CN110196219A - One kind being based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action - Google Patents
One kind being based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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Abstract
The present invention relates to one kind to be based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action, includes the following steps: (1) Bacteria Culture, and the bacterium is Salmonella Pullorm S06004 and S06004 ΔspiC；(2) the immune and sampling of chicken；(3) target cell-macrophage preparation；(4) effector cell-chicken spleen CD8+The preparation of T cell: preparation step and magnetic bead sorting chicken spleen CD8 including chicken splenic lymphocytes suspension+The step of T cell；(5) CTL specific killing macrophage.Compared with the existing technology, the invention has the following beneficial effects: macrophage (target cell) purity of the adherent acquisition of the present invention is higher, and chicken spleen CD8+T cell separating effect is obvious, and disengaging time is short；Using detection of Salmonella host cell-macrophage as target cell, it can more accurately reflect host to the Scavenging activity of detection of Salmonella；Flow cytometry is used for chicken spleen CD8+T cell specific killing action can detect chicken spleen CD8 in individual cell level+The CTL effect of T cell.
Description
Technical field
The present invention relates to one kind to be based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action.
Background technique
Cellullar immunologic response is mediated by T lymphocyte, may advantageously facilitate the generation of body antibody, it helps removing is posted
It is born in pathogenic microorganism intracellular (such as bacterium, virus).In addition, cellullar immunologic response is in poultry immunosurveillance and antitumor
Also play a significant role in (such as Marek's disease) immunologic process.Poultry is by after cause pathogeny imcrobe infection, and T cell is by generating rush
Inflammatory cytokine adjusts locally and systemically immune response, activated macrophage and removing intracellular bacteria etc., in immune response
Vital effect is played in the process.Wherein, cytotoxic T cell (CTL) has dissolution activity, to abnormal cell
Play a crucial role in the identification and removing of (cell of such as tumour and pathogen infection), mainly by perforin/granzyme, it is dead by
Both approach of body kill target cell, and therefore, the measurement of cytotoxic activity all has in fundamental research and production practices
Significance.
Presently, there are it is more detection cytotoxicity method: MTT reduction method, LDH method for releasing, reporter gene infection protocol,
ELISPOT test and radioactive chromium (51Cr) release measurement etc..Wherein, the assessment most popular detection method of cytotoxicity is radiation
Property chromium (51Cr) release measurement, this method are considered as " goldstandard " of the cell-mediated cytotoxicity of detection, have repeatability high
The advantages of with relatively easy execution, but sensitivity is low, and radioactivity is strong, in the cracking of aggregate level detection cell, rather than in list
On cellular level, and the data of sxemiquantitative can only be provided.Therefore, it is necessary to establish, safety is good, can quantify, high-throughput detection is thin
The method of born of the same parents' poison.
With the discovery of the universal and new fluorescent marker of fluoremetry instrument, fluorimetry would be possible to replace and pass
System51Cr method for releasing, defect described above promote the development of flow-cytometry method.Flow cytometry cytotoxicity assay
Have many advantages, such as "dead", detection individual cell level cytotoxicity and assesses all stages of cytotoxic process.Mesh
It is preceding that there has been no be based on Flow cytometry chicken spleen CD8+The relevant report of T cell specific killing action.
Summary of the invention
The purpose of the present invention is for radioactive chromium (51Cr) that there are sensitivity is low for release measurement cytotoxicity method, radiation
Property it is strong the deficiencies of, establish it is a kind of update, method more reliable and that cytotoxicity can be detected with individual cell level it is (i.e. a kind of based on stream
Formula cell art detects chicken spleen CD8+The method of T cell specific killing action).
In order to achieve the above-mentioned object of the invention, the technical scheme adopted by the invention is as follows: one kind be based on Flow cytometry
Chicken spleen CD8+The method of T cell specific killing action, which is characterized in that chicken CD8 can be detected with individual cell level+T cell
Specific killing action includes the following steps:
(1) Bacteria Culture, the bacterium are Salmonella Pullorm S06004 and S06004 ΔspiC；
(2) the immune and sampling of chicken；
(3) target cell-macrophage preparation, for the first time using primary macrophage as target cell, closer to body nature shape
State provides more accurate result；
(4) effector cell-chicken spleen CD8+The preparation of T cell: preparation step and magnetic bead including chicken splenic lymphocytes suspension
Sort chicken spleen CD8+The step of T cell；
(5) CTL specific killing macrophage can react the specific killing of individual cell level by flow cytometer detection apoptosis
Ability.
