) and induction time length.
Inducer and method of the invention is suitable for external evoked NETs and is formed, have it is simple, conveniently, it is reproducible excellent
Point can be used for making the kit that NETs is quickly detected.
It should be noted that unless otherwise prescribed, being related to the " comprising " of group timesharing in this application, meaning is related to closing
Formula " by ... is formed " or its equivalent definition and open "include", "comprise" etc. or its equivalent definition, and cannot limit
In its simple literal meaning.
Detailed description of the invention
Fig. 1 is the confocal microscopy view after NETs fluorescent staining obtained by the embodiment of the present invention 1.
Fig. 2 is the high-resolution Laser Scanning Confocal Microscope figure of NETs local fiber net obtained by the embodiment of the present invention 1.
Specific embodiment
Below by specific embodiment, the present invention is described in detail, but the purposes of these exemplary embodiments and
Purpose is only used to enumerate the present invention, not constitutes any type of any restriction to real protection scope of the invention, more non-to incite somebody to action
Protection scope of the present invention is confined to this.
Embodiment 1
(1) human peripheral neutrophil leucocyte is separated:
The fresh EDTA of subject or citrate anticoagulation peripheric venous blood 5ml is taken, with PBS according to 1:1 dilution proportion.50ml from
10 ml separating liquid Polymorphprep are added in heart pipeTM, then by diluted anticoagulation slowly along tube wall blow slowly drip be added
To separating liquid upper layer.Then under room temperature, 700 × g horizontal centrifugal 30min.Different layerings is presented after centrifugation, from up to
Lower is serum layer, nucleus layer, separation liquid layer, leucocyte (wherein most is neutrophil leucocyte) layer and red blood cell respectively
Layer.It is extended in leukocytic cream with capillary, whole leucocytes is gently sucked out along tube wall to a new EP pipe.Then it is washed with 1ml PBS
It washs, 1500rpm is centrifuged 10min, leaves and takes precipitating, is repeated 1 times.Cell, 3g/L platform is resuspended with the RPMI culture medium containing 5% FBS
Expect blue dyeing, tally differential counting and identification of cell purity.
(2) external evoked neutrophil leucocyte forms NETs:
(2.1) neutrophil leucocyte of extraction is diluted with the above-mentioned RPMI culture medium for containing 5% FBS, adjusting to cell density is 1
× 106A/ml；
(2.2) cell is pressed 5 × 105The number in a/hole is seeded in 24 orifice plates for being covered with slide, every 0.5 ml of hole；It is removed in plate
Outside experimental group, it is additionally provided with control group, experimental group adds inducer, and isometric PBS buffer, every group of sample is added in control group
Respectively set 3 multiple holes；
(2.3) neutrophil leucocyte is induced stage by stage using IgG2, APD, PMA and lipopolysaccharides, detailed process are as follows:
S1: pretreatment cell: IgG2 being added into hole, in 37 DEG C, 5 %CO2Pretreatment cell 15min is stood in incubator；Its
In, the final concentration of 10nM of IgG2；
S2: rapid induction: APD and PMA is added in above-mentioned solution after pretreatment, while temperature being made to be rapidly heated from 37 DEG C
To 42 DEG C, 10min is kept the temperature, is then cooled to 37 DEG C again with 1 DEG C of speed per minute, the temperature is kept to continue to induce 15min；Its
In, APD induces final concentration of 20nM, PMA to induce final concentration 5nM；
S3: extend induction: the lipopolysaccharides of final concentration of 20nM is added in the solution after above-mentioned rapid induction, is kept for 37 DEG C of temperature,
Extend induction 30min, to enhance core extension, to form the NETs of conspicuousness.
