CN102735841B - Method for detecting content of soluble CD28 in blood of patients suffering Graves disease - Google Patents
Method for detecting content of soluble CD28 in blood of patients suffering Graves disease Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of soluble CD28 in blood of patients suffering Graves disease. The method comprises: preparing materials, reagents and blood specimens, then sequentially conducting biotin labeling of a monoclonal antibody, identification of the biotin-labeled monoclonal antibody, drawing of an sCD28 standard work curve, separation and purification of T cells in Graves-suffering patient blood, phenotypic analysis of the T cells of the Graves-suffering patients, determination of the content of soluble CD28 in the Graves-suffering patient blood, immunoblotting analysis of the soluble CD28 in the patient blood, determination of the influence of the soluble CD28 on cytokines secreted by dendritic cells, determination of the influence of the soluble CD28 on dendritic cell NF-kB nuclear translocation and other determination steps, as well as statistical analysis. The method for detecting the content of soluble CD28 in blood of patients suffering Graves disease in the invention provides an experimental basis and theoretical base for further investigating the establishment of an optimized in vitro detection scheme by combining other soluble costimulatory molecules and the promotion significance in clinical diagnosis, treatment and prognosis assessment of autoimmune diseases, and for searching new immunologic intervention means.
Description
Technical field
The present invention relates to medical domain, be specifically related to the method for sCD28 content in the sick patient's blood of a kind of Graves of mensuration.
Background technology
Graves disease claims again diffuse goiter companion hyperthyroidism (hyperthyroidism) or graves disease, is modal type in hyperthyroidism.Also be a kind of organ specificity autoimmune disease of accompanying thyroid hormone secretion to increase, its etiology and pathogenesis is still not clear.Think that at present its immune mechanism of causing a disease is the effect of simulating TSH due to the antibody (TRAb) that thyroid cell surface thyrotropic hormone (TSH) acceptor produces as antigenic stimulus body, be combined with tsh receptor, make thyroid follicular cells continuation excite, form and secrete excessive thyroxine and cause hyperthyroidism.In recent years, people more and more pay attention to the effect of immune mechanism of causing a disease in the sick morbidity of Graves, and hope can be found the new way of immunologic intervention means as treatment Graves disease.
Autoantibody TRAb is important effector, it is the link of swimming more on the lower in morbidity, it not the startup factor of the sick morbidity of Graves, for this reason, Recent study person start to invest upstream link---the activation of autoreactive T cell and the research of associated adjustment molecule of replying in opening thyroid gland abnormal immune sight.Large quantity research shows, the different phase that the receptor-ligand molecule of multipair costimulatory molecules is replied at immunopathogenesis has participated in the pathologic process of the autoimmune disease such as rheumatoid arthritis, lupus erythematosus and experimental self cerebrospinal meningitis in unique separately mode.The reported first such as Schmidt in the peripheral blood of patient with rheumatoid arthritis, there is the CD4 of a group uniqueness
+t cell, its surface lacks the expression of costimulatory molecule CD28 completely.People find successively in recent years, under various chronic inflammatory states, as all having abnormal CD4 in the patients' such as multiple sclerosis, unstable angina, Wegener granulomatosis and ankylosing spondylitis peripheral blood
+cD28
-the high frequency of T cell exists.In addition, in being greater than the elderly's the peripheral blood of 65 years old, the age also finds that there is this crowd of special CD4
+cD28
-t cell exists.Already studies confirm that, in rheumatoid arthritis people, outside there is rheumatoid nodules and joint in patient when constitutional symptom, CD4
+cD28
-t cell quantity significantly increases; Equally, in coronary syndrome, CD4
+cD28
-the danger that acute coronary event occurs for the quantity of T cell and patient is obvious positive correlation, and local visible this group of cells of struvite atheromatous plaque that occur in acute coronary artery syndrome patient body are the amplification of clone's property
[12].Further studies confirm that CD4
+cD28
-t cell is the autoreactive T cell that a group has unique biological characteristics and function in essence.Research both at home and abroad confirmed already, and costimulatory molecules, no matter acceptor or part all exist with membranous type and two kinds of forms of solubility, is expressed in respectively on cell membrane and secretes in body fluid, has participated in mediation and the adjusting of costimulatory signal.Have bibliographical information, sCD28 molecule be mainly derived from membrane CD28 molecule coming off and the horizontal montage of mRNA, translation after direct secretion.But also have document to confirm through RT-PCR, the sCD28 molecule in lupus erythematosus patients blood is that full-length gene is coded, that is to say, the sCD28 molecule in patient blood is due to cell membrane CD28 molecule comes off.Separately have experiment in vitro result to show, the order of severity of the autoimmune diseases such as sCD28 molecule and multiple sclerosis, chorionitis is closely related, and has the function of suppressor T cell propagation.But bring into play which kind of effect in the diagnostic value of sCD28 molecule in Graves disease and in vivo immune response and immunological regulation, there is again what meaning in the generation of the diseases such as tumour, immunologic deficiency disease, autoimmune disease and organ transplant, in developing, lapsing to, still unclear at present.
Based on above reason, invent mensuration and the mechanism of action method of sCD28 content in a kind of effective Graves patient blood, become assistant officer's problem to be solved in the art.
Summary of the invention
The object of the present invention is to provide the method for sCD28 content in a kind of Graves of mensuration patient blood, participate in this disease pathogenesis for inquiring into the laboratory study of Graves disease and finding sCD28 molecule, the biological parameter with important foundation researching value and potential clinical value is provided.