The step (1) specifically: picking detection of Salmonella S06004, S06004 ΔspiCSingle colonie is inoculated in the training of LB liquid
It supports culture in base and adjusts bacterial concentration to logarithmic growth phase.
The step (2) specifically: 3 Japanese instar chicklings are randomly divided into 2 groups, respectively intramuscular injection be immunized PBS and
S06004ΔspiC。
The step (3) specifically: separate chicken peripheral blood lymphocytes with lymphocyte separation medium, adhere-wall culture obtains
Macrophage takes the adherent macrophage in part to be marked with the anti-chicken macrophage antibody of mouse, carries out purity testing, remaining macrophage
It is inoculated in 96 orifice plates, overnight incubation.Target cell by the host cell of detection of Salmonella (macrophage) as CTL for the first time, Ke Yigeng
Scavenging activity of the accurate reflection host to bacterium detection of Salmonella intracellular.
The preparation step of chicken splenic lymphocytes suspension specifically: chicken spleen is ground into filtering and obtains lymphocyte suspension,
2 removal some red blood cells of low-speed centrifugal separate through lymphocyte separation medium and obtain chicken lymphocyte.
Magnetic bead sorting chicken spleen CD8+T cell step specifically: with the magnetic bead of CD8 α-APC and anti-mouse IgG respectively as one
Anti-, secondary antibody marks good chicken lymphocyte prepared above, carries out separation by LS column and obtains chicken spleen CD8+T cell takes part
Chicken spleen CD8+T cell carries out purity testing.
Step (5) specifically: bacterium S06004 is added in the macrophage being incubated overnight, centrifugation, 1 h of culture are added
1 h of culture medium culture containing 100 μ g/mL gentamicins kills the bacterium outside macrophage；Replacement contains 10 μ g/mL gentamicins
Culture medium inhibit the proliferation of the outer bacterium of macrophage, continue to cultivate 5 h, chicken spleen CD8 is then added+T cell, co-incubation
24 h collect cell, detect the apoptosis situation of target cell (i.e. macrophage), can evaluate chicken CD8 with individual cell level+T cell
Specific killing action.
Compared with the existing technology, the invention has the following beneficial effects:
1. target cell (i.e. macrophage) purity obtained by adhere-wall culture used in the present invention is higher, compared to other
CTL detection method, closer to body nature, provides more accurate result using primary cell as target cell；
2. macrophage can more accurately reflect host to sand for detection of Salmonella host cell for the first time as the target cell of CTL
The removing of door bacterium.
3. the present invention use by magnetic bead sorting chicken spleen CD8+T cell obvious, disengaging time with separating effect
Short, the advantages that cell activity is high.
4. flow cytometry is used for chicken spleen CD8 for the first time by the present invention+T cell specific killing action, and demonstrate and exempt from
CD8 after epidemic disease in chicken spleen+T cell has stronger CTL lethal effect.CTL specific killing ability is detected compared to other
Method, flow cytometry can individual cell level detect CTL effect, and have higher sensibility (because it detects that early stage
Marker (Apoptosis)).
Detailed description of the invention
Fig. 1 adhere-wall culture obtains macrophage Purity:
(A) unmarked group of adherent preceding negative control；(B) purity of adherent preceding macrophage；(C) adherent rear negative control is unmarked
Group；(D) purity of adherent rear macrophage；
Fig. 2 magnetic bead sorting chicken spleen CD8+T cell Purity:
(A) unmarked group of negative control；(B) CD8 before sorting+The purity of T cell；(C) CD8 after sorting+The purity of T cell；
Fig. 3 flow cytometry CTL specific killing ability:
(A) non-immune group chicken spleen CD8+Induced t cell target cell apoptosis situation；(B) immune group chicken spleen CD8+Induced t cell
Target cell apoptosis situation；(C) after CTL specific killing each cell ratio.
Specific embodiment
The present invention is described in further detail below in conjunction with the drawings and specific embodiments.
1. material
1.1 experimental animal
3 age in days SPF chickens are purchased from Jinan Si Pafasi poultry company.
1.2 bacterial strain
Salmonella Pullorm S06004 (NalR) saved by the separation of this laboratory, people: Geng Shizhong is acquired, people's contact method is acquired
0514-87991556, collecting location: Xuzhou, acquisition time in March, 2006；Salmonella Pullorm mutant strain S06004 ΔspiCMutant strain is saved the (building of Liu Nannan Salmonella Pullorm S06004 Δ spiC mutant strain and its immune by the building of this room
Biological characteristics Primary Study [D] Yangzhou University, 2012.).
