CN104422761A - Application of fibroblast-like synoviocytes in preparation of rheumatoid arthritis (RA) diagnosis reagent - Google Patents
Application of fibroblast-like synoviocytes in preparation of rheumatoid arthritis (RA) diagnosis reagent Download PDFInfo
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Abstract
The invention provides the application of fibroblast-like synoviocytes in preparation of a rheumatoid arthritis (RA) diagnosis reagent such as a kit or a cell chip. The application proves that a specific antibody which can be combined with the fibroblast-like synoviocytes exists in the serum of a RA patient. An indirect immunofluorescence method taking the fibroblast-like synoviocytes as a substrate antigen and a cell chip detection method are established, the application of the fibroblast-like synoviocytes in preparation of the RA diagnosis reagent has higher sensitivity and specificity for RA diagnosis, and a new diagnosis reagent and a new diagnosis method are provided for the RA diagnosis.
Description
Technical field
The present invention relates to Diagnosis of Rheumatoid Arthritis reagent technique, specifically, be fibroblast-like synoviocyte preparing the application in Diagnosis of Rheumatoid Arthritis reagent, belong to medical domain.
Background technology
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of systemic autoimmune disease, mainly involves periarticular, shows as chronic, symmetry, Progressive symmetric erythrokeratodermia arthritis.The pathological change of RA shows as synovium of joint hyperplasia at first, and gradually to cartilage, bone and tendon infringement around, finally causes destruction of joint, joint deformity and afunction.RA is mainly in female middle-aged, and morbidity rate is 0.32% ~ 0.36% in China, in American-European countries then up to 1%.The cause of disease of RA it be not immediately clear, mainly change relevant with infective agent, inherent cause and hormonal readiness, its clinical manifestation, mainly based on joint symptoms, shows as deadlock in morning, arthralgia, arthroncus and joint deformity.Extra-articular manifestation mainly comprises the damage of rheumatoid nodules, rheumatoid vasculitis and lung, kidney damages and heart damage.The diagnosis Main Basis Americanism diseases caused by dampness association RA criteria for classification in 1987 of RA: refuse to budge (1) morning continuous 1 hour, the course of disease at least 6 week; (2) swollen joint of more than 3 or 3 is had, at least 6 weeks; (3) wrist, the palm refer to, proximal joint swell, at least 6 week; (4) symmetry swollen joint, at least 6 weeks; (5) subcutaneous nodule is had; (6) hand x-ray changes; (7) serum rheumatoid factor (SRF) (RF) raises; Meet more than 4 or 4 and get rid of other arthritis and can be diagnosed as RA.The Testing index that with the addition of cyclic citrullinated peptid (antiCCP antibody) in up-to-date Americanism diseases caused by dampness association RA criteria for classification and diagnose as RA for 2012.From above-mentioned RA diagnostic criteria, the diagnosis of this disease mainly relies on the inspection of clinical manifestation binding radioactivity and laboratory parameters.In the middle of the autoantibody that known RA is relevant, rheumatoid factor susceptibility is 60% ~ 70%, and specificity is 86%; Antikeratin antibody (AKA) susceptibility is 44% ~ 73%, and specificity is 90%; Antiperinuclear factor antibody (APF) susceptibility is 48% ~ 66%, and specificity is 92%; Antifilaggrin antibody (AFA) susceptibility is 47% ~ 69%, and specificity is 93%; AntiCCP antibody susceptibility is 47% ~ 82%, and specificity is 96%.Although these RA laboratory parameters have reached quite high Sensitivity and Specificity, but according to statistics, in the middle of RA, still have about 20% patient to be that RF and CCP is double-negative, so, seek the higher RA Testing index of Sensitivity and Specificity and method, be still the emphasis problem of this Disease Clinical research.
Above-mentioned autoantibody detect be all with specific protein or polypeptide for antigen substrate, detect the antibody for this albumen or polypeptide in serum by the principle design of antigen-antibody reaction.The source of these antigen substrates is mainly through separating-purifying, recombinant expressed or iii vitro chemical synthesis (polypeptide) acquisition, in the antigen substrate obtained by said method and body, there is some difference on physicochemical property, biologically active or space conformation for naturally occurring antigen, applies the antigen substrate obtained like this and carry out detection to its serum antibody and inevitably can cause error.For avoiding the problems referred to above, researcher adopts IIF method to carry out etiologic diagnosis to disease.The method is that substrate antigen is fixed on solid phase carrier by certain specific cells, as microslide, by test serum and its reaction, be combined by the antibody of anti-human igg two anti-energy and the cell effect in serum of band fluorescence again, thus judge in serum how many containing this cell antibody anti-in fluorescence microscopy Microscopic observation fluorescence intensity, compare with standard positive serum or negative serum, thus judge the yin and yang attribute of test serum.Because the method is easy and simple to handle, judges directly perceived, be used widely clinically.
