CN102735841A - 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 solubility CD28 content in the sick patient's blood of a kind of Graves of mensuration.
Background technology
The Graves disease is claimed diffuse goiter companion's hyperthyroidism (hyperthyroidism) or graves disease again, is modal type in the hyperthyroidism.Also be a kind of organ specificity autoimmune disease of accompanying thyroid hormone secretion to increase, its cause of disease and pathogenesis are still indeterminate.Think that at present its immune mechanism of causing a disease is because the antibody (TRAb) that thyroid cell surface thyrotropic hormone (TSH) acceptor is produced as the antigenic stimulus body is simulated the effect of TSH; Combine with tsh receptor, make the thyroid follicular cells continuation excite, form and secrete excessive thyroxine and cause hyperthyroidism.In recent years, People more and more is paid attention to the effect of immune mechanism of causing a disease in the sick morbidity of Graves, and hope can be sought the immunologic intervention means as the sick new way of treatment Graves.
Autoantibody TRAb is important effector; It is the link of leaning on downstream in the morbidity; It or not the startup factor of the sick morbidity of Graves; For this reason, Recent study person begin sight to invest in opening upper reaches link---the activation and the associated adjustment molecular studies of autoreactive T cell that the thyroid gland abnormal immune is replied.Big quantity research shows, the different phase that many receptor-ligand molecules to costimulatory molecules are replied at immunopathogenesis has been participated in the pathologic process of autoimmune diseases such as rheumatoid arthritis, lupus erythematosus and experimental self cerebrospinal meningitis with unique mode separately.Reported first such as Schmidt in the peripheral blood of patient with rheumatoid arthritis, have a unique CD4 of a group
+The T cell, its surface lacks the expression of costimulatory molecules CD28 fully.People find successively in recent years, under various chronic inflammatory states, as in the peripheral blood of patients such as multiple sclerosis, unstable angina, Wegener granulomatosis and ankylosing spondylitis unusual CD4 being arranged all
+CD28
-The high frequency of T cell exists.In addition, also find to have the special CD4 of this group at the age in greater than 65 years old the elderly's peripheral blood
+CD28
-The T cell exists.There had been research to confirm already, at the rheumatoid arthritis philtrum, outside rheumatoid nodules and joint appear in the patient during constitutional symptom, CD4
+CD28
-The T cell quantity significantly increases; Equally, in coronary syndrome, CD4
+CD28
-The danger that acute coronary event takes place for the quantity of T cell and patient is obvious positive correlation, and local visible this group cell of struvite atheromatous plaque that takes place in the acute coronary artery syndrome patient body is the amplification of clone's property
[12]Further research confirms CD4
+CD28
-The 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 on the cell membrane respectively and secretes in body fluid, has participated in the mediation and the adjusting of costimulatory signal.Bibliographical information is arranged, solubility CD28 molecule be mainly derived from membranous type CD28 molecule coming off with the horizontal montage of mRNA, the translation after direct secretion.But also have document to confirm through RT-PCR, the solubility CD28 molecule in the systemic loupus erythematosus patient blood is that full-length gene is coded, that is to say, the solubility CD28 molecule in the patient blood is due to cell membrane CD28 molecule comes off.Other has the experiment in vitro result to show, the order of severity of autoimmune diseases such as solubility CD28 molecule and multiple sclerosis, chorionitis is closely related, and has the function of suppressor T cell propagation.But diagnostic value and in vivo in immune response and immunological regulation performance which kind of effect thereof of solubility CD28 molecule in the Graves disease; In the generation of diseases such as tumour, immunologic deficiency disease, autoimmune disease and organ transplant, in developing, lapsing to what meaning is arranged again, still unclear at present.
Based on above reason, invent solubility CD28 Determination on content and mechanism of action method in a kind of effective Graves patient blood, become assistant officer's problem to be solved in the present technique field.
Summary of the invention
The object of the present invention is to provide the method for solubility CD28 content in a kind of Graves of mensuration patient blood; For inquire into the sick laboratory study of Graves with seek that solubility CD28 molecule participates in should the disease pathogenesis, the biological parameter with important foundation researching value and potential clinical value is provided.
