CN103983785A - Novel specific cell immune response in-vitro detection method and its clinic application - Google Patents

Novel specific cell immune response in-vitro detection method and its clinic application Download PDF

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CN103983785A
CN103983785A CN201410122051.1A CN201410122051A CN103983785A CN 103983785 A CN103983785 A CN 103983785A CN 201410122051 A CN201410122051 A CN 201410122051A CN 103983785 A CN103983785 A CN 103983785A
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陈仁奋
魏秀妹
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    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]

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Abstract

The invention discloses a novel specific cell immune response in-vitro detection method and its clinic application. The method comprises the following steps: synthesizing and screening a Sm synthetic peptide antigen, carrying out mixed culturing on blood to be detected and the Sm synthetic peptide antigen, and detecting the level of specific cytokines in the supernatant of the above obtained cultured blood. The detection method can be applied in the clinic auxiliary diagnosis and treatment monitoring of systemic lupus erythematosus. The method has the many clinic application functions, has the characteristics of simplicity, rapidness, easy operation, flexible utilization and the like, and has a very good application prospect.

Description

Novel specificity cellular immunity response external detection method and clinical practice thereof
Technical field
The invention belongs to clinical immunity and medical experiment diagnostic field, be specifically related to a kind of novel cellullar immunologic response vitro detection technology.This experimental technique is based on detecting the cytokine levels being produced by Smith (Sm) antigenic synthetic peptide stimulated in vitro periphery blood T cell, for laboratory diagnosis and the treatment monitoring of systemic loupus erythematosus (SLE) provide reference.
Background technology
Systemic loupus erythematosus (SLE) be affect crowd the most extensively, one of the most serious autoimmunity disease, be conventionally apt to occur in women at reproduction age, can cause the inflammatory disorders of whole body Various Tissues and organ.Because this seizure of disease is hidden, course of disease delay increases the weight of repeatedly and constantly, and complicated clinical manifestation is various, and diagnosis and treatment difficulty are large, and the World Health Organization (WHO) classifies it as new century medical domain fort anxious to be captured as.
The cause of disease of this disease it be not immediately clear.Traditional viewpoint is the multiple autoantibody that B cellular abnormality activation that SLE patient's many organs inflammation infringement mainly causes owing to humoral immunity disorder produces, and deposit also the approach such as activating complement by antigenantibody complex and cause.Therefore, clinical diagnosis, the reference experiment index of state of an illness monitoring and treatment nearly all relies on autoantibodies in serum.And large quantity research in recent years shows, cell-mediated immune response and the cell factor of generation thereof, particularly struvite cell factor etc. may be another key reason (YAP that causes lupus erythematosus pathology inflammation extremely, D. Y. & LAI, K. N. Cytokines and their roles in the pathogenesis of systemic lupus erythematosus:from basics to recent advances. J Biomed Biotechnol, 2010, 365083.), and interact with humoral immunity, (DAVIS plays an important role in the generation of whole disease and evolution, L. S., HUTCHESON, J. & MOHAN, C. The role of cytokines in the pathogenesis and treatment of systemic lupus erythematosus. J Interferon Cytokine Res, 2011, 31, 781-9.).Iwata has reported that the pathogenesis (IWATA taking as the leading factor with the disorder of T cellular immunity even appears in some patients SLE recently, S., SAITO, K., TOKUNAGA, M. & TANAKA, Y. B cell or T cell-dominant recurrence after rituximab therapy in patients with SLE. Ann Rheum Dis. 2012.).Like this, the product (as cell factor) that detects cellullar immunologic response is the same with autoantibody, and to the diagnosis of SLE, monitoring is most important with treatment.
Weighing the level of cellullar immunologic response can weigh by detecting the cell factor being produced by immunogene stimulated in vitro whole blood leucocyte.Although cell factor can reflect the cell immune response of body sensitively, but because multiple physiology or pathological factor all can affect the release of cell factor, so cell factor itself is not had a disease specific (PICCOLI, L., MERONI, V., GENCO, F., TAMAROZZI, F., TINELLI, C., FILICE, C. & BRUNETTI, E. Serum cytokine profile by ELISA in patients withechinococcal cysts of the liver:a stage-specific approach to assess their biological activity. Clin Dev Immunol, 2012, 483935.).Up to now, not yet realize in the world detecting cell factor and carry out the diagnosis of autoimmune disease experimental technique of (comprising SLE).