Anticellular antibody is applied clinically more has following detection: it is take Human umbilical vein endothelial cells as substrate antigen that (1) AECA detects, for the auxiliary diagnosis of leukoplakia (BD); Its susceptibility in BD is 54.8%, and specificity is 87.2%; And be respectively 8%, 9% and 10% in systemic loupus erythematosus (SLE), systemic sclerosis (SSc) and the positives rate of RA; (2) anti-megacaryocyte antibody test be with people's megacaryocyte for substrate antigen, be mainly used in the auxiliary diagnosis of SLE; (3) anti-human collecting duct antibody test is mainly used in the auxiliary diagnosis of kidney trouble; (4) anti-parietal cell anti-body detects the auxiliary diagnosis being mainly used in pernicious anaemia.
Protein chip has the advantages such as high flux, Parallel testing, robotization, for solution that is easy, that detect multiple autoantibody problem brings hope fast, simultaneously, has played important effect in the fields such as clinical disease diagnosis.The sample that protein chip is previously put in preparation process is all specific antigen (carrying out detection specificity antibody with it), and its source great majority are for form by gene engineering method is recombinant expressed.Recombinant expressed proteantigen, no matter be protokaryon or eukaryotic expression, how many its protein products there are differences in physicochemical property, biologic activity or immunological properties and between naturally occurring albumen, can cause like this producing error with when this Protein Detection human body specific antibody.Directly with the cell of original cuiture as substrate, albumen and cells in vivo due to cells compare and differ minimum, having more advantage for when detecting this cell protein antibody than recombinant expressed albumen.There is no in prior art and adopt the fibroblast-like synoviocyte point sample of some to detect corresponding antibodies content in serum thus RA to be carried out to the report of laboratory diagnosis.
RA pathogenesis is still not clear so far, may participate in the pathogenetic cell of RA disease according to research reports and reach more than ten kinds.Wherein, what gain public acceptance at present is CD4+T cell, and especially Th17 cell is considered to play a major role in RA morbidity.And other effector cells, as fibroblast-like synoviocyte, cartilage cell, mast cell, macrophage etc. also play the part of different role in RA pathogenesis.But with regard to RA, in prior art, which kind of cell protein unexposed can reach the object of auxiliary diagnosis as antigen substrate by detecting antibody in patients serum, and a lot of cell participates in the generation of RA disease not by antibody exerts effect.The kind of known human inner cell is countless, in RA, how to find a kind of cell, carries out autoantibody detect and have to set up the difficult point that high susceptibility and specific aided diagnosis method are still this area for its protein substrate.
Normal synovial backing layer cell comprises macrophage-like synovial cell, fibroblast-like synoviocyte (fibroblast-like synovial cells is called for short FLS) and dendron shape synovial cell.In RA pathogenesis, unanimously think fibroblast-like synoviocyte hyperplasia and be the basic reason causing joint pathology to change to the destruction of cartilage, it is the effector cell participating in the reaction of RA synovial membrane inflammation, but have no report with regard to the application of fibroblast-like synoviocyte in preparation RA diagnostic reagent, to those skilled in the art, fibroblast-like synoviocyte can be used for diagnosing RA to be also non-obvious.
Summary of the invention
The object of the invention is for deficiency of the prior art, provide fibroblast-like synoviocyte preparing the application in Diagnosis of Rheumatoid Arthritis reagent.
Of the present invention again one object be provide a kind of fibroblast-like synoviocyte preparing the application in Diagnosis of Rheumatoid Arthritis kit.
Another object of the present invention provides a kind of fibroblast-like synoviocyte preparing the application in Diagnosis of Rheumatoid Arthritis cell chip.
For achieving the above object, the technical scheme that the present invention takes is:
Fibroblast-like synoviocyte is preparing the application in Diagnosis of Rheumatoid Arthritis reagent.
Described fibroblast-like synoviocyte is the human desmocyte sample synovial cell of in vitro culture.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
Fibroblast-like synoviocyte is preparing the application in Diagnosis of Rheumatoid Arthritis kit.
Described diagnostic kit comprises the human desmocyte sample synovial cell of in vitro culture and indirect immunofluorescene assay method other reagent used.
The step of described indirect immunofluorescene assay method is:
(1) the human desmocyte sample synovial cell of in vitro culture is fixed;
(2) close with the PBS confining liquid containing BSA;
(3) get test serum and hatch fixing fibroblast-like synoviocyte;
(4) goat anti-human igg two of Cy3 mark is anti-hatches;
(5) DAPI solution dye core;
(6) fluorescence microscope, the fibroblast-like synoviocyte endochylema uniform coloring of rheumatoid arthritis patients sera incubation, the fibroblast-like synoviocyte endochylema that other patients and normal human serum are hatched is all not painted.
Described fluorescence microscope condition is: use chromium optical filter, excitation wavelength 554nm, emission wavelength 570nm.
For realizing above-mentioned 3rd object, the technical scheme that the present invention takes is:
Fibroblast-like synoviocyte is preparing the application in Diagnosis of Rheumatoid Arthritis cell chip.
The human desmocyte sample synovial cell of in vitro culture is fixed with in described cell chip.