(3) it post-processes: the quantitative analysis and identification of NETs
(3.1) quantitative analysis: after induction, solution supernatant is collected, 4 DEG C, 100g centrifugation 10min remove cell precipitation, take
Upper final proof detects quantitative analysis NETs content using dissociative DNA；The specific steps are using PicoGreen dsDNA kit
Kit detects the DNA content that dissociates in cell conditioned medium with fluorescence microplate reader by its operating procedure, to quantify to NET
Analysis；With reference to kit specification dilution standard product liquid storage and sample；It is added in 96 orifice plates by 50 holes μ l/, 50 μ are added in every hole
The diluted PicoGreen of l, standard items final concentration be followed successively by 1000 ng/ml, 100 ng/ml, 50 ng/ml, 10 ng/ml,
2.5 ng/ml, 1 ng/ml, 0.1ng/ml and 0 ng/ml detect signal strength, 480 nm of exciting light, hair with Fluorescent reader
Penetrate 520 nm of light.
(3.2) 4% paraformaldehyde, 0.5 ml, fixed cell climbing sheet Morphological Identification: are added into the hole after absorption supernatant
Cell climbing sheet is taken out after 10min from 24 orifice plates, successively embathes 3 times with 0.01 mol/L PBS, every time 5 min；It uses later
The penetrating 1-2 min of the PBS of the X-100 containing 0.5%Triton, 0.5 mg/L DAPI dye liquor are protected from light dyeing 5 min, 0.01 mol/L
PBS embathes 2 times, and mounting liquid (is configured) mounting, Laser Scanning Confocal Microscope imaging by isometric glycerol and water.
Embodiment 2
(1) human peripheral neutrophil leucocyte is separated:
With 1 step of embodiment (1).
(2) induction neutrophil leucocyte forms NETs:
(2.1) neutrophil leucocyte of extraction is diluted with the RPMI culture medium containing 5% FBS, adjust to cell density be 1 ×
106A/ml；
(2.2) cell is pressed 5 × 105The number in a/hole is seeded in 24 orifice plates for being covered with slide, every 0.5 ml of hole；
(2.3) neutrophil leucocyte is induced stage by stage using IgG4, APD and lipopolysaccharides, detailed process are as follows:
S1: pretreatment cell: IgG4 being added into hole, makes its final concentration of 20nM, in 37 DEG C, 5 %CO2It is stood in incubator
Pretreatment cell 30min；
S2: rapid induction: APD, final concentration of 10nM are added in above-mentioned solution after pretreatment, while making temperature from 37 DEG C
40 DEG C are warming up to, heat preservation induction stimulation 15min is then cooled to 38 DEG C again with 0.5 DEG C of speed per minute, heat preservation induction
30min；
S3: extend induction: the lipopolysaccharides of final concentration of 30nM is added in the solution after above-mentioned rapid induction, is kept for 38 DEG C of temperature,
Continue to extend induction 30min, to form the NETs of conspicuousness.
(3) it post-processes: the quantitative analysis and identification of NETs
Operating method obtains the NETs of conspicuousness with 1 step of embodiment (3)；Laser scanning confocal microscopy, under high power lens
High-visible extracellular fiber reticular structure.
Embodiment 3
(1) human peripheral neutrophil leucocyte is separated:
With 2 step of embodiment (1).
(2) induction neutrophil leucocyte forms NETs:
(2.1) neutrophil leucocyte of extraction is diluted with the RPMI culture medium containing 5% FBS, adjusting to cell density is 1 x 106
A/ml；
(2.2) cell is pressed 5 × 105The number in a/hole is seeded in 24 orifice plates for being covered with slide, every 0.5 ml of hole；
(2.3) neutrophil leucocyte is directly induced using IgG4, APD and lipopolysaccharides co-induction object, detailed process are as follows: to
IgG4, APD and lipopolysaccharides are disposably directly added into hole, making its final concentration is respectively 20nM, 10nM, 30nM, in 37 DEG C, 5 %
CO2Inducing cell 90min in incubator.
(3) it post-processes: the quantitative analysis and identification of NETs:
Operating method is the same as 2 step of embodiment (3), Laser scanning confocal microscopy visible cell outer fiber reticular structure.