Diagnosis and monitoring SLE are the criteria for classifications of the Americanism diseases caused by dampness association (ACR) based on comprehensive clinical manifestation and experimental check, it is larger that clinician is affected by subjective factor in the time judging 11 indexs of this standard regulation, diagnostic accuracy is poor, add both responsive and special experimental index of shortage, often occur mistaken diagnosis or fail to pinpoint a disease in diagnosis.And the treatment of SLE is relied on to all kinds of immunodepressant substantially, drug side-effect is large, often causes secondary infection.Although continue to bring out out novel medicine (cytokine antagonist) in recent years, clinician relies on personal experience to their selection more and makes subjective judgement, has blindness.And the current experimental technique (KALUNIAN that can instruct cell factor individualized treatment that still lacks in the world, K. & JOAN, T. M. New directions in the treatment of systemic lupus erythematosus. Curr Med Res Opin. 2009.; RONNBLOM, L. & ELKON, K. B. Cytokines as therapeutic targets in SLE. Nat Rev Rheumatol, 2010,6,339-347.).
Smith (Sm) antigen has been proved to be the most special antigen of systemic loupus erythematosus (SLE).Anti-Sm antibody except occurring that height is special in SLE patient body, also there is the Specific T cell immunity reaction (FRITSCH-STORK for Sm antigen, R., MULLEGGER, D., SKRINER, K., JAHN-SCHMID, B., SMOLEN, J. S. & STEINER, G. The spliceosomal autoantigen heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) is a major T cell autoantigen in patients with systemic lupus erythematosus. Arthritis Res Ther, 2006, 8, R118.).Compared with double-stranded DNA, Sm belongs to proteantigen, has advantages of and can directly stimulate T cell activation propagation the release cells factor.
Based on above background, the present invention is the high specific to SLE and can directly stimulate in vitro T cell activation to produce the hypersensitivity of a large amount of cell factors in conjunction with Sm antigen, develop a kind of experimental technique of novel cellullar immunologic response vitro detection, provide reference in order to auxiliary diagnosis SLE and for its treatment and monitoring.
Summary of the invention
The object of the invention is to set up a kind of novel cellullar immunologic response external detection method, and monitor for auxiliary diagnosis and the treatment of SLE.
Technical scheme of the present invention is as follows:
The present invention utilizes Sm antigen can stimulate specifically SLE patient T cell activation and produces the phenomenon of cytokine profiles, first filters out the Sm synthetic peptide that these can be identified by T cell-specific, and is mixed the antigen as stimulated in vitro complete blood cell.Memory t cell based in SLE patient's whole blood can identify fast and specifically Sm antigen and activation produces cell factor, and the present invention is controlled at 18-24 hour (37 DEG C) by blood to be checked and Sm hybrid peptide antigen Mixed culture time.Then detect the level of cultivating the specific cells factor in blood supernatant, to judge the cellullar immunologic response ability of body to specific antigen and patient's whole cell immunologic function situation.Background contrast (only adding physiological saline) and positive control (mitogen: phytohemagglutin phytolectin (PHA)) are set in experiment simultaneously.
Wherein, comprise five synthetic peptides for the Sm hybrid peptide antigen that stimulates peripheral blood, automatic or manual synthetic by FMOC solid phase synthesis process, to analyze and purifying through HPLC, antigen purity is more than 90%.Concrete amino acid sequence is as follows:
Sm1： RGNNIRYFILPDSLPLDTLL Sm2 ：SHETVTIELKNGTQVHGTIT
Sm3：GVDVSMNTHLKAVKMT Sm4：TVGKSSKMLQHIDYRMRCI
Sm5： PMGIPPGRGTPMGMPPPGM
The result that this experiment detects can have various clinical purposes: 1. and stimulate and produce the content of IL-6 through Sm hybrid peptide by measuring blood to be checked, judge whether patient exists high-caliber specificity cellular immunity response to the stimulation of Sm antigen, can laboratory diagnosis SLE; 2. the cell factor spectrum producing after Sm hybrid peptide stimulates by analyzing SLE patient, provides laboratory reference for patient while selecting cytokine antagonist to carry out individualized treatment; 3. the ability of the cell factor by observation SLE peripheral blood in patients, mitogenstimulated being discharged, both whole cell immunologic function situation that can evaluating patient, also can be used to judge the reliability (suppressing as whether patient exists stronger immunologic function) of this experimental result, to get rid of false negative result.