A kind of method of measuring solubility CD28 content in the Graves patient blood of the present invention, concrete steps are following:
Step 1) is prepared 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; The 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 blood preparation that many cases just send out the Graves patient as experimental group, prepare many parts of healthy human blood's samples as the normal control group;
Step 2) biotin labeling of monoclonal antibody
With carbonate buffer solution the concentration of the 2D5 monoclonal antibody of purifying is adjusted into 200 μ g/ml; Get its 1.0ml 4 ℃ of dialysed overnight in 50ml CBS; Move into the Eppendof pipe, add the biotin 40 μ l of 1mg/ml, lucifuge concussion 4h; 0.01mol/L 4 ℃ of dialysis 72h are subsequent use in-20 ℃ of preservations after the packing among the pH7.2 PBS;
The evaluation of step 3) biotin labeling monoclonal antibody
With the CD28-T transgenic cell with after the PBS washing, with 5 * 10
5The dose of/pipe is loaded in the small test tube, and the dosage of managing with 20 μ l/ adds biotin labeled monoclonal antibody 2D5,4 ℃ of reaction 45min; Fully the washing back adds avidin-PE with the dosage of 10 μ l/ pipe; In 4 ℃ of reaction 45min, fully flow cytometry analysis is used in the washing back, and negative control is set simultaneously again;
The drafting of step 4) sCD28 standard working curve
Encapsulate through the pretreated enzyme joint inspection of 0.01% poly-D-lysine drafting board with 2F5mAb, 4 ℃ are spent the night, contain the PBS washing of 0.01%Tween-20 after; Spend the night with the 3%BSA sealing, the washing back adds the gradient dilution liquid of the standard items rhCD28/Fc recombinant protein of 0～16ng/ml, reaction 2h; Fully after the washing, add biotin labeling monoclonal antibody and HRP labelled streptavidin more respectively, 37 ℃ of reaction 1h; After the washing, add tmb substrate room temperature reaction 15min, measure A with said method
450, each gradient is done 3 multiple holes, is horizontal ordinate with the concentration of sCD28/Fc, A
450Value is ordinate, draws the standard working curve of sCD28;
The separation of T cell and purifying in the step 5) Graves patient blood
Extract the Graves patient or the fresh peripheral blood 10ml of healthy subjects of anticoagulant heparin, with the no Ca of pH7.2
2+, Mg
2+Hank ' s liquid gently is suspended from the lymphocyte separation medium after doing dilution in 1: 2, with the centrifugal 30min of the rotating speed of 1400rpm, abandons supernatant; Draw interface cloud confluent monolayer cells, add Hank ' the s liquid of 5 times of volumes, mixing, 2000rpm, centrifugal 10min abandons supernatant; Rotating speed with 1400rpm changes 10min, and repeated washing is once counted, and using the RPMI1640 complete medium adjustment cell concentration that contains 10%FCS is 3 * 10
6/ ml puts 6 hole plastic culture plates in 37 ℃, 5%CO
2Incubator is hatched 2h to remove adherent monocyte and B cell etc., collects the non-adherent cell suspension, through E garland combined techniques enrichment T cell, and through flow cytometry analysis, CD3
+The T cell reaches more than 90%, and liquid nitrogen cryopreservation is subsequent use;
The phenotype analytical of step 6) Graves patient T cell
The frozen patient of recovery and the purifying T cell of normal healthy controls group in the liquid nitrogen, wash 2 times with PBS after, with 5 * 10
5The dose of/pipe is loaded in the small test tube; The straight labeling antibody 10 μ l of PE and the mouse IgG-PE 10 μ l that add CD3, CD4, CD8, CD28 and ICOS molecule respectively are as negative control; In 4 ℃ of lucifuge reaction 45min, after the PBS washing that contains 5% calf serum, behind the adding 0.5ml PBS; With flow cytometry analysis T cell phenotype, image processing utilization EXPO is cytometer software analysis software v.2.
Solubility CD28 Determination on content in the step 7) Graves patient blood
Collect in T cells and supernatant, the Graves ' patient blood and control group normal health human plasma 0.5ml.Get the check-out console that coated antibody 2F5 has encapsulated in advance, add above-mentioned collection testing sample 100 μ l, 37 ℃ of water-bath 2h; Fully after the washing, add biotin labeled monoclonal antibody 2D5, again after 37 ℃ of water-bath 1h fully wash; Add streptavidin-HRP, after the reaction washing, add the substrate TMB of interim preparation; Behind the room temperature reaction 15min, use 2mol/L H
2SO
4Stop the reaction of enzyme-to-substrate, measure A in ELIASA
450, each sample is provided with three multiple holes, makes linear standard curve with rhCD28/Fc recombinant protein standard items simultaneously, to analyze solubility CD28 content in patient's Graves blood plasma;
The step 8) Western blot is analyzed solubility CD28 in the blood samples of patients
Collect the T cells and supernatant respectively, through PHA activated T cells and supernatant, healthy subjects 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, constant voltage is carried out SDS-PAGE electrophoresis 3h for 100 volts, after electrophoresis finishes; The 3M filter paper and the nitrocellulose membrane of clip and the identical size of gel piece, and correctly arrange filter paper, nitrocellulose membrane and gel piece as requested, constant current 100mA electricity changes nitrocellulose membrane 2h; With the PBS sealing nitrocellulose membrane that contains 5% skimmed milk power and 0.3% Tween-20, shaking table spends the night, and next day, TBS washing back added the mouse-anti people CD28 antibody 8G8 (10 μ g/ml) of dilution; Sealing 2h; TBS fully washs, and adds sheep anti mouse I gG two anti-(dilution in 1: 1000), incubated at room 2h; TBS fully washs the back and adds colour developing liquid BCIP colour developing, observes the specific proteins band with the chemiluminescence method of ECL detection system;