NETs's quantifies in separation human peripheral neutrophil leucocyte step and post-processing in following comparative example 1-4
Analysis and authentication step are identical as the method in embodiment 1-2, and sample source is identical, only neutrophil leucocyte are induced to form NETs's
Step is different.
Comparative example 1
The induction neutrophil leucocyte of no pretreatment cell process forms NETs:
In addition to induction period is without pretreatment cell step (S1 step), remaining step is same as Example 1, experiment NETs knot
Fruit is labeled as D1.
Comparative example 2
NETs is formed without the neutrophil leucocyte for extending Induction Process:
In addition to induction period is without induction step (S3 step) is extended, remaining step is same as Example 1, NETs experimental result
Labeled as D2.
Comparative example 3
No pretreatment cell process and the neutrophil leucocyte for extending Induction Process form NETs and (that is: contain only rapid induction step
Suddenly):
Except induction period without pretreatment cell step (S1 step) and extend induction step (S3 step) in addition to, remaining step and
Embodiment 1 is identical, and NETs experimental result is labeled as D3.
Comparative example 4
The neutrophil leucocyte of no alternating temperature Induction Process forms NETs:
Except 37 DEG C of constant temperature that the heat temperature raising Induction Process in S2 step and S3 step is substituted for identical induction time induce it
Outside, remaining step is same as Example 1, and NETs experimental result is labeled as D4.
The quantitative analysis of NETs in separation human peripheral neutrophil leucocyte step and post-processing in following comparative example
And authentication step is identical as the method in embodiment 3, and sample source is identical, and neutrophil leucocyte is only induced to form the inducer of NETs
It is different.
Comparative example 5
The neutrophil leucocyte for containing only PMA inducer forms NETs:
In addition to IgG4, APD and lipopolysaccharides co-induction object are replaced with the PMA of 60nM, remaining step is same as Example 3,
NETs experimental result is labeled as D5.
Comparative example 6
The neutrophil leucocyte for containing only LPS inducer forms NETs:
In addition to IgG4, APD and lipopolysaccharides co-induction object are replaced with the LPS of 60nM, remaining step is same as Example 3,
NETs experimental result is labeled as D6.
Comparative example 7
The neutrophil leucocyte for containing only IgG inducer forms NETs:
In addition to IgG4, APD and lipopolysaccharides co-induction object are replaced with the IgG4 of 60nM, remaining step is same as Example 3,
NETs experimental result is labeled as D7.
Effect data
As described in Example 1, NETs content detects quantitative analysis using dissociative DNA, specially uses PicoGreen
DsDNA kit kit by its operating procedure with fluorescence microplate reader detection cell conditioned medium in dissociate DNA content, to NETs into
Row quantitative analysis.Upper final proof is added in 96 orifice plates by 50 holes μ l/, and the 50 diluted PicoGreen of μ l, standard items are added in every hole
Final concentration is followed successively by 1000 ng/ml, 100 ng/ml, 50 ng/ml, 10 ng/ml, 2.5 ng/ml, 1 ng/ml, 0.1ng/ml
With 0 ng/ml.Signal strength is detected with Fluorescent reader.As a result from 3 repeated sample values.Sample uses single factor test
Variance analysis is compared, and thinks that difference is statistically significant with P < 0.05.
Replace the NETs control group of inducer as blank control using PBS buffer solution in embodiment 1.