advantage of the present invention:
The present invention applies one group of Sm antigenic synthetic peptide to SLE high special originally stimulates the T cell in periphery whole blood, then by measuring the cytokine levels producing after T cell activation, set up in the world first a kind of external test method of the cellullar immunologic response for disease specific antigen, and be applied to the laboratory diagnosis of SLE, immunologic function is monitored and is instructed individualized treatment, has filled up the blank in this field.This novel cellular immune function detection means possesses various clinical application function, and easy fast, easily operation, the feature such as can apply in a flexible way.To complement one another with the humoral immunity detection method such as anti-Sm and anti-dsDNA antibody of current widespread use, and can more fully assess SLE patient's immune state, improved widely the test diagnostic level of SLE.The comprehensive cytokine profiles being produced by Sm antigenic synthetic peptide and mitogenstimulated of analyzing, the treatment that can be SLE provides the experimental basis of science, for reducing the spinoff for the treatment of, the subjectivity and the blindness that reduce in current lupus treatment play important effect, have good application prospect.
Brief description of the drawings
Fig. 1. vitro detection Sm hybrid peptide antigenic stimulus peripheral blood leucocyte produces the experimental technique of cell factor.
Fig. 2. the IL-6 level that systemic loupus erythematosus (SLE) patient produces after Sm antigenic stimulus with the peripheral blood contrasting.The positive dividing value of dotted line.Detected object comprises SLE patient's 41 examples, and disease contrast (Disease control) 61 examples (comprising rheumatoid arthritis 10 examples, Sjogren syndrome 7 examples, chorionitis 6 examples, mixed connective tissue disease 6 examples, Other diseases 32 examples), normal healthy controls (Healthy control) 42 examples.
Fig. 3. experimenter's performance curve (ROC curve) is analyzed diagnostic value of the present invention.A:SLE distinguishes the ROC curve of disease contrast (disease control); B:SLE distinguishes the ROC curve of normal healthy controls (healthy control); C:SLE distinguishes the ROC curve of all contrasts (controls).Area under AUC:ROC curve regions.
Fig. 4. the cell factor spectrum that different SLE patients (A, B, C) peripheral blood leucocyte is stimulated by Sm synthetic peptide hybrid antigen (Sm peptide cocktail) and produces compares.Nil and PHA(phytohemagglutin phytolectin) be self background and positive control.
Fig. 5. follow up a case by regular visits to SLE patient's peripheral blood leucocyte variation to PHA stimulation responses level in use immunodepressant (metacortandracin: prednisone) therapeutic process.The peripheral blood leucocyte that is illustrated as same patient is (day 0) before medication, use after the metacortandracin one week (day 7) of 20mg/d, continue to use the 30th day of 10mg/d metacortandracin, and use the various cytokine levels that produce that PHA stimulated in the 60th day of low dosage 5mg/ metacortandracin.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described, but the present invention is not limited only to this.
embodiment 1:vitro detection Sm hybrid peptide antigenic stimulus patient leucocyte produces cell factor
1. experiment material
(1) sterile blood culture tube: contain 30 μ l physiological saline (background contrast), (every kind of each 5 μ of Sm synthetic peptide g) and 10 μ g PHA positive controls for 30 μ g Sm hybrid peptide antigens;
(2) cytokines measurement: can freely select business-like kit.As need be detected cytokine profiles simultaneously, available the liquid chip technology (Luminex xMAP multiplex beads assay) that experiment adopts.Detect single cell factor (as IL-6) content, available ELISA method as only needed.
2. experimental procedure
(1) Sm synthetic peptide sequence and synthetic
For stimulating the Sm hybrid peptide antigen of peripheral blood to comprise five synthetic peptides, automatic or manual synthetic by FMOC solid phase synthesis process, to analyze and purifying through HPLC, antigen purity is more than 90%.Concrete amino acid sequence is as follows:
Sm1： RGNNIRYFILPDSLPLDTLL Sm2 ：SHETVTIELKNGTQVHGTIT
Sm3：GVDVSMNTHLKAVKMT Sm4：TVGKSSKMLQHIDYRMRCI
Sm5： PMGIPPGRGTPMGMPPPGM
(2) blood culture: as shown in Figure 1, each 1 ml of periphery heparin anti-coagulating adds three kinds to contain respectively 30 μ l physiological saline (blank pipe), 6 × 5 μ g Sm mix the sterile tube of synthetic peptide and 10 μ g mitogen phytohemagglutin phytolectins (PHA), put into 37 DEG C of incubation 20-24 hour (spending the night) after fully mixing.