2. method
2.1 Bacteria Culture
Picking S06004, S06004 ΔspiCSingle colonie is inoculated in 4 mL LB liquid mediums, and 37 DEG C, 180 r/min shaking tables
After 12 h of shaken cultivation, bacterium solution is inoculated in expand in conical flask with the volume of 1:500 and is cultivated, 14 h of shaken cultivation, after taking-up
Stationary culture 3-4 h again.It is washed 3 times with sterile PBS, adjusts bacterium (S06004, S06004 ΔspiC) concentration.
The immune and sampling of 2.2 chickens
3 Japanese instar chicklings are randomly divided into 2 groups, PBS and S06004 Δ is immunized in intramuscular injection respectivelyspiC(1 × 107 CFU/ plumage),
100 μ L/ plumages.
The preparation of 2.3 target cells-macrophage
The chicken peripheral blood being collected in anticoagulant tube is diluted in equal volume with DPBS；The peripheral blood diluted is added in equal volume
In Histopaque-1083, in 20 DEG C, 2000 r/min are centrifuged 20 min, take intermediate white haze confluent monolayer cells；Again with isometric DPBS
The white haze confluent monolayer cells obtained are diluted, are added in isometric lymphocyte separation medium after mixing, the same terms separate again,
Ensure to obtain the leucocyte of high-purity；The cell of acquisition is resuspended with DPBS, is washed twice, with containing 5% fetal calf serum, 5% chicken blood
Clearly, 1640 culture medium of RPMI of 100 U/mL penicillin and streptomycins adjusts cell concentration to 1 × 107A/mL takes 10 mL to be added to carefully
In born of the same parents' culture dish；In 41 DEG C, 5% CO2Incubator stationary culture discards culture medium and non-attached cell after 24 h, replaces fresh
Culture medium continues to cultivate 24 h；Then discard culture medium and suspension cell, adherent cell is macrophage in culture dish.
The macrophage of acquisition is washed twice with PBS；And 3 min are digested with the pancreatin of no EDTA, add containing 5% chicken blood
Clearly, the complete RPMI 1640 of 5% FBS, 100 U/mL penicillin and streptomycins, 0.1% beta -mercaptoethanol and 25% HEPES terminate pancreatin
Reaction；Attached cell is blown and beaten, and in 4 DEG C, 1000 r/min are centrifuged 10 min；Discard supernatant；Part cell is taken to use mouse
Anti- chicken macrophage antibody label (Mouse Anti-Chicken Monocyte/Macroage-PE) (only use after label by cell
In purity testing), purity testing is carried out by FACSAria SORP flow cytometer；Remaining macrophage (is used for subsequent examination
Test: 2.5 CTL specific killing macrophages) it is resuspended with complete 1640 culture medium of RPMI, adjustment cell concentration to 5 × 105
A/mL；It is inoculated in 96 orifice plates, every 100 μ L of hole, in 37 DEG C, 5% CO2Overnight incubation.
2.4 effector cells-spleen CD8+The preparation of T cell
2.4.1 the preparation of chicken splenic lymphocytes suspension
After putting to death chicken by injection of heart air, spleen is taken, is placed in the 15 mL centrifuge tubes containing DPBS；2 are washed with DPBS
Time, spleen is transferred in plate, the Gentle MACS C pipe containing 6 mL DPBS is shredded and be transferred to sterile scissors, is passed through
The full-automatic tissue processor of Gentle MACS is ground；It is filtered with 70 μm of filter, takes filtrate；600 r/min from
5 min of the heart draws supernatant in new centrifuge tube, is repeated 2 times, to remove most of red blood cell；Cell suspension is slowly added to
In the lymphocyte separation medium of volume, 20 DEG C, 1800 r/min of horizontal rotor is centrifuged 20 min；It is thin to draw intermediate white haze layer lymph
Born of the same parents, and add 4 DEG C of complete 1640 culture medium of RPMI of 6 mL (containing 10% FBS, 100 U/mL penicillin and streptomycins), 1000 r/min from
10 min of the heart；Supernatant is abandoned, lymphocyte is obtained.