Step 9) solubility CD28 is to the influence of the cell factor of BMDC secretion
Aseptic extraction normal person anticoagulant heparin peripheral blood, conventional Ficoll separate and obtain mononuclearcell, wash 2 times after, adjust cell density to 3 * 10 with RPMI-16 40
6/ ml adds in 6 well culture plates (2ml/ hole), cultivates 2h for 37 ℃, sucking-off suspension cell gently, and-80 ℃ are frozen subsequent use.In culture plate, add the RPMI-1640 that contains GM-CSF (100ng/ml), IL-4 (50ng/ml), 100IU/ml penicillin, 100 μ g/ml streptomysins then, 37 ℃ of cultivations, every 3d changes liquid once.6d cultivating selects for use lipopolysaccharides (LPS) to induce its maturation, adds the CD28 recombinant protein then, and two days later, the collecting cell culture supernatant is measured cell factor IL-2 and IL-6 concentration, and negative control group is set simultaneously;
Step 10) solubility CD28 is to the influence of BMDC NF-κ B nuclear translocation
Collecting ripe BMDC and adjusting cell concentration is 1 * 10
7/ ml, packing 0.5ml adds the CD28 fusion and cultivates 30min, 60min and 120min respectively in aseptic Eppendoff pipe, then with PBS washing three times; Use 4% paraformaldehyde fixed cell 20min of precooling then, after the PBS room temperature washing three times, 0.1% Triton handles 10min, after the PBS room temperature washing three times; Blocking buffer room temperature sealing 1h adds NF-κ B monoclonal antibody with the dosage of 2 μ g/ pipe, and room temperature reaction 2h is after the PBS room temperature washing three times; Add the anti-mouse two anti-room temperatures continuation reaction 1h of rabbit of cy5 mark, after the PBS washing, the dosage of managing with 100 μ l/ adds nuclear staining agent PI and adds the RNase enzyme with the dosage that 10 μ l/ manage; Cultivate dyeing 30min for 37 ℃, after the PBS room temperature washing three times, fluorescence mountant mounting; Through the Laser Scanning Confocal Microscope observations, control group is human IgG antibody's a Fc section, in addition then; Collect the BMDC that LPS stimulates,, analyze the maturity of BMDC through the positive percentage of flow cytometer detection cell CD83, CD86 and CD80;
The step 11) statistical analysis
Expression that all data are all used
; The data Kolmogorov-Smirnov check; The nonparametric statistics analysis is looked the sample type difference and is adopted Mann-Whitney, Kruskal-Wallis and Wilcoxon ranking check respectively; The Pearson correlation analysis is adopted in correlation analysis; Part index number adopts sample average relatively with the t check, adopts the SPSS10.0 statistical software to carry out statistical study.
Beneficial effect: through measuring the method for solubility CD28 content in the sick patient's blood of Graves; Can obtain; Solubility CD28 content increases unusually in the Graves patient peripheral blood; And with the losing of membranous type CD28 molecule on the T cell, solubility CD28 molecule can also promote NF-κ B molecular core transposition of antigen presenting cell BMDC and the sex character such as secretion of cell factor IL-6, further other solubility costimulatory moleculeses of discussion associating is set up the prioritization scheme of vitro detection; With the promotion meaning in clinical diagnosis, treatment and the prognosis evaluation of autoimmune disease, experimental basis and theoretical foundation are provided for seeking 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 the DMEM basal medium (Gibco, the U.S.), add tire ox or calf serum 100ml, L-glutaminate 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.)
Sample source: choose 59 examples and be in hospital first the Graves patient (17 of men, women 42,43.7 ± 15.8 years old age) that make a definite diagnosis with outpatient service as experimental group in Suzhou No.2 Renmin Hospital from year Dec in May, 2004 to 2006.Meet Graves patient diagnostic criteria (hypermetabolism syndrome; Palpation and ultrasound diagnosis confirm to have diffuse goiter; Companion or do not accompany expophthalmos, hyperthyroxinemia, thyrotrophin receptor antibody (TRAb) positive or radioisotope scanning show that the thyroid iodine uptake diffusivity strengthens).Blood station, center, Suzhou City provides 55 parts of healthy human blood's samples (23 of men, 32 of woman, 21～48 years old age) as the normal control group.All research objects all do not have other autoimmune diseases of merging, lung's illness, tumour and infectious diseases etc.; Also do not have and use the medicine that influences immunologic function; Equal 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, and the normal control group is 4.6 ± 3.2U/L (P＜0.01).
2. experimental technique
The biotin of step 1) monoclonal antibody (Biotin) mark
(0.01M CBS, PH9.3) concentration with the 2D5 monoclonal antibody of purifying is adjusted into 200 μ g/ml, gets its 1.0ml 4 ℃ of dialysed overnight in 50ml CBS with carbonate buffer solution.Move into the Eppendof pipe, add 1mg/ml biotin (Biotin) 40 μ l, lucifuge concussion 4h, 4 ℃ of dialysis 72h (changed liquid 4 times in first day, later every 24h changes liquid 2～3 times) are subsequent use in-20 ℃ of preservations after the packing among the 0.01mol/L pH7.2PBS.