(3) cytokines measurement: take out centrifugal (3000 turn 10 minutes) separated plasma in second day, then adopt liquid chip technology to detect cytokine profiles in blood plasma simultaneously or detect single cell factor (e.g. IL-6) content with ELISA.
3. result is calculated and is judged
Release of cytokines level=cytokine content (antigen or positive control)-cytokine content (self background contrast)
(1) analyzing blood to be checked stimulates the level of IL-6 producing to Sm hybrid peptide, in order to laboratory diagnosis SLE: if person to be checked is subject to Sm hybrid peptide to stimulate the IL-6 >=2000pg/ml producing, result is the SLE positive, otherwise negative.
(2) if positive control pipe produce IL-6 lower than 1000 pg/ml, show that patient is in moderate or hyperimmunization holddown (as used the immunodepressant of high dose), test findings is undecidable, must after Cellular Immunologic Function In Patients returns to certain level (IL-6 >=1000 pg/ml that positive control pipe produces), re-start detection again.
embodiment 2:the laboratory diagnosis of cellullar immunologic response detection method to SLE
Stimulate T cell to produce IL-6 level for diagnosing the method for SLE to carry out clinical verification to application Sm hybrid peptide, 41 routine SLE patients are detected respectively, 61 routine disease collators are (comprising rheumatoid arthritis 10 examples, Sjogren syndrome 7 examples, chorionitis 6 examples, mixed connective tissue disease 6 examples, Other diseases 32 examples) and normal healthy controls 41 examples.The results are shown in Figure 2.To produce IL-6 2000pg/ml as the positive dividing value (dotted line in Fig. 2) of diagnosis, show that this method is 73% to the diagnostic sensitivity of SLE, specificity is 92%.
Adopt experimenter's performance curve (receiver operating characteristic curve, be called for short ROC curve) assess the diagnostic value of this method, taking ROC curve lower zone area (AUC) as reference index, Fig. 3 shows that SLE is to disease contrast (disease control), normal healthy controls (healthy control), and comprehensive two contrast AUC be respectively 0.86,0.94 and 0.90, show that this method has higher diagnostic value to SLE.
embodiment 3:sLE patient stimulates the cell factor spectrum producing to have obvious individual difference through Sm hybrid peptide
Detect seven kinds of cell factors of cultivating in blood supernatant by liquid chip technology, result shows that the cell factor spectrum that different SLE patients' immunocyte produces after Sm hybrid peptide antigen (Sm peptide cocktail) stimulates exists obvious individual difference simultaneously.Taking 3 patients SLE shown in Fig. 4 as example: the struvite cell factor that patient SLE A produces is taking IL-6 and IL-1 β as main, patient B is mainly taking IL-6 and TNF-α as main, and patient C only takes as the leading factor with IL-6.This result has also reflected same cell factor simultaneously, and in different lupus patients' Inflammation development, role may be different.Therefore, analyze the cell factor spectrum of immunocyte generation in the special Sm antigenic stimulus experiment of this disease of SLE patient, clinician can suppress pointedly to prevailing cell factor wherein, in the time selecting cytokine antagonist treatment Inflammation, just there is like this scientific experiment foundation, thereby avoid blindly medication, optimize result for the treatment of.In addition, the cell factor type of detection can convert according to clinical needs, to provide guide for the individualized treatment of SLE.
This experiment shows to be subject to Sm hybrid peptide to stimulate the cell factor producing to compose by analyzing SLE patient periphery whole blood, can be laboratory reference is provided when patient carries out the individualized treatment of cytokine antagonist.
embodiment 4:the responsibility that peripheral blood leucocyte stimulates PHA reflects clinical immunosupress level
SLE patient is in use immunodepressant (as metacortandracin: prednisone) therapeutic process, and its peripheral blood leucocyte stimulates the cytokine levels producing to occur significantly fluctuating to PHA.Taking a SLE patient following up a case by regular visits in Fig. 5 as example, before medication (day 0), this patient's peripheral blood leucocyte is very high to PHA stimulation responses level, follow the use of heavy dose of metacortandracin (20mg/ days), after one week (day 7), it obviously declines to PHA stimulation responses level, again along with the continuation of relative high dose (10mg/ days) metacortandracin is used, at the 30th day, occur that significant cellular immune function suppresses, show as reactionless to PHA stimulation responses.Lower in time subsequently metacortandracin dosage to 5mg/ days, at the 60th day that follows up a case by regular visits to, PHA stimulation responses is started to react to some extent (IL-6 is bottom out first), show that cellular immune function starts to recover.Above result shows the increase using along with the immunodepressant for the treatment of lupus inflammation and continues, significantly suppressing appears in the normal lymphocyte function of body (should have strong immune response to the stimulation of PHA), illustrates that the chance that the spinoffs such as scabies secondary infection occur is increasing.And using after low dosage immunodepressant a period of time, normal lymphocyte function gos up gradually.Therefore clinician should be according to physical signs of patient, and immune response ability to mitogenstimulated, and the security of follow-up immunization suppression therapy is assessed, and the decision of the dosage of making adjustment in time, to reduce drug side-effect, improves curative effect.