2.4.2 magnetic bead sorting chicken spleen CD8+T cell
The lymphocyte that will be prepared washes 2 times with the MACS buffer of pre-cooling and cell is resuspended, and CD8 α-APC is added and is marked
Note, is protected from light 30 min of effect by 4 DEG C；The MACS buffer of 8 mL pre-cooling is added, 4 DEG C, 1500 r/min are centrifuged 10 min removal
Unbonded antibody；Every 107A cell is resuspended with 100 μ L MACS buffer, and the magnetic bead of the anti-mouse IgG of 20 μ L is added
(Anti-Mouse IgG1-Microbeads) is protected from light 15 min of effect in 4 DEG C；It is added the MACS buffer of 8 mL pre-cooling, 4
DEG C, 1500 r/min are centrifuged the unbonded magnetic bead of 10 min removal；With the MACS buffer of 500 μ L before carrying out magnetic separation
It is resuspended (about 108A cell)；LS column is placed on MACS separator (a superpower magnet), and with 3 mL MACS buffer
It is rinsed；Cell suspension is added in column, the chicken spleen CD8 of magnetic mark+T cell is attracted in column, and unlabelled
Cell is flowed out from pillar；It is rinsed pillar 3 times, is then removed LS column from separator, and add with 3 mL MACS buffer
Suitable MACS buffer goes out the cell of marked by magnetic bead into column, through piston；It is resuspended and is counted with PBS, pass through streaming
Cell instrument carries out purity testing.
2.5 CTL specific killing macrophages
Macrophage is inoculated in 96 orifice plates, every hole 5 × 104A cell, 37 DEG C, 5% CO2Overnight incubation is to cell monolayer；It connects
1 h before kind bacterium S06004, is changed to the RPMI 1640 containing 5% fetal calf serum, 5% chicken serum, antibiotic-free for cell culture medium and trains
Support base；S06004 is cultivated to logarithmic growth phase, 4500 r/min are centrifuged 5 min, abandon supernatant, are washed 3 times with PBS, by bacterium
S06004 infects macrophage with the ratio of MOI=10；20 DEG C, 1000 r/min are centrifuged 10 min, make bacterium S06004 and macrophage
Cell comes into full contact with；37 DEG C of 1 h of culture；Supernatant is abandoned, and is washed macrophage 3 times with PBS, is added and contains 5% fetal calf serum, 5% chicken
1640 culture medium of RPMI of serum and 100 μ g/mL gentamicins, 37 DEG C of 1 h of culture, sufficiently kills extracellular bacterium；With
PBS is washed 3 times, changes 1640 culture medium of RPMI containing 5% fetal calf serum, 5% chicken serum and 10 μ g/mL gentamicins into inhibit
The proliferation of extracellular bacterium；Continue to cultivate 5 h, every hole is added 1 × 106A chicken spleen CD8+T cell；37 DEG C, 5% CO2Co-incubation
24 h take out non-attached cell, and digest adherent macrophage with no EDTA pancreatin；According to apoptosis detection kit manipulator
Volume detection target cell (i.e. macrophage) apoptosis situation, step approximately as: with 4 DEG C be pre-chilled PBS wash cell twice, with knot
It closes buffer and cell is resuspended, adjustment concentration is 1 × 107A/mL；100 μ L cells are taken to add 5 μ L Annexin V/Alexa
The μ L propidium iodide solution of Fluor 488 and 10；15 min of effect are protected from light, flow cytometry analysis is used.
3 results
The adherent preparation result of macrophage in 3.1 peripheral bloods
It by the macrophage of adherent preparation, is digested with no EDTA pancreatin, is marked through the anti-chicken macrophage antibody of mouse, it is thin using streaming
Born of the same parents' art carries out purity testing.As seen from Figure 1, before not adherent, macrophage purity is 9.76%；The macrophage of adherent acquisition
Cell purity reaches 97.7%, illustrates that the macrophage purity of adherent acquisition is higher, and method is simple, can preferably be used for the later period
Experiment.
CD8 in 3.2 spleens+T lymphocyte magnetic bead sorting result
The lymphocyte that will be prepared is marked through primary antibody secondary antibody, then carries out magnetic separation by LS column, and the cell of sorting is utilized stream
Formula cell art carries out purity testing.As seen from Figure 2, CD8 before sorting+T lymphocyte proportion is 39.5%, sorting
Purity reaches 93.5% later, and separating effect is obvious, and disengaging time is shorter.