Step 2) evaluation of biotin labeling monoclonal antibody
The CD28-T transgenic cell with after the PBS washing, is sub-packed in the small test tube (5 * 10
5/ pipe), add biotin labeled monoclonal antibody 2D5 (20 μ l/ pipe), 4 ℃ of reaction 45min, fully the washing back adds avidin-PE (10 μ l/ pipe), in 4 ℃ of reaction 45min, fully uses flow cytometry analysis after the washing again.Negative control is set simultaneously.
The drafting of step 3) sCD28 standard working curve
Encapsulate (2 μ g/ml, 100 μ l/ holes) through the pretreated enzyme joint inspection of 0.01% poly-D-lysine drafting board with 2F5mAb, 4 ℃ are spent the night.After PBS (the containing 0.01%Tween-20) washing, the 3%BSA sealing is spent the night.(0～16ng/ml), reaction 2h is fully after the washing for the gradient dilution liquid of washing back adding standard items rhCD28/Fc recombinant protein; Add biotin labeling monoclonal antibody and HRP labelled streptavidin more respectively, 37 ℃ of reaction 1h are after the washing; Add tmb substrate room temperature reaction 15min, measure A with said method
450Each gradient is done 3 multiple holes.Concentration with sCD28/Fc is horizontal ordinate, A
450Value is ordinate, draws the standard working curve of sCD28.
The separation of T cell and purifying in the step 4) Graves patient blood
Extract the Graves patient or the fresh peripheral blood 10ml of healthy subjects of anticoagulant heparin, with the no Ca of pH7.2
2+, Mg
2+Hank ' s liquid gently is suspended from the lymphocyte separation medium (Ficoll) after doing dilution in 1: 2,1400rpm, and centrifugal 30min abandons supernatant; Draw interface cloud confluent monolayer cells, add Hank ' the s liquid of 5 times of volumes, mixing, 2000rpm, centrifugal 10min abandons supernatant; 1400rpm, 10min repeated washing once count, and using the RPMI1640 complete medium adjustment cell concentration that contains 10%FCS is 3 * 10
6/ ml puts 6 hole plastic culture plates in 37 ℃, 5%CO
2Incubator is hatched 2h to remove adherent monocyte and B cell etc.Collect the non-adherent cell suspension, through E garland combined techniques enrichment T cell.Through flow cytometry analysis, CD3
+The T cell reaches more than 90%, and liquid nitrogen cryopreservation is subsequent use.
The phenotype analytical of step 5) Graves patient T cell
The frozen patient of recovery and the purifying T cell of normal healthy controls group in the liquid nitrogen, wash 2 times with PBS after, be sub-packed in the small test tube (5 * 10
5/ pipe); The straight labeling antibody 10 μ l of PE and the mouse IgG-PE 10 μ l that add CD3, CD4, CD8, CD28 and ICOS molecule respectively are as negative control; In 4 ℃ of lucifuge reaction 45min, and after the PBS that contains 5% calf serum fully washs (1400r/min, 5min); After adding 0.5ml PBS, with flow cytometry analysis T cell phenotype.Image processing utilization EXPO is cytometer software analysis software v.2.
Solubility CD28 Determination on content in the step 6) Graves patient blood
Collect in T cells and supernatant, the Graves ' patient blood and control group normal health human plasma 0.5ml.Get the check-out console that coated antibody 2F5 has encapsulated in advance, add above-mentioned collection testing sample 100 μ l, 37 ℃ of water-bath 2h; Fully after the washing, add biotin labeled monoclonal antibody 2D5, again after 37 ℃ of water-bath 1h fully wash; Add streptavidin-HRP, after the reaction washing, add the substrate TMB of interim preparation; Behind the room temperature reaction 15min, use 2mol/L H
2SO
4Stop the reaction of enzyme-to-substrate, measure A in ELIASA
450Each sample is provided with three multiple holes, makes linear standard curve with rhCD28/Fc recombinant protein standard items simultaneously, to analyze solubility CD28 content in patient's Graves blood plasma.
The step 7) Western blot is analyzed solubility CD28 in the blood samples of patients
Collect the T cells and supernatant respectively, through PHA activated T cells and supernatant, healthy subjects 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, constant voltage is carried out SDS-PAGE electrophoresis 3h for 100 volts.After electrophoresis finishes, the 3M filter paper and the nitrocellulose membrane of clip and the identical size of gel piece, and correctly arrange filter paper, nitrocellulose membrane and gel piece as requested, constant current 100mA electricity commentaries on classics nitrocellulose membrane 2h.With the PBS sealing nitrocellulose membrane that contains 5% skimmed milk power and 0.3% Tween-20, shaking table spends the night, and next day, TBS washing back added the mouse-anti people CD28 antibody 8G8 (10 μ g/ml) of dilution; Sealing 2h; TBS fully washs, and adds sheep anti-mouse igg two anti-(dilution in 1: 1000), incubated at room 2h; TBS fully washs the back and adds colour developing liquid BCIP colour developing, observes the specific proteins band with the chemiluminescence method of ECL detection system.