A kind of method of measuring sCD28 content in Graves patient blood of the present invention, concrete steps are as follows:
Step 1) preparation material, reagent and blood preparation:
Cow's serum; RPMI 1640 or DMEM basal medium; CD28 monoclonal antibody 2D5,2F5,3B6,3F8 and 8G8; Affinity column: Protein G immune affinity chromatographic column; RhCD28/Fc recombinant protein; Succinyl hydroxyl biotin; Dimethyl sulfoxide (DMSO); 4% poly-D-lysine; Streptavidin-HRP; Avidin-PE; Enzyme mark assay plate; Enzyme mark analyzer; Culture flask; Experimental apparatus: CO
2incubator, hydro-extractor; Inverted microscope and fluorescent microscope; Flow cytometer; Tmb substrate; H-TSH (hTSH) kit; Lymphocyte separation medium; The direct mark CD3 of mouse-anti people PE, CD4, CD8, CD28, NF-κ B monoclonal antibody;
Blood preparation: choose many cases onset Graves patient's blood preparation as experimental group, prepare many parts of healthy human blood samples as Normal group;
Step 2) biotin labeling of monoclonal antibody
The concentration of the 2D5 monoclonal antibody of purifying is adjusted into 200 μ g/ml with carbonate buffer solution, get its 1.0ml 4 DEG C of dialysed overnight in 50ml CBS, move into Eppendof pipe, add the biotin 40 μ l of 1mg/ml, lucifuge concussion 4h, in 0.01mol/L pH7.2 PBS, 4 DEG C of dialysis 72h, save backup in-20 DEG C after packing;
Step 3) qualification of biotin labeling monoclonal antibody
After CD28-T transgenic cell is washed with PBS, with 5 × 10
5the dosage of/pipe is sub-packed in small test tube, adds biotin labeled monoclonal antibody 2D5 with the dosage of 20 μ l/ pipes, 4 DEG C of reaction 45min, fully after washing, add avidin-PE with the dosage of 10 μ l/ pipes, in 4 DEG C of reaction 45min, fully after washing, use flow cytometry analysis again, negative control is set simultaneously;
Step 4) drafting of sCD28 standard working curve
Coated through the pretreated enzyme joint inspection of 0.01% poly-D-lysine drafting board with 2F5mAb, 4 DEG C are spent the night, and after the PBS washing containing 0.01%Tween-20, spend the night with 3%BSA sealing, after washing, add the gradient dilution liquid of the standard items rhCD28/Fc recombinant protein of 0～16ng/ml, reaction 2h, after fully washing, then adds respectively biotin labeling monoclonal antibody and HRP labelled streptavidin, 37 DEG C of reaction 1h, after washing, add tmb substrate room temperature reaction 15min, measure A with said method
450, each gradient is done 3 multiple holes, taking the concentration of sCD28/Fc as horizontal ordinate, and A
450value is ordinate, draws the standard working curve of sCD28;
Step 5) separation and the purifying of T cell in Graves patient blood
Extract Graves patient or the fresh peripheral blood 10ml of Healthy People of anticoagulant heparin, with pH7.2 without Ca
2+, Mg
2+hank ' s liquid is done, after dilution in 1: 2, to be gently suspended from lymphocyte separation medium, with the centrifugal 30min of rotating speed of 1400rpm, abandons supernatant; Draw interface cloud confluent monolayer cells, add Hank ' the s liquid of 5 times of volumes, mix, 2000rpm, centrifugal 10min, abandons supernatant; Rotating speed with 1400rpm turns 10min, and repeated washing once, is counted, and adjusting cell concentration with the RPMI1640 complete medium containing 10%FCS is 3 × 10
6/ ml, puts 6 hole plastic culture plates in 37 DEG C, 5%CO
2incubator is hatched 2h to remove adherent monocyte and B cell etc., collects non-adherent cell suspension, through E garland combined techniques enrichment T cell, and through flow cytometry analysis, CD3
+t cell reaches more than 90%, and liquid nitrogen cryopreservation is for subsequent use;
Step 6) the sick human T-cell's of Graves phenotype analytical
The purifying T cell of frozen patient and the normal healthy controls group of recovering in liquid nitrogen, with after PBS washing 2 times, with 5 × 10
5the dosage of/pipe is sub-packed in small test tube, add respectively the straight labeling antibody 10 μ l of PE of CD3, CD4, CD8, CD28 and ICOS molecule and mouse IgG-PE 10 μ l as negative control, in 4 DEG C of lucifuge reaction 45min, after the PBS washing containing 5% calf serum, add after 0.5ml PBS, with flow cytometry analysis T cell phenotype, v.2 cytometer software analysis software of EXPO is used in image processing.