Above-described embodiment and comparative example formed in NETs the DNA content that dissociates it is as shown in table 1 below (ng/ml, P <
0.05):
Table 1
| Project | Dissociative DNA content (× 100, ng/ml) |
| Blank control group | 3.61 |
| Embodiment 1 | 31.2 |
| Embodiment 2 | 29.5 |
| Embodiment 3 | 17.7 |
| D1 | 23.8 |
| D2 | 21.3 |
| D3 | 18.2 |
| D4 | 26.6 |
| D5 | 11.3 |
| D6 | 10.8 |
| D7 | 8.9 |
As seen from the above table, free DNA content (NETs content) is significantly higher than blank control group in embodiment 1-2 supernatant.As reality
Test the comparison between group, embodiment 3, comparative example 1-7 sample induction result also have compared with the control group conspicuousness improve or
Different degrees of certain raising, difference are statistically significant；Embodiment 1-2 is compared with comparative example, before identical stimulation time
It puts, there is higher dissociative DNA content relative to single inducer, illustrate that the stimulating method result induced stage by stage is obviously excellent
In directly induction, it was demonstrated that under the premise of short time induction, rapid induction method of the present invention obtains unexpected technical effect.
In conclusion specific inducer component through the invention and stage by stage in abductive approach technological parameter etc. collaboration
Combination and coordinative role, so that, conspicuousness NETs high with shaping rate has been obtained under low concentration, short time inductive condition,
It is of great significance to the screening for promoting or inhibiting NETs related drugs or fast medical detection.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to limit protection model of the invention
It encloses.In addition, it should also be understood that, after reading the technical contents of the present invention, those skilled in the art can make the present invention each
Kind change, modification and/or variation, all these equivalent forms equally fall within guarantor defined by the application the appended claims
Within the scope of shield.
Claims (9)
1. the inducer that a kind of rapid induction neutrophil leucocyte in vitro generates extracellular trap baiting net NETs, which is characterized in that include
Following components: pretreatment component A, selected from one or both of IgG2, IgG4；Inducing component B is selected from APD (12-O- acetyl
Phorbol -13- caprinoyl ester) or APD and PMA composition mixture；Lipopolysaccharides.
2. the abductive approach that a kind of rapid induction neutrophil leucocyte in vitro generates extracellular trap baiting net NETs, which is characterized in that packet
Include following steps:
(1) peripheral blood neutrophil is separated；
(2) neutrophil leucocyte is induced using the inducer comprising component A, component B and lipopolysaccharides, to form NETs；
Wherein, component A, component B define with defined described in claim 1 it is identical.
3. method according to claim 2, which is characterized in that it further comprises the post-processing of NETs identification and quantitative analysis
Step.
4. method according to claim 2, which is characterized in that induction in the step (2) is to induce stage by stage, including pre-
Processing, rapid induction extend induction three phases.
5. method according to claim 2, which is characterized in that the final concentration of 1-20 nM of the induction of IgG in the component A；
The final concentration of 5-20 nM of the induction of APD；PMA induction is final concentration of to be not higher than 20 nM；Lipopolysaccharide-induced final concentration of 10-50
nM。
6. the method as described in claim 2-5, which is characterized in that detailed process is as follows for step (1):
(a) anticoagulant peripheric venous blood 5-10 ml is taken, with PBS according to the dilution proportion of 1:1；
(b) in centrifuge tube be added 10-20 ml separating liquid, then by diluted anticoagulation slowly along tube wall blow slowly drip be added
To separating liquid upper layer；
(c) 700-900 × g horizontal centrifugal 30-40 min at room temperature；Different layerings is presented after centrifugation, uses capillary
Pipe extends in leukocytic cream, and whole leucocytes are sucked out to a new EP pipe along tube wall；
(d) leucocyte is washed with 1-2 ml PBS, 1500-2500 rpm is centrifuged 10-15 min, leaves and takes precipitating, repeats 1-2 times；
(e) cell, 3-4g/L Trypan Blue is resuspended with the RPMI culture medium containing 5% FBS, tally differential counting is simultaneously identified
Cell purity；When purity is lower, repeat step (d).