3.3 CTL specific killing macrophages analyze result
Apoptosis detects CTL specific killing result as shown in figure 3, as can be seen from Figure 3A, non-immune group chicken spleen CD8+T
After cell and target cell co-culture 24 h, the live cell fraction of macrophage (target cell) is 86.9%, apoptotic cell 5.52%,
Dead cell is 7%；As can be seen from Figure 3B, immune group chicken spleen CD8+After T cell and target cell co-culture 24 h, target cell
Live cell fraction be 67.4%, apoptotic cell 21.7%, dead cell 10.5%；Fig. 3 C is the results show that with control group phase
Than, in immune group target cell live cell fraction significantly reduce (P< 0.05), apoptotic cell ratio significantly increase (P <
0.05), dead cell ratio is also risen, but there was no significant difference, illustrates the CD8 in immune rear chicken spleen+T cell has
Stronger CTL lethal effect.
Basic principles and main features and advantages of the present invention of the invention have been shown and described above.The skill of the industry
Art personnel it should be appreciated that the present invention is not limited to the above embodiments, the above embodiments and description only describe
The principle of the present invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these
Changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent thereof.
Claims (8)
1. one kind is based on Flow cytometry chicken spleen CD8+The method of T cell specific killing action, which is characterized in that can
Chicken CD8 is detected with individual cell level+The specific killing action of T cell, includes the following steps:
(1) Bacteria Culture, the bacterium are Salmonella Pullorm S06004 and S06004 ΔspiC；
(2) the immune and sampling of chicken；
(3) target cell-macrophage preparation, using isolated primary macrophage as target cell, closer to body nature shape
State provides more accurate result；
(4) effector cell-chicken spleen CD8+The preparation of T cell: preparation step and magnetic bead including chicken splenic lymphocytes suspension
Sort chicken spleen CD8+The step of T cell；
(5) CTL specific killing macrophage can be detected special by Apoptosis by Flow Cytometry with individual cell level
Anisotropic killing ability.
2. according to claim 1 a kind of based on Flow cytometry chicken spleen CD8+T cell specific killing action
Method, which is characterized in that the step (1) specifically: picking Salmonella Pullorm S06004, S06004 ΔspiCSingle colonie
It is inoculated in culture in LB liquid medium and adjusts bacterial concentration to logarithmic growth phase.
3. according to claim 1 a kind of based on Flow cytometry chicken spleen CD8+T cell specific killing action
Method, which is characterized in that the step (2) specifically: 3 Japanese instar chicklings are randomly divided into 2 groups, intramuscular injection is immune respectively
PBS and S06004 ΔspiC。
4. according to claim 1 a kind of based on Flow cytometry chicken spleen CD8+T cell specific killing action
Method, which is characterized in that the step (3) specifically: chicken peripheral blood lymphocytes is separated with lymphocyte separation medium, it is adherent
Culture obtains macrophage, and the adherent macrophage in part is taken to be marked with the anti-chicken macrophage antibody of mouse, carries out purity testing, remaining
Macrophage is inoculated in 96 orifice plates, overnight incubation.
5. according to claim 1 a kind of based on Flow cytometry chicken spleen CD8+T cell specific killing action
Method, which is characterized in that the preparation step of chicken splenic lymphocytes suspension specifically: it is thin that chicken spleen is ground into filtering acquisition lymph
Born of the same parents' suspension, 2 removal some red blood cells of low-speed centrifugal separate through lymphocyte separation medium and obtain chicken lymphocyte.
6. according to claim 1 a kind of based on Flow cytometry chicken spleen CD8+T cell specific killing action
Method, which is characterized in that magnetic bead sorting chicken spleen CD8+T cell step specifically: with the magnetic bead of CD8 α-APC and anti-mouse IgG point
Not Zuo Wei primary antibody, secondary antibody mark chicken lymphocyte, by LS column carry out separation obtain chicken spleen CD8+T cell takes part chicken spleen
Dirty CD8+T cell carries out purity testing.
7. according to claim 1 a kind of based on Flow cytometry chicken spleen CD8+T cell specific killing action
Method, which is characterized in that step (5) specifically: bacterium S06004 is added in the macrophage being incubated overnight, centrifugation, culture 1
The bacterium outside 1 h of the culture medium culture kill macrophage containing 100 μ g/mL gentamicins is added in h；Replacement is celebrated containing 10 μ g/mL
The culture medium of big mycin inhibits the proliferation of extracellular bacterium, continues to cultivate 5 h, and spleen CD8 is then added+T cell, co-incubation 24
H collects cell, detects the apoptosis situation of target cell, evaluates chicken spleen CD8+T cell specific killing action.
8. being based on Flow cytometry chicken spleen CD8 as described in claim 1-7+The method of T cell specific killing action can
Evaluation for detection of Salmonella immune effect of vaccine and cellullar immunologic response.
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