Step 7) mensuration of sCD28 content in Graves patient blood
Collect in T cells and supernatant, Graves ' patient blood and control group normal health human plasma 0.5ml.Get pre-coated check-out console of coated antibody 2F5, add above-mentioned collection testing sample 100 μ l, 37 DEG C of water-bath 2h, fully, after washing, add biotin labeled monoclonal antibody 2D5, again after 37 DEG C of water-bath 1h fully wash, add streptavidin-HRP, after reaction washing, add the substrate TMB of interim preparation, after room temperature reaction 15min, use 2mol/L H
2sO
4stop the reaction of enzyme-to-substrate, measure A in microplate reader
450, each sample arranges three multiple holes, makes linear standard curve, to analyze sCD28 content in patient's Graves blood plasma with rhCD28/Fc recombinant protein standard items simultaneously;
Step 8) Western blot analyzes sCD28 in blood samples of patients
Collect respectively T cells and supernatant, through the T cells and supernatant of PHA activation, Healthy Human Serum and Graves ' patients serum, in above-mentioned protein example, add 1 × sds gel sample loading buffer, in boiling water, boil 3min and make protein denaturation, the spacer gel of preparation 8% and 5% separation gel, 100 volts of constant voltages are carried out SDS-PAGE electrophoresis 3h, after electrophoresis finishes, 3M filter paper and the nitrocellulose membrane of clip and gel piece formed objects, and correctly arrange as requested filter paper, nitrocellulose membrane and gel piece, constant current 100mA electricity turns nitrocellulose membrane 2h, with the PBS sealing nitrocellulose membrane containing 5% skimmed milk power and 0.3% Tween-20, shaking table spends the night, after TBS washing, add the mouse-anti people CD28 antibody 8G8 (10 μ g/ml) of dilution next day, sealing 2h, TBS fully washs, add sheep anti mouse I gG bis-anti-(dilution in 1: 1000), incubated at room 2h, TBS adds nitrite ion BCIP colour developing after fully washing, with the chemiluminescence method observation specific proteins band of ECL detection system,
Step 9) impact of the cell factor of sCD28 on dendritic cell secretion
Aseptic extraction normal person anticoagulant heparin peripheral blood, conventional Ficoll separates and obtains mononuclearcell, washs after 2 times, adjusts cell density to 3 × 10 with RPMI-16 40
6/ ml, adds in 6 well culture plates (2ml/ hole), cultivates 2h for 37 DEG C, sucking-off suspension cell gently, and-80 DEG C are frozen for subsequent use.Then in culture plate, add the RPMI-1640 containing GM-CSF (100ng/ml), IL-4 (50ng/ml), 100IU/ml penicillin, 100 μ g/ml streptomysins, 37 DEG C of cultivations, every 3d changes liquid once.At the 6d cultivating, select lipopolysaccharides (LPS) to induce its maturation, then add CD28 recombinant protein, two days later, collecting cell culture supernatant, measures cell factor IL-2 and IL-6 concentration, and negative control group is set simultaneously;
Step 10) impact of sCD28 on dendritic cell NF-κ B nuclear translocation
Collecting ripe dendritic cell and adjusting cell concentration is 1 × 10
7/ ml, packing 0.5ml is in aseptic Eppendoff pipe, add CD28 fusion to cultivate respectively 30min, 60min and 120min, then with PBS washing three times, then use 4% paraformaldehyde fixed cell 20min of precooling, after PBS room temperature washing three times, 0.1% Triton processes 10min, after PBS room temperature washing three times, Blocking buffer room temperature sealing 1h, add NF-κ B monoclonal antibody with the dosage of 2 μ g/ pipes, room temperature reaction 2h, after PBS room temperature washing three times, add the anti-room temperature of the anti-mouse two of rabbit of cy5 mark to continue reaction 1h, after PBS washing, add nuclear staining agent PI and add RNase enzyme with the dosage of 10 μ l/ pipes with the dosage of 100 μ l/ pipes, cultivate dyeing 30min for 37 DEG C, after PBS room temperature washing three times, fluorescence mountant mounting, then by confocal microscopy result, control group is human IgG antibody's Fc section, in addition, collect the dendritic cell that LPS stimulates, detect cell CD83 by flow cytometer, the positive percentage of CD86 and CD80, analyze the maturity of dendritic cell,
Step 11) statistical analysis
All data are all used
represent, data acquisition is checked with Kolmogorov-Smirnov, nonparametric statistics is analyzed depending on sample type difference and is adopted respectively Mann-Whitney, Kruskal-Wallis and Wilcoxon ranking inspection, correlation analysis adopts Pearson correlation analysis, part index number adopts sample average relatively with t inspection, adopts SPSS10.0 statistical software to carry out statistical study.
Beneficial effect: the method for sCD28 content in the sick patient's blood of Graves after measured, can obtain, in Graves patient peripheral blood, sCD28 content increases extremely, and with the loss of membrane CD28 molecule on T cell, sCD28 molecule can also promote the sex character such as NF-κ B molecular core transposition and the secretion of cell factor IL-6 of antigen presenting cell dendritic cell, further discussion is combined other solubility costimulatory moleculeses and is set up the prioritization scheme of vitro detection, with the clinical diagnosis in autoimmune disease, promotion meaning in treatment and prognosis evaluation, provide experimental basis and theoretical foundation for finding new immunologic intervention means.
Embodiment
Embodiment 1
1. main material and reagent
Cow's serum (Hyclone, the U.S.); Full nutrient culture media: in every liter of RPMI1640 or DMEM basal medium (Gibco, the U.S.), add tire ox or calf serum 100ml, Glu 0.15g, NaHCO
32.0g, Sodium Pyruvate 0.11g, glucose 3.6g, HEPES 4.766g, 2 mercapto ethanol 10.0ml (5 × 10
-3mol/L); CD28 monoclonal antibody 2D5,2F5,3B6,3F8 and 8G8 (these section office develop voluntarily); Affinity column: Protein G immune affinity chromatographic column (Pharmacia, Sweden); RhCD28/Fc recombinant protein (R & D, the U.S.); Succinyl hydroxyl biotin (Sigma, the U.S.); Dimethyl sulfoxide (DMSO) (Sigma, the U.S.); 4% poly-D-lysine (Sigma, the U.S.); Streptavidin-HRP (Roche, Switzerland); Avidin-PE (Immunotech, France); Enzyme mark assay plate (8 × 12 holes, Costar, the U.S.); Enzyme mark analyzer (Bio-Rad, the U.S.); Culture flask: 50ml, 500ml plastic culture bottle (Nunc, Denmark); Experimental apparatus: CO
2incubator, hydro-extractor (Jouan, France); Inverted microscope and fluorescent microscope (Olympus, Japan); Flow cytometer (Beckman-Coulter, the U.S.); Tmb substrate (KPL company, Britain).H-TSH (hTSH) kit (Biological company, the U.S.), lymphocyte separation medium (Ficoll, Shanghai reagent two factories), the direct mark CD3 of mouse-anti people PE, CD4, CD8, CD28, NF-κ B monoclonal antibody (ebioscience company, the U.S.)