Step 8) solubility CD28 is to the influence of the cell factor of BMDC secretion
Aseptic extraction normal person anticoagulant heparin peripheral blood, conventional Ficoll separate and obtain mononuclearcell, wash 2 times after, adjust cell density to 3 * 10 with RPMI-1640
6/ ml adds in 6 well culture plates (2ml/ hole), cultivates 2h for 37 ℃, sucking-off suspension cell gently, and-80 ℃ are frozen subsequent use.In culture plate, add the RPMI-1640 that contains GM-CSF (100ng/ml), IL-4 (50ng/ml), 100IU/ml penicillin, 100 μ g/ml streptomysins then, 37 ℃ of cultivations, every 3d changes liquid once.6d cultivating selects lipopolysaccharide-induced its maturation for use, adds the CD28 recombinant protein then, and two days later, the collecting cell culture supernatant is measured cell factor IL-2 and IL-6 concentration.Negative control group is set simultaneously.
Step 9) solubility CD28 is to the influence of BMDC NF-κ B nuclear translocation
Collecting ripe BMDC and adjusting cell concentration is 1 * 10
7/ ml, packing 0.5ml adds CD28 fusion (R&D company, the U.S.) and cultivates 30min, 60min and 120min respectively in aseptic Eppendoff pipe; With PBS washing three times (1400rpm * 5min, down together), use 4% paraformaldehyde fixed cell 20min of precooling then then; After the PBS room temperature washing three times, 0.1% Triton handles 10min, after the PBS room temperature washing three times; Blocking buffer room temperature sealing 1h adds NF-κ B monoclonal antibody (2 μ g/ pipe) room temperature reaction 2h, after the PBS room temperature washing three times; The anti-mouse two anti-room temperatures of rabbit that add the cy5 mark continue reaction 1h, after the PBS washing, add 37 ℃ of nuclear staining agent PI (100 μ l/ pipe) and RNase enzymes (10 μ l/ pipe) and cultivate dyeing 30min; After the PBS room temperature washing three times, fluorescence mountant mounting is then through the Laser Scanning Confocal Microscope observations.Control group is human IgG antibody's a Fc section.In addition, collect the BMDC that LPS stimulates,, analyze the maturity of BMDC through the positive percentage of flow cytometer detection cell CD83, CD86 and CD80.
The step 10) statistical analysis
Expression that all data are all used
; The data Kolmogorov-Smirnov check; The nonparametric statistics analysis is looked the sample type difference and is adopted Mann-Whitney, Kruskal-Wallis and Wilcoxon ranking check respectively; The Pearson correlation analysis is adopted in correlation analysis; Part index number adopts sample average relatively with the t check, adopts the SPSS10.0 statistical software to carry out statistical study.
3. mensuration interpretation of result
1) solubility CD28 content significantly raises in the Graves patient blood
With the sandwich solubility CD28 detection method of two monoclonal antibodies of above-mentioned foundation; Analyze solubility CD28 concentration in the Graves patient blood; Mensuration result shows; Solubility CD28 content in the Graves patient blood (2.16 ± 1.15ng/ml) apparently higher than the normal healthy controls group (0.83 ± 1.35ng/ml) (P＜0.01), and find to have thyromegaly, solubility CD28 concentration surpasses 5ng/ml in the individual patients body of simultaneous phenomenon such as eyeball is outstanding.Results suggest, solubility CD28 obviously increases in the Graves patient body, possibly participate in the sick pathologic processes such as incidence and development of Graves.
2) solubility CD28 expresses with T cell activation state and is proportionate
Detect solubility CD28 content in the PHA activated T cells and supernatant apparently higher than control group (0.67 ± 0.15 vs, 0.34 ± 0.11ng/ml, P＜0.05), the generation of prompting solubility CD28 maybe be relevant with T cell activation state.Then further confirm through immunoblot experiment (Western blotting); On the nitrocellulose membrane that concentrated culture supernatant and patient's Graves blood plasma of activating T cell are printed and dyed, the solubility CD28 protein molecular band of specific stain occurs, and in static T cells and supernatant and healthy subjects control group serum, specific dyeing band do not occurred.This shows that the active state of the generation of solubility CD28 and T cell has certain correlativity.