This description of test is analyzed the immune functional state that cytokine levels that SLE patient periphery whole blood produces mitogenstimulated can monitor patients.
SEQUENCE LISTING
<110> Chen Ren puts forth energy
Specificity cellular immunity response external detection method and clinical practice thereof that <120> is novel
<130> 2014
<160> 5
<170> PatentIn version 3.3
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<211> 20
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<213> artificial sequence
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Arg Gly Asn Asn Ile Arg Tyr Phe Ile Leu Pro Asp Ser Leu Pro Leu
1 5 10 15
Asp Thr Leu Leu
20
<210> 2
<211> 20
<212> PRT
<213> artificial sequence
<400> 2
Ser His Glu Thr Val Thr Ile Glu Leu Lys Asn Gly Thr Gln Val His
1 5 10 15
Gly Thr Ile Thr
20
<210> 3
<211> 16
<212> PRT
<213> artificial sequence
<400> 3
Gly Val Asp Val Ser Met Asn Thr His Leu Lys Ala Val Lys Met Thr
1 5 10 15
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<213> artificial sequence
<400> 4
Thr Val Gly Lys Ser Ser Lys Met Leu Gln His Ile Asp Tyr Arg Met
1 5 10 15
Arg Cys Ile
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<211> 19
<212> PRT
<213> artificial sequence
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Pro Met Gly Ile Pro Pro Gly Arg Gly Thr Pro Met Gly Met Pro Pro
1 5 10 15
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Claims (4)

1. a novel cellullar immunologic response external detection method, is characterized in that comprising the following steps:
(1) Smith (Sm) synthetic peptide that screening mixing energy are identified by T cell-specific as the antigen that stimulates peripheral blood leucocyte to be checked, sets up mitogen PHA to make positive control simultaneously;
(2) blood to be checked is mixed with antigen, cultivate 20-24 hour for 37 DEG C;
(3) detect the level of cultivating the specific cells factor in blood supernatant, to judge the cellullar immunologic response level of body to specific antigen Sm hybrid peptide and the former mitogen PHA of nospecific immunity.
2. cellullar immunologic response external detection method as claimed in claim 1, is characterized in that: described Sm hybrid peptide antigen comprises five synthetic peptides, is respectively Sm1, Sm2, Sm3, Sm4, Sm5, and concrete amino acid sequence is as follows:
Sm1： RGNNIRYFILPDSLPLDTLL；
Sm2 ：SHETVTIELKNGTQVHGTIT；
Sm3：GVDVSMNTHLKAVKMT；
Sm4：TVGKSSKMLQHIDYRMRCI；
Sm5： PMGIPPGRGTPMGMPPPGM。
3. the cellullar immunologic response external detection method as described in claim 1,2, is characterized in that: described Sm antigenic synthetic peptide is automatic or manual synthetic by FMOC solid phase synthesis process, analyzes and purifying through HPLC, and antigen purity is more than 90%.
4. a clinical practice for the cellullar immunologic response external detection method as described in claim 1,2,3, is characterized in that comprising:
(1) stimulate by measuring blood to be checked the content that produces IL-6 through Sm hybrid peptide, judge whether patient exists high-caliber cellullar immunologic response to the stimulation of Sm hybrid peptide antigen, with laboratory diagnosis SLE;
(2) the cell factor spectrum producing after Sm hybrid peptide antigen-specific stimulation by analyzing SLE patient, provides laboratory reference for patient selection cytokine antagonist carries out individualized treatment;
(3), by observing the ability of the cell factor that SLE peripheral blood in patients discharges mitogenstimulated, whole cell immune functional state that both can evaluating patient, also can be used to judge the reliability of this experimental result, to get rid of false negative result.
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