Specimen origin: choose 59 examples from year Dec in May, 2004 to 2006 Suzhou No.2 Renmin Hospital be in hospital and the onset Graves patient that makes a definite diagnosis of outpatient service (male 17,42 of female, 43.7 ± 15.8 years old age) as experimental group.Meet Graves patient diagnostic criteria (hypermetabolism syndrome, palpation and ultrasound diagnosis confirm to have diffuse goiter, accompany or do not accompany expophthalmos, hyperthyroxinemia, thyrotrophin receptor antibody (TRAb) positive or radioisotope scanning show that thyroid iodine uptake diffusivity strengthens).Blood station, center, Suzhou City provides 55 parts of healthy human blood's samples (23 of men, 32 of female, 21～48 years old age) as Normal group.All research objects are all without merging other autoimmune diseases, Pulmonary Diseases, tumour and infectious diseases etc., also without using the medicine that affects immunologic function, all conventional determining thyroid function (robotics luminescent system detection kit, Bayer company, Germany) and TRAb elisa kit for detecting (Diagnostika company, the U.S.).GD organizes thyroid function: FT3 29.4 ± 15.2pmol/L (normal value 3.5-5.5pmol/L), FT477.8 ± 44.9pmol/L (normal value 11.5-22.7pmol/L) and sTSH 0.13 ± 0.4 μ IU/mL (normal value 0.335-5.5 μ IU/mL).TRAb measured value: GD group is 26.2 ± 1.2U/L, Normal group is 4.6 ± 3.2U/L (P < 0.01).
2. experimental technique
Step 1) biotin (Biotin) mark of monoclonal antibody
The concentration of the 2D5 monoclonal antibody of purifying is adjusted into 200 μ g/ml with carbonate buffer solution (0.01M CBS, PH9.3), gets its 1.0ml 4 DEG C of dialysed overnight in 50ml CBS.Move into Eppendof pipe, add 1mg/ml biotin (Biotin) 40 μ l, lucifuge concussion 4h, in 0.01mol/L pH7.2PBS, (first day changes liquid 4 times to 4 DEG C of dialysis 72h, later every 24h changes liquid 2～3 times), after packing, save backup in-20 DEG C.
Step 2) qualification of biotin labeling monoclonal antibody
CD28-T transgenic cell, with after PBS washing, is sub-packed in small test tube to (5 × 10
5/ pipe), add biotin labeled monoclonal antibody 2D5 (20 μ l/ pipe), 4 DEG C of reaction 45min, fully add avidin-PE (10 μ l/ pipe), then in 4 DEG C of reaction 45min, fully after washing, use flow cytometry analysis after washing.Negative control is set simultaneously.
Step 3) drafting of sCD28 standard working curve
With 2F5mAb coated (2 μ g/ml, 100 μ l/ holes), through the pretreated enzyme joint inspection of 0.01% poly-D-lysine drafting board, 4 DEG C are spent the night.After PBS (containing 0.01%Tween-20) washing, 3%BSA sealing is spent the night.After washing, add the gradient dilution liquid (0～16ng/ml) of standard items rhCD28/Fc recombinant protein, reaction 2h, fully after washing, add respectively again biotin labeling monoclonal antibody and HRP labelled streptavidin, 37 DEG C of reaction 1h, after washing, add tmb substrate room temperature reaction 15min, measure A with said method
450.Each gradient is done 3 multiple holes.Taking the concentration of sCD28/Fc as horizontal ordinate, A
450value is ordinate, draws the standard working curve of sCD28.
Step 4) separation and the purifying of T cell in Graves patient blood
Extract Graves patient or the fresh peripheral blood 10ml of Healthy People of anticoagulant heparin, with pH7.2 without Ca
2+, Mg
2+hank ' s liquid is done, after dilution in 1: 2, to be gently suspended from lymphocyte separation medium (Ficoll) upper, 1400rpm, and centrifugal 30min, abandons supernatant; Draw interface cloud confluent monolayer cells, add Hank ' the s liquid of 5 times of volumes, mix, 2000rpm, centrifugal 10min, abandons supernatant; 1400rpm, 10min repeated washing once, is counted, and adjusting cell concentration with the RPMI1640 complete medium containing 10%FCS is 3 × 10
6/ ml, puts 6 hole plastic culture plates in 37 DEG C, 5%CO
2incubator is hatched 2h to remove adherent monocyte and B cell etc.Collect non-adherent cell suspension, through E garland combined techniques enrichment T cell.Through flow cytometry analysis, CD3
+t cell reaches more than 90%, and liquid nitrogen cryopreservation is for subsequent use.
Step 5) the sick human T-cell's of Graves phenotype analytical
The purifying T cell of frozen patient and the normal healthy controls group of recovering in liquid nitrogen, with after PBS washing 2 times, is sub-packed in small test tube (5 × 10
5/ pipe), add respectively the straight labeling antibody 10 μ l of PE of CD3, CD4, CD8, CD28 and ICOS molecule and mouse IgG-PE 10 μ l as negative control, in 4 DEG C of lucifuge reaction 45min, (1400r/min after fully washing containing the PBS of 5% calf serum, 5min), add after 0.5ml PBS, with flow cytometry analysis T cell phenotype.V.2 cytometer software analysis software of EXPO is used in image processing.