3) the CD28 positive expression rate significantly reduces on patient's Graves periphery blood T cell
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.The result shows that the positive expression rate of costimulatory molecules CD28 significantly descends on patient's Graves periphery blood T cell, not only CD4
+The CD28 molecule is lost to some extent on the T cell subsets, and CD8
+The positive expression rate of CD28 molecule also significantly is lower than normal healthy controls group (being respectively 9.46 ± 8.58% and 17.55 ± 5.28%, P＜0.01) on the T cell subsets.Further discover Graves patient's peripheral blood CD4
+CD28
-T cell subsets quantity is significantly higher than the normal control group and (is respectively 10.2 ± 8.6% and 2.3 ± 1.9%; P＜0.01); And the positive percentage of CD3T cell also is starkly lower than normal healthy controls group (51.43 ± 7.54% and 69.37 ± 9.21% in patient's body; P＜0.05), cell count is also pointed out, and the relative number of T cell reduces in the blood samples of patients.But the unusual up-regulated expression ICOS molecule (11.2 ± 9.46%) of patient's periphery blood T cell, costimulatory molecules ICOS expresses (1.32 ± 0.6%) hardly in the normal healthy controls group.After giving patient's drug therapy; Analyze patient T cell phenotype once more and show, and compare before the treatment, the positive percentage of CD28 has obvious rise (to be respectively 26.83 ± 7.35% and 49.54 ± 7.81% in the patient's body after the drug therapy; P＜0.01), no matter is CD4
+T cell subsets, or CD8
+The CD28 molecule on T cell subsets surface all obviously presents restorative rise, point out the expression percent and the state of an illness of patient T cell surface CD28 molecule that confidential relation is arranged thus, the CD28 molecule possibly participate in should disease pathologic processes such as incidence and development.
4) solubility CD28 content is relevant with the sick Clinical detection index of Graves
The correlation analysis of the important clinical lab index of solubility CD28 concentration and medical diagnosis on disease shows in disease patient's body; Solubility CD28 level and FT3, FT4 and TRAb all are proportionate in patient's body; Related coefficient γ is respectively 0.663,0.624 and 0.728; Solubility CD28 concentration and serum TSH are remarkable negative correlation, and related coefficient γ is-0.726, and the concentration of prompting solubility CD28 is the sick important biomolecule mathematic(al) parameter of Graves.
5) solubility CD28 concentration and clinical sign are proportionate
In order further to understand the correlativity of the peripheral blood solubility CD28 and the state of an illness; Whether the patient exists by the degree of its thyromegaly and exophthalmos and divides into groups; Employing nonparametric Kruskal-Wallis check is compared results suggest, solubility CD28 and Graves patient's two big main physical signs in the peripheral blood between organizing; Thyroid enlargement degree (P＜0.05) and exophthalmos (P＜0.01) all have significant positive correlation, and the order of severity of prompting solubility CD28 and disease is obvious positive correlation.
6) solubility CD28 promotes BMDC secretion IL-6
ELI SA through to cell factor in solubility CD28 fusion and the interactional culture supernatant of BMDC detects confirmation; Solubility CD28 can obviously promote the secretion of IL-6, and (870.52 ± 73.61pg/mlvs 4.25 ± 2.83pg/ml); And compare the concentration there was no significant difference of IL-2 with the normal control group; But IL-6 content among the Graves patients serum is measured the back to be found; 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 the blood samples of patients; (11.54 ± 1.72pg/ml vs 15.36 ± 1.48pg/ml), prompting IL-6 possibly participate in the sick pathology pathogenic process of Graves ' to the then significantly minimizing of IL-2 content in patient's body.
7) solubility CD28 promotes the nuclear translocation of BMDC NF-κ B
In order to inquire into solubility CD28 the BMDC in monokaryon source is expressed the influence of molecules of interest; The employing Laser Scanning Confocal Microscope is observed; Solubility CD28 molecular action is behind the BMDC 30min in external evoked monokaryon source; NF-κ B has begun nuclear translocation, and most of NF-κ B molecule is transferred in the nucleus from endochylema behind the 60min, and nearly all NF-κ B has transferred to nucleus inside behind the 120min; The above results shows; After the molecules of interest that solubility CD28 molecule and BMDC are expressed interacted, through reverse signal mediation BMDC signaling molecule NF-κ B nuclear translocation, prompting solubility CD28 had the effect of regulating dendritic cell function.
4 conclusions
This assay method tentative confirmation; Solubility CD28 content increases unusually in the Graves patient peripheral blood; And losing with membranous type CD28 molecule on the T cell; Experiment in vitro confirms that solubility CD28 molecule can also promote NF-κ B molecular core transposition of antigen presenting cell BMDC and the secretion of cell factor IL-6, further inquires into the prioritization scheme that other solubility costimulatory moleculeses of associating are set up vitro detection; With the meaning in clinical diagnosis, treatment and the prognosis evaluation of autoimmune disease, will experimental basis and theoretical foundation be provided for seeking new immunologic intervention means.
The foregoing description just is to let the one of ordinary skilled in the art can understand content of the present invention and enforcement according to this in order technical conceive of the present invention and characteristics to be described, to be its objective is, can not limit protection scope of the present invention with this.The variation or the modification of every equivalence that the essence of content has been done according to the present invention all should be encompassed in protection scope of the present invention.