7. the method as described in claim 2-5, which is characterized in that the external evoked neutrophil leucocyte of the step (2) is formed
Detailed process is as follows by NETs:
(1) neutrophil leucocyte of extraction is diluted with the above-mentioned RPMI culture medium for containing 5% FBS, adjust to cell density be 1 ×
106A/ml；
(2) cell is pressed 5 × 105The number in a/hole is seeded in 24 orifice plates for being covered with slide, every 0.5 ml of hole；Except experimental group
Outside, it is additionally provided with control group, experimental group adds inducer, and isometric PBS buffer is added in control group, and every group of sample sets 3
Multiple holes；
(3) neutrophil leucocyte is induced stage by stage using the inducer comprising component A, component B and lipopolysaccharides, detailed process
Are as follows:
S1: pretreatment cell: pretreatment component A is added into hole, in 37 DEG C, 5 %CO2Pretreatment cell is stood in incubator
15-30 min；Wherein, the final concentration of 10-20 nM of pretreatment component A；
S2: rapid induction: in above-mentioned solution after pretreatment be added component B, while make temperature from 37 DEG C be rapidly heated to
39-42 DEG C, after keeping the temperature 5-15 min, then with 0.5-1 DEG C per minute of speed it is cooled to 37-38 DEG C, heat preservation induction 15-30
min；Wherein, the final concentration of 10-20 nM of APD in component B, PMA final concentration are not higher than 10 nM；
S3: extend induction: the lipopolysaccharides of final concentration of 10-30 nM is added in the solution after above-mentioned rapid induction, keeps temperature
37-39 DEG C, continue to extend induction 30-60min, to enhance core extension, to form the NETs of conspicuousness；
Wherein, component A, B definition is the same as above claim 1 definition.
8. method as claimed in claim 3, which is characterized in that the NETs quantitative analysis and authentication step are specific as follows:
(1) after cell induction, solution supernatant is collected, 4 DEG C, 100-200 × g centrifugation 5-10 min remove cell precipitation,
Final proof is taken, quantitative analysis NETs content is detected using dissociative DNA；
(2) 4% paraformaldehyde, 0.5 ml is added into the hole after absorption supernatant, fixes after 10 min of cell climbing sheet from 24 orifice plates
Cell climbing sheet is taken out, successively embathes 3 times with 0.01 mol/L PBS, every time 5 min；Later with containing 0.5% Triton X-
The penetrating 1-2 min of 100 PBS, 0.5 mg/L DAPI dye liquor are protected from light dyeing 5-10 min, 0.01 mol/L PBS and embathe 2
It is secondary, the observation of mounting fluid-tight piece.
9. a kind of kit for the extracellular trap baiting net detection of neutrophil leucocyte comprising inducing component described in claim 1.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112941025A (en) * | 2021-03-25 | 2021-06-11 | 四川大学华西医院 | Method and kit for separating neutrophils from blood |
| CN113238050A (en) * | 2021-02-20 | 2021-08-10 | 吴炜 | Rapid detection method of neutrophil extracellular trapping net |
| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| CN113789300A (en) * | 2021-09-10 | 2021-12-14 | 江苏大学附属医院 | Method for extracting and quantifying neutrophil extracellular trapping net |
| CN115404216A (en) * | 2022-08-24 | 2022-11-29 | 中山大学附属第五医院 | Inducer, induction method and inhibitor of neutrophil extracellular trap |
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2018
- 2018-08-30 CN CN201811005880.6A patent/CN109112103A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113238050A (en) * | 2021-02-20 | 2021-08-10 | 吴炜 | Rapid detection method of neutrophil extracellular trapping net |
| CN112941025A (en) * | 2021-03-25 | 2021-06-11 | 四川大学华西医院 | Method and kit for separating neutrophils from blood |
| CN112941025B (en) * | 2021-03-25 | 2023-05-02 | 四川大学华西医院 | Method and kit for separating neutrophils in blood |
| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| CN113310963B (en) * | 2021-05-31 | 2023-12-01 | 四川大学华西医院 | Improved neutrophil NETs immunofluorescence detection method |
| CN113789300A (en) * | 2021-09-10 | 2021-12-14 | 江苏大学附属医院 | Method for extracting and quantifying neutrophil extracellular trapping net |
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