Step 6) mensuration of sCD28 content in Graves patient blood
Collect in T cells and supernatant, Graves ' patient blood and control group normal health human plasma 0.5ml.Get pre-coated check-out console of coated antibody 2F5, add above-mentioned collection testing sample 100 μ l, 37 DEG C of water-bath 2h, fully, after washing, add biotin labeled monoclonal antibody 2D5, again after 37 DEG C of water-bath 1h fully wash, add streptavidin-HRP, after reaction washing, add the substrate TMB of interim preparation, after room temperature reaction 15min, use 2mol/L H
2sO
4stop the reaction of enzyme-to-substrate, measure A in microplate reader
450.Each sample arranges three multiple holes, makes linear standard curve, to analyze sCD28 content in patient's Graves blood plasma with rhCD28/Fc recombinant protein standard items simultaneously.
Step 7) Western blot analyzes sCD28 in blood samples of patients
The T cells and supernatant, Healthy Human Serum and the Graves ' patients serum that collect respectively T cells and supernatant, activate through PHA, in above-mentioned protein example, add 1 × sds gel sample loading buffer, in boiling water, boil 3min and make protein denaturation, preparation 8% spacer gel and 5% separation gel, 100 volts of constant voltages are carried out SDS-PAGE electrophoresis 3h.After electrophoresis finishes, 3M filter paper and the nitrocellulose membrane of clip and gel piece formed objects, and correctly arrange as requested filter paper, nitrocellulose membrane and gel piece, constant current 100mA electricity turns nitrocellulose membrane 2h.With the PBS sealing nitrocellulose membrane containing 5% skimmed milk power and 0.3% Tween-20, shaking table spends the night, after TBS washing, add the mouse-anti people CD28 antibody 8G8 (10 μ g/ml) of dilution next day, sealing 2h, TBS fully washs, and adds sheep anti-mouse igg two anti-(dilution in 1: 1000), incubated at room 2h, TBS adds nitrite ion BCIP colour developing after fully washing, with the chemiluminescence method observation specific proteins band of ECL detection system.
Step 8) impact of the cell factor of sCD28 on dendritic cell secretion
Aseptic extraction normal person anticoagulant heparin peripheral blood, conventional Ficoll separates and obtains mononuclearcell, washs after 2 times, with RPMI-1640 adjustment cell density to 3 × 10
6/ ml, adds in 6 well culture plates (2ml/ hole), cultivates 2h for 37 DEG C, sucking-off suspension cell gently, and-80 DEG C are frozen for subsequent use.Then in culture plate, add the RPMI-1640 containing GM-CSF (100ng/ml), IL-4 (50ng/ml), 100IU/ml penicillin, 100 μ g/ml streptomysins, 37 DEG C of cultivations, every 3d changes liquid once.At the 6d cultivating, select lipopolysaccharide-induced its maturation, then add CD28 recombinant protein, two days later, collecting cell culture supernatant, measures cell factor IL-2 and IL-6 concentration.Negative control group is set simultaneously.
Step 9) impact of sCD28 on dendritic cell NF-κ B nuclear translocation
Collecting ripe dendritic cell and adjusting cell concentration is 1 × 10
7/ ml, packing 0.5ml is in aseptic Eppendoff pipe, add CD28 fusion (R & D company, the U.S.) cultivate respectively 30min, 60min and 120min, then with three (1400rpm × 5min of PBS washing, lower same), then use 4% paraformaldehyde fixed cell 20min of precooling, after PBS room temperature washing three times, 0.1% Triton processes 10min, after PBS room temperature washing three times, Blocking buffer room temperature sealing 1h, add NF-κ B monoclonal antibody (2 μ g/ pipe) room temperature reaction 2h, after PBS room temperature washing three times, add the anti-room temperature of the anti-mouse two of rabbit of cy5 mark to continue reaction 1h, after PBS washing, add 37 DEG C of nuclear staining agent PI (100 μ l/ pipe) and RNase enzymes (10 μ l/ pipe) to cultivate dyeing 30min, after PBS room temperature washing three times, fluorescence mountant mounting, then by confocal microscopy result.Control group is human IgG antibody's Fc section.In addition, collect the dendritic cell that LPS stimulates, detect the positive percentage of cell CD83, CD86 and CD80 by flow cytometer, analyze the maturity of dendritic cell.
Step 10) statistical analysis
All data are all used
represent, data acquisition is checked with Kolmogorov-Smirnov, nonparametric statistics is analyzed depending on sample type difference and is adopted respectively Mann-Whitney, Kruskal-Wallis and Wilcoxon ranking inspection, correlation analysis adopts Pearson correlation analysis, part index number adopts sample average relatively with t inspection, adopts SPSS10.0 statistical software to carry out statistical study.
3. measurement result analysis
1) in Graves patient blood, sCD28 content significantly raises
By the sandwich sCD28 detection method of two monoclonal antibodies of above-mentioned foundation, analyze sCD28 concentration in Graves patient blood, measurement result shows, in Graves patient blood, sCD28 content (2.16 ± 1.15ng/ml) is apparently higher than normal healthy controls group (0.83 ± 1.35ng/ml) (P < 0.01), and finds to have sCD28 concentration in indivedual patient bodies of the simultaneous phenomenons such as thyroid gland enlargement, exophthalmos ' and exceed 5ng/ml.Results suggest, sCD28 obviously increases in Graves patient body, may participate in the pathologic processes such as the generation development of Graves disease.