Claims (5)
1. method of measuring solubility CD28 content in the sick patient's blood of Graves is characterized in that concrete steps are:
Step 1) is prepared 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; The 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 blood preparation that many cases just send out the Graves patient as experimental group, prepare many parts of healthy human blood's samples as the normal control group;
Step 2) biotin labeling of monoclonal antibody
With carbonate buffer solution the concentration of the 2D5 monoclonal antibody of purifying is adjusted into 200 μ g/ml; Get its 1.0ml 4 ℃ of dialysed overnight in 50ml CBS; Move into the Eppendof pipe, add the biotin 40 μ l of 1mg/ml, lucifuge concussion 4h; 0.01mol/L 4 ℃ of dialysis 72h are subsequent use in-20 ℃ of preservations after the packing among the pH7.2PBS;
The evaluation of step 3) biotin labeling monoclonal antibody
With the CD28-T transgenic cell with after the PBS washing, with 5 * 10
5The dose of/pipe is loaded in the small test tube, and the dosage of managing with 20 μ l/ adds biotin labeled monoclonal antibody 2D5,4 ℃ of reaction 45min; Fully the washing back adds avidin-PE with the dosage of 10 μ l/ pipe; In 4 ℃ of reaction 45min, fully flow cytometry analysis is used in the washing back, and negative control is set simultaneously again;
The drafting of step 4) sCD28 standard working curve
Encapsulate through the pretreated enzyme joint inspection of 0.01% poly-D-lysine drafting board with 2F5mAb, 4 ℃ are spent the night, contain the PBS washing of 0.01%Tween-20 after; Spend the night with the 3%BSA sealing, the washing back adds the gradient dilution liquid of the standard items rhCD28/Fc recombinant protein of 0～16ng/ml, reaction 2h; Fully after the washing, add biotin labeling monoclonal antibody and HRP labelled streptavidin more respectively, 37 ℃ of reaction 1h; After the washing, add tmb substrate room temperature reaction 15min, measure A with said method
450, each gradient is done 3 multiple holes, is horizontal ordinate with the concentration of sCD28/Fc, A
450Value is ordinate, draws the standard working curve of sCD28;
The separation of T cell and purifying in the step 5) Graves patient blood
Extract the Graves patient or the fresh peripheral blood 10ml of healthy subjects of anticoagulant heparin, with the no Ca of pH7.2
2+, Mg
2+Hank ' s liquid gently is suspended from the lymphocyte separation medium after doing dilution in 1: 2, with the centrifugal 30min of the rotating speed of 1400rpm, abandons supernatant; Draw interface cloud confluent monolayer cells, add Hank ' the s liquid of 5 times of volumes, mixing, 2000rpm, centrifugal 10min abandons supernatant; Rotating speed with 1400rpm changes 10min, and repeated washing is once counted, and using the RPMI1640 complete medium adjustment cell concentration that contains 10%FCS is 3 * 10
6/ ml puts 6 hole plastic culture plates in 37 ℃, 5%CO
2Incubator is hatched 2h to remove adherent monocyte and B cell etc., collects the non-adherent cell suspension, through E garland combined techniques enrichment T cell, and through flow cytometry analysis, CD3
+The T cell reaches more than 90%, and liquid nitrogen cryopreservation is subsequent use;
The phenotype analytical of step 6) Graves patient T cell
The frozen patient of recovery and the purifying T cell of normal healthy controls group in the liquid nitrogen, wash 2 times with PBS after, with 5 * 10
5The dose of/pipe is loaded in the small test tube; The straight labeling antibody 10 μ l of PE and the mouse IgG-PE 10 μ l that add CD3, CD4, CD8, CD28 and ICOS molecule respectively are as negative control; In 4 ℃ of lucifuge reaction 45min, after the PBS washing that contains 5% calf serum, behind the adding 0.5ml PBS; With flow cytometry analysis T cell phenotype, image processing utilization EXPO is cytometer software analysis software v.2.
Solubility CD28 Determination on content in the step 7) Graves patient blood
Collect in T cells and supernatant, the Graves ' patient blood and control group normal health human plasma 0.5ml.Get the check-out console that coated antibody 2F5 has encapsulated in advance, add above-mentioned collection testing sample 100 μ l, 37 ℃ of water-bath 2h; Fully after the washing, add biotin labeled monoclonal antibody 2D5, again after 37 ℃ of water-bath 1h fully wash; Add streptavidin-HRP, after the reaction washing, add the substrate TMB of interim preparation; Behind the room temperature reaction 15min, use 2mol/L H
2SO
4Stop the reaction of enzyme-to-substrate, measure A in ELIASA
450, each sample is provided with three multiple holes, makes linear standard curve with rhCD28/Fc recombinant protein standard items simultaneously, to analyze solubility CD28 content in patient's Graves blood plasma;
The step 8) Western blot is analyzed solubility CD28 in the blood samples of patients