2) sCD28 is expressed with T cell activation state and is proportionate
In the T cells and supernatant of PHA activation, detect sCD28 content apparently higher than control group (0.67 ± 0.15 vs 0.34 ± 0.11ng/ml, P < 0.05), the generation of prompting sCD28 may be relevant with T cell activation state.Then further confirm through immunoblot experiment (Western blotting), on the concentrated culture supernatant of activating T cell and the nitrocellulose membrane of patient's Graves blood plasma printing and dyeing, there is the sCD28 protein molecular band of specific stain, and do not occurred specific dyeing band in static T cells and supernatant and Healthy People control group serum.As can be seen here, the active state of the generation of sCD28 and T cell has certain correlativity.
3) on patient's Graves periphery blood T cell, CD28 positive expression rate significantly reduces
Adopted immunofluorescence label technology and flow cytometry analysis patient Graves and control group healthy human peripheral blood CD3
+, CD8
+, CD4
+, CD28
+, CD8
+cD28
+, CD4
+cD28
+the percent of T cell.Result demonstration, on patient's Graves periphery blood T cell, the positive expression rate of costimulatory molecule CD28 significantly declines, not only CD4
+on T cell subsets, CD28 molecule is lost to some extent, and CD8
+on T cell subsets, the positive expression rate of CD28 molecule is also significantly lower than normal healthy controls group (be respectively 9.46 ± 8.58% and 17.55 ± 5.28%, P < 0.01).Further research is found, Graves peripheral blood in patients CD4
+cD28
-t cell subsets quantity is significantly higher than Normal group and (is respectively 10.2 ± 8.6% and 2.3 ± 1.9%, P < 0.01), and in patient body, the positive percentage of CD3T cell is also starkly lower than normal healthy controls group (51.43 ± 7.54% and 69.37 ± 9.21%, P < 0.05), cell count is also pointed out, and in blood samples of patients, the relative number of T cell reduces.But peripheral blood in patients T cell is abnormal up-regulated expression ICOS molecule (11.2 ± 9.46%), and costimulatory molecules ICOS expresses (1.32 ± 0.6%) hardly in normal healthy controls group.Giving after patient's drug therapy, again analyzing patient T cell phenotype shows, with treatment before compare, in patient body after drug therapy, the positive percentage of CD28 has obvious rise (to be respectively 26.83 ± 7.35% and 49.54 ± 7.81%, P < 0.01), no matter be CD4
+t cell subsets, or CD8
+the CD28 molecule on T cell subsets surface all obviously presents restorative rise, points out thus expression percent and the state of an illness of patient T cell surface CD28 molecule to have close relationship, and CD28 molecule may participate in the pathologic processes such as the generation development of this disease.
4) sCD28 content is relevant to the sick clinical detection index of Graves
In Disease body, the correlation analysis of the important clinical lab index of sCD28 concentration and medical diagnosis on disease shows, in patient body, sCD28 level and FT3, FT4 and TRAb are all proportionate, related coefficient γ is respectively 0.663,0.624 and 0.728, sCD28 concentration and serum TSH are remarkable negative correlation, related coefficient γ is-0.726, and the concentration of prompting sCD28 is the important biomolecule mathematic(al) parameter of Graves disease.
5) sCD28 concentration and clinical sign are proportionate
In order further to understand the correlativity of peripheral blood sCD28 and the state of an illness, whether patient exists and divides into groups by the degree of its thyroid gland enlargement and exophthalmos, employing nonparametric Kruskal-Wallis inspection is compared between organizing, results suggest, sCD28 and Graves patient's two large main physical signs in peripheral blood, thyroid enlargement degree (P < 0.05) and exophthalmos (P < 0.01) all have significant positive correlation, and the order of severity of prompting sCD28 and disease is obvious positive correlation.
6) sCD28 promotes dendritic cell secretion IL-6
Detect and confirm by the ELI SA to cell factor in sCD28 fusion and the interactional culture supernatant of dendritic cell, sCD28 can obviously promote the secretion (870.52 ± 73.61pg/mlvs, 4.25 ± 2.83pg/ml) of IL-6, and compared with Normal group, the concentration there was no significant difference of IL-2; But IL-6 content in Graves patients serum is measured to rear discovery, in blood samples of patients, IL-6 concentration is also apparently higher than normal population normal healthy controls group (73.46 ± 9.18pg/ml vs, 8.27 ± 3.24pg/ml), in patient body, IL-2 content significantly reduces (11.54 ± 1.72pg/ml vs, 15.36 ± 1.48pg/ml), and prompting IL-6 may participate in the sick pathology pathogenic process of Graves '.
7) sCD28 promotes the nuclear translocation of dendritic cell NF-κ B
In order to inquire into the impact of the 1 expressed by dendritic cells molecules of interest of sCD28 on monokaryon source, employing confocal microscopy arrives, sCD28 molecular action is after the dendritic cell 30min in external evoked monokaryon source, NF-κ B has started nuclear translocation, after 60min, most of NF-κ B molecule is transferred in nucleus from endochylema, NF-κ B nearly all after 120min has transferred to nucleus inside, the above results shows, after the molecules of interest of sCD28 molecule and 1 expressed by dendritic cells interacts, mediate dendritic cell signaling molecule NF-κ B nuclear translocation by reverse signal, prompting sCD28 has the effect that regulates dendritic cell function.
4 conclusions
This assay method tentative confirmation, in Graves patient peripheral blood, sCD28 content increases extremely, and with the loss of membrane CD28 molecule on T cell, experiment in vitro confirms, sCD28 molecule can also promote the NF-κ B molecular core transposition of antigen presenting cell dendritic cell and the secretion of cell factor IL-6, further inquire into other solubility costimulatory moleculeses of associating and set up the prioritization scheme of vitro detection, with the clinical diagnosis in autoimmune disease, meaning in treatment and prognosis evaluation, to provide experimental basis and theoretical foundation for finding new immunologic intervention means.
Above-described embodiment is just to allow one of ordinary skilled in the art can understand content of the present invention and implement according to this for technical conceive of the present invention and feature being described, its objective is, can not limit the scope of the invention with this.Every equivalent variation or modification that according to the present invention, the essence of content has been done, all should be encompassed in protection scope of the present invention.
Claims (5)
1. a method of measuring sCD28 content in the sick patient's blood of Graves, is characterized in that, concrete steps are:
Step 1) preparation material, reagent and blood preparation:
Cow's serum; RPMI1640 or DMEM basal medium; CD28 monoclonal antibody 2D5,2F5,3B6,3F8 and 8G8; Affinity column: Protein G immune affinity chromatographic column; RhCD28/Fc recombinant protein; Succinyl hydroxyl biotin; Dimethyl sulfoxide (DMSO); 4% poly-D-lysine; Streptavidin-HRP; Avidin-PE; Enzyme mark assay plate; Enzyme mark analyzer; Culture flask; Experimental apparatus: CO
2incubator, hydro-extractor; Inverted microscope and fluorescent microscope; Flow cytometer; Tmb substrate; H-TSH's kit; Lymphocyte separation medium; The direct mark CD3 of mouse-anti people PE, CD4, CD8, CD28, NF-κ B monoclonal antibody;
Blood preparation: choose many cases onset Graves patient's blood preparation as experimental group, prepare many parts of healthy human blood samples as Normal group;
Step 2) biotin labeling of monoclonal antibody
The concentration of the 2D5 monoclonal antibody of purifying is adjusted into 200 μ g/ml with carbonate buffer solution, get its 1.0ml 4 DEG C of dialysed overnight in 50ml CBS, move into Eppendof pipe, add the biotin 40 μ l of 1mg/ml, lucifuge concussion 4h, in 0.01mol/L pH7.2PBS, 4 DEG C of dialysis 72h, save backup in-20 DEG C after packing;
Step 3) qualification of biotin labeling monoclonal antibody
After CD28-T transgenic cell is washed with PBS, with 5 × 10
5the dosage of/pipe is sub-packed in small test tube, adds biotin labeled monoclonal antibody 2D5 with the dosage of 20 μ l/ pipes, 4 DEG C of reaction 45min, fully after washing, add avidin-PE with the dosage of 10 μ l/ pipes, in 4 DEG C of reaction 45min, fully after washing, use flow cytometry analysis again, negative control is set simultaneously;
Step 4) drafting of sCD28 standard working curve
Coated through the pretreated enzyme joint inspection of 0.01% poly-D-lysine drafting board with 2F5mAb, 4 DEG C are spent the night, and after the PBS washing containing 0.01%Tween-20, spend the night with 3%BSA sealing, after washing, add the gradient dilution liquid of the standard items rhCD28/Fc recombinant protein of 0～16ng/ml, reaction 2h, after fully washing, then adds respectively biotin labeling monoclonal antibody and HRP labelled streptavidin, 37 DEG C of reaction 1h, after washing, add tmb substrate room temperature reaction 15min, measure A with said method
450, each gradient is done 3 multiple holes, taking the concentration of sCD28/Fc as horizontal ordinate, and A
450value is ordinate, draws the standard working curve of sCD28;
Step 5) separation and the purifying of T cell in Graves patient blood
Extract Graves patient or the fresh peripheral blood 10ml of Healthy People of anticoagulant heparin, with pH7.2 without Ca
2+, Mg
2+hank ' s liquid is done, after dilution in 1: 2, to be gently suspended from lymphocyte separation medium, with the centrifugal 30min of rotating speed of 1400rpm, abandons supernatant; Draw interface cloud confluent monolayer cells, add Hank ' the s liquid of 5 times of volumes, mix, 2000rpm, centrifugal 10min, abandons supernatant; Rotating speed with 1400rpm turns 10min, and repeated washing once, is counted, and adjusting cell concentrations with RPMI 1640 complete mediums containing 10%FCS is 3 × 10
6/ ml, puts 6 hole plastic culture plates in 37 DEG C, 5%CO
2incubator is hatched 2h to remove adherent monocyte and B cell, collects non-adherent cell suspension, through E garland combined techniques enrichment T cell, and through flow cytometry analysis, CD3
+t cell reaches more than 90%, and liquid nitrogen cryopreservation is for subsequent use;
Step 6) the sick human T-cell's of Graves phenotype analytical
The purifying T cell of frozen patient and the normal healthy controls group of recovering in liquid nitrogen, with after PBS washing 2 times, with 5 × 10
5the dosage of/pipe is sub-packed in small test tube, add respectively the straight labeling antibody 10 μ l of PE of CD3, CD4, CD8, CD28 and ICOS molecule and mouse IgG-PE 10 μ l as negative control, in 4 DEG C of lucifuge reaction 45min, after the PBS washing containing 5% calf serum, add after 0.5ml PBS, with flow cytometry analysis T cell phenotype, v.2 cytometer software analysis software of EXPO is used in image processing;
Step 7) mensuration of sCD28 content in Graves patient blood