Collect the T cells and supernatant respectively, through PHA activated T cells and supernatant, healthy subjects 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, constant voltage is carried out SDS-PAGE electrophoresis 3h for 100 volts, after electrophoresis finishes; The 3M filter paper and the nitrocellulose membrane of clip and the identical size of gel piece, and correctly arrange filter paper, nitrocellulose membrane and gel piece as requested, constant current 100mA electricity changes nitrocellulose membrane 2h; With the PBS sealing nitrocellulose membrane that contains 5% skimmed milk power and 0.3% Tween-20, shaking table spends the night, and next day, TBS washing back added the mouse-anti people CD28 antibody 8G8 (10 μ g/ml) of dilution; Sealing 2h; TBS fully washs, and adds sheep anti-mouse igg two anti-(dilution in 1: 1000), incubated at room 2h; TBS fully washs the back and adds colour developing liquid BCIP colour developing, observes the specific proteins band with the chemiluminescence method of ECL detection system;
Step 9) solubility CD28 is to the influence of the cell factor of BMDC secretion
Aseptic extraction normal person anticoagulant heparin peripheral blood, conventional Ficoll separate and obtain mononuclearcell, wash 2 times after, adjust cell density to 3 * 10 with RPMI-1640
6/ ml adds in 6 well culture plates (2ml/ hole), cultivates 2h for 37 ℃, sucking-off suspension cell gently, and-80 ℃ are frozen subsequent use.In culture plate, add the RPMI-1640 that contains GM-CSF (100ng/ml), IL-4 (50ng/ml), 100IU/ml penicillin, 100 μ g/ml streptomysins then, 37 ℃ of cultivations, every 3d changes liquid once.6d cultivating selects for use lipopolysaccharides (LPS) to induce its maturation, adds the CD28 recombinant protein then, and two days later, the collecting cell culture supernatant is measured cell factor IL-2 and IL-6 concentration, and negative control group is set simultaneously;
Step 10) solubility CD28 is to the influence of BMDC NF-κ B nuclear translocation
Collecting ripe BMDC and adjusting cell concentration is 1 * 10
7/ ml, packing 0.5ml adds the CD28 fusion and cultivates 30min, 60min and 120min respectively in aseptic Eppendoff pipe, then with PBS washing three times; Use 4% paraformaldehyde fixed cell 20min of precooling then, after the PBS room temperature washing three times, 0.1% Triton handles 10min, after the PBS room temperature washing three times; Blocking buffer room temperature sealing 1h adds NF-κ B monoclonal antibody with the dosage of 2 μ g/ pipe, and room temperature reaction 2h is after the PBS room temperature washing three times; Add the anti-mouse two anti-room temperatures continuation reaction 1h of rabbit of cy5 mark, after the PBS washing, the dosage of managing with 100 μ l/ adds nuclear staining agent PI and adds the RNase enzyme with the dosage that 10 μ l/ manage; Cultivate dyeing 30min for 37 ℃, after the PBS room temperature washing three times, fluorescence mountant mounting; Through the Laser Scanning Confocal Microscope observations, control group is human IgG antibody's a Fc section, in addition then; Collect the BMDC that LPS stimulates,, analyze the maturity of BMDC through the positive percentage of flow cytometer detection cell CD83, CD86 and CD80;
The step 11) statistical analysis
Expression that all data are all used
; The data Kolmogorov-Smirnov check; The nonparametric statistics analysis is looked the sample type difference and is adopted Mann-Whitney, Kruskal-Wallis and Wilcoxon ranking check respectively; The Pearson correlation analysis is adopted in correlation analysis; Part index number adopts sample average relatively with the t check, adopts the SPSS10.0 statistical software to carry out statistical study.
2. the method for solubility CD28 content in the sick patient's blood of mensuration Graves according to claim 1; It is characterized in that; In the said step 1), in every liter of RPMI1640 or DMEM basal medium, add tire ox or calf serum 100ml, L-glutaminate 0.15g, NaHCO
32.0g, Sodium Pyruvate 0.11g, glucose 3.6g, HEPES 4.766g, concentration be 5 * 10
-3The 2 mercapto ethanol 10.0ml of mol/L.
3. the method for solubility CD28 content is characterized in that said step 2 in the sick patient's blood of mensuration according to claim 1 Graves) in the 72h of dialysis changed liquid in first day 4 times, later every 24h changes liquid 2～3 times.
4. the method for solubility CD28 content is characterized in that in the sick patient's blood of mensuration according to claim 1 Graves, said step 6) through the washing of the PBS that contains 5% calf serum for the centrifugal commentaries on classics of the rotating speed of 1400r/min 5min.
5. the method for solubility CD28 content is characterized in that the washing of PBS is with the centrifugal commentaries on classics of the speed of 1400r/min 5min in the said step 10) in the sick patient's blood of mensuration Graves according to claim 1.
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| CN113692286A (en) * | 2019-03-14 | 2021-11-23 | 比昂生物制剂公司 | A method for immunosuppression |
| CN113981031A (en) * | 2021-11-01 | 2022-01-28 | 山西中医药大学 | Novel T cell function detection method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105372318A (en) * | 2014-08-28 | 2016-03-02 | 山东省肿瘤医院 | Cell fixation method combining cell in-situ electrophoresis with flow cytometer |
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| CN113981031A (en) * | 2021-11-01 | 2022-01-28 | 山西中医药大学 | Novel T cell function detection method |
| CN114057897A (en) * | 2021-12-21 | 2022-02-18 | 中国海洋大学 | Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application |
| CN114057897B (en) * | 2021-12-21 | 2023-01-13 | 中国海洋大学 | Sebastes schlegeli IL-6 recombinant protein, soluble expression method and application |
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