Collect in T cells and supernatant, Graves ' patient blood and control group normal health human plasma 0.5ml, get pre-coated check-out console of coated antibody 2F5, add above-mentioned collection testing sample 100 μ l, 37 DEG C of water-bath 2h, fully after washing, add biotin labeled monoclonal antibody 2D5, then after 37 DEG C of water-bath 1h fully wash, add streptavidin-HRP, after reaction washing, add the substrate TMB of interim preparation, after room temperature reaction 15min, use 2mol/L H
2sO
4stop the reaction of enzyme-to-substrate, measure A in microplate reader
450, each sample arranges three multiple holes, makes linear standard curve, to analyze sCD28 content in patient's Graves blood plasma with rhCD28/Fc recombinant protein standard items simultaneously;
Step 8) Western blot analyzes sCD28 in blood samples of patients
Collect respectively T cells and supernatant, through the T cells and supernatant of PHA activation, Healthy Human Serum and Graves ' patients serum, in above-mentioned protein example, add 1 × sds gel sample loading buffer, in boiling water, boil 3min and make protein denaturation, the spacer gel of preparation 8% and 5% separation gel, 100 volts of constant voltages are carried out SDS-PAGE electrophoresis 3h, after electrophoresis finishes, 3M filter paper and the nitrocellulose membrane of clip and gel piece formed objects, and correctly arrange as requested filter paper, nitrocellulose membrane and gel piece, constant current 100mA electricity turns nitrocellulose membrane 2h, with the PBS sealing nitrocellulose membrane containing 5% skimmed milk power and 0.3%Tween-20, shaking table spends the night, add concentration after TBS washing next day is the mouse-anti people CD28 antibody 8G8 of 10 μ g/ml, sealing 2h, TBS fully washs, add the sheep anti-mouse igg two of dilution in 1: 1000 anti-, incubated at room 2h, TBS adds nitrite ion BCIP colour developing after fully washing, with the chemiluminescence method observation specific proteins band of ECL detection system,
Step 9) impact of the cell factor of sCD28 on dendritic cell secretion
Aseptic extraction normal person anticoagulant heparin peripheral blood, conventional Fico11 separates and obtains mononuclearcell, washs after 2 times, with RPMI-1640 adjustment cell density to 3 × 10
6/ ml, add in 6 well culture plates, every hole adds 2ml, cultivate 2h for 37 DEG C, sucking-off suspension cell gently,-80 DEG C frozen for subsequent use, then in culture plate, adds the RPMI-1640 containing 100ng/mlGM-CSF, 50ng/ml IL-4,100IU/ml penicillin, 100 μ g/ml streptomysins, 37 DEG C of cultivations, every 3d changes liquid once, at the 6d cultivating, select lipopolysaccharides (LPS) to induce its maturation, then add CD28 recombinant protein, two days later, collecting cell culture supernatant, measures cell factor IL-2 and IL-6 concentration, and negative control group is set simultaneously;
Step 10) impact of sCD28 on dendritic cell NF-κ B nuclear translocation
Collecting ripe dendritic cell and adjusting cell concentration is 1 × 10
7/ ml, packing 0.5ml is in aseptic Eppendoff pipe, add CD28 fusion to cultivate respectively 30min, 60min and 120min, then with PBS washing three times, then use 4% paraformaldehyde fixed cell 20min of precooling, after PBS room temperature washing three times, 0.1%Triton processes 10min, after PBS room temperature washing three times, confining liquid room temperature sealing 1h, add NF-κ B monoclonal antibody with the dosage of 2 μ g/ pipes, room temperature reaction 2h, after PBS room temperature washing three times, add the anti-room temperature of the anti-mouse two of rabbit of cy5 mark to continue reaction 1h, after PBS washing, add nuclear staining agent PI and add RNase enzyme with the dosage of 10 μ l/ pipes with the dosage of 100 μ l/ pipes, cultivate dyeing 30min for 37 DEG C, after PBS room temperature washing three times, fluorescence mountant mounting, then by confocal microscopy result, control group is human IgG antibody's Fc section, in addition, collect the dendritic cell that LPS stimulates, detect cell CD83 by flow cytometer, the positive percentage of CD86 and CD80, analyze the maturity of dendritic cell,
Step 11) statistical analysis
All data are all used
represent, data acquisition is checked with Kolmogorov-Smirnov, nonparametric statistics is analyzed depending on sample type difference and is adopted respectively Mann-Whitney, Kruskal-Wallis and Wilcoxon ranking inspection, correlation analysis adopts Pearson correlation analysis, part index number adopts sample average relatively with t inspection, adopts SPSS10.0 statistical software to carry out statistical study.
2. the method for sCD28 content in the sick patient's blood of mensuration Graves according to claim 1; it is characterized in that; described step 1) in, in every liter of RPMI1640 or DMEM basal medium, add tire ox or calf serum 100ml, Glu 0.15g, NaHCO
32.0g, Sodium Pyruvate 0.11g, glucose 3.6g, HEPES4.766g, concentration are 5 × 10
-3the 2 mercapto ethanol 10.0ml of mol/L.
3. the method for sCD28 content in the sick patient's blood of mensuration according to claim 1 Graves, is characterized in that described step 2) in the 72h first day of dialysis change liquid 4 times, later every 24h changes liquid 2～3 times.
4. the method for sCD28 content in the sick patient's blood of mensuration according to claim 1 Graves, is characterized in that described step 6) the washing through the PBS containing 5% calf serum be the centrifugal 5min that turns of rotating speed with 1400r/min.
5. the method for sCD28 content in the sick patient's blood of mensuration according to claim 1 Graves, is characterized in that described step 10) in the washing of PBS be the centrifugal 5min that turns of speed with 1400r/min.
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