The preparation method of described cell chip is:
(1) apply point sample instrument Software for Design matrix, determine dot spacing, point sample order, point sample coordinate, each sample arranges 3 multiple holes;
(2) selecting pretreated slide as substrate, is 1 × 10 by concentration
3the fibroblast-like synoviocyte suspension application point sample instrument of/mL is added in substrate, dried overnight;
(3) be placed in wet box by the substrate after point sample, 37 DEG C of waters bath with thermostatic control fix 2 hours;
(4) close and wash substrate, being placed in 4 DEG C and saving backup.
The using method of described cell chip is: get test serum and add the reaction of cell chip matrix, through washing, then anti-human igg two anti-reflective marked with Cy3 should, again after washing, scanning of fluorescent intensity is carried out with laser confocal scanner, finally analyze anti-fibroblast-like synoviocyte antibody concentration in test serum according to typical curve Quant Anlysis, then compare with cutoff value, judge whether test serum is positive.
The invention has the advantages that:
The invention provides fibroblast-like synoviocyte and prepare Diagnosis of Rheumatoid Arthritis reagent as the application in kit, cell chip; Confirm in RA patients serum the antibody that there is energy specificity and be combined with fibroblast-like synoviocyte; Establishing with fibroblast-like synoviocyte is indirect immunofluorescence method and the cell chip detection method of substrate antigen, possesses the feature of high flux, automation mechanized operation, have higher Sensitivity and Specificity to the diagnosis of RA, the diagnosis for RA provides new diagnostic reagent and method.
Accompanying drawing explanation
Accompanying drawing 1 is the fibroblast-like synoviocyte identifying original cuiture with specific marker CD55, and figure A is CD55 dyeing, shows as cytoplasm painted; Figure B is DAPI dyeing showed cell core; Figure C is the picture after A+B superposition, simultaneously showed cell core and CD55 colour developing.
Accompanying drawing 2 is that different patient and normal human serum and fibroblast-like synoviocyte albumen Western-Blot scheme, and is RA patients serum (RA1-RA8), SLE patients serum (SLE1-SLE2), OA patients serum (OA1-OA2) and normal human serum (NC1-NC4) respectively.
Accompanying drawing 3 is RA patients, SLE patient, OA patient and normal human serum hatch fibroblast-like synoviocyte indirect immunofluorescence figure.
Accompanying drawing 4 is the linear relationships between human IgG standard concentration and fluorescence intensity.
Accompanying drawing 5 is the cartilage cells identifying original cuiture with specific marker CII, and figure A is CII dyeing, shows as cell cytosol painted; Figure B is DAPI dyeing showed cell core; Figure C is the picture after A+B superposition, simultaneously showed cell core and CII colour developing.
Accompanying drawing 6 is that the Western-Blot of RA patient and normal human serum and chondrocyte proteins schemes, and A figure represents RA patients serum, and B figure represents normal human serum.
Accompanying drawing 7 is cartilage cell's endochylema coloring case that RA patients serum and normal human serum are hatched, and A figure represents RA patients serum, and B figure represents normal human serum.
Accompanying drawing 8 is that the Western-Blot of RA patient and normal human serum and Th17 cell protein schemes, and A figure represents RA patients serum, and B figure represents normal human serum.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
embodiment 1
One, the separation qualification of human desmocyte sample synovial cell and original cuiture
1, synovial origin
The RA patient of knee prosthesis or villusectomy taken from by synovial membrane, and patient diagnosis meets Americanism diseases caused by dampness association criteria for classification in 1987.
2, synovial cell is separated cultivation
The synovial tissue obtained in art is shredded with aseptic, at 37 DEG C, digests 4h, centrifugal collecting cell with 1.0 mg/mL NTx enzymes (GIBCO), add the DMEM nutrient solution (GIBCO) containing 20% hyclone, be placed in 37 DEG C, 5%CO
2adherent growth in incubator.Time bottom Growth of Cells to 80% culture flask, digest with 0.25% pancreatin (Sigma), calf serum stops, and 0.01M PBS washs centrifugal, collecting cell, Secondary Culture.After going down to posterity, synovial cell breeds rapidly, after 1 generation, fibroblast-like synoviocyte ratio increases gradually, visual field inner cell more than 95% (Zhang Peng can be accounted for the 3rd generation, Shiguan paulownia. knee osteoarthritis synovial cell in vitro culture and Observation of biological characteristics. Chinese bone fracture, 2006,19(11): 656-658).This experiment all adopt the 3rd generation fibroblast-like synoviocyte.
3, the qualification of fibroblast-like synoviocyte
Double dish sucks supernatant, fixes 10 minutes with methyl alcohol at-20 DEG C.Wash 3 times with 0.01M PBS, sheep blood serum closes 30 minutes, and 0.01M PBS washs 3 times, each 5 minutes.With fibroblast-like synoviocyte specific marker CD55(M D Smith, E Barg, H Weedon. Microarchitecture and protective mechanisms in synovial tissue from clinically and arthroscopically normal knee joints. Ann Rheum Dis 2003,62:303-307) monoclonal antibody (Abcam) dilutes rear incubated at room 1 hour according to 1:1000,0.01M PBS washs 3 times, each 5 minutes.Anti-according to 1:2000 dilution with the anti-mouse IgG bis-of Cy3 mark, incubated at room 30 minutes, 0.01M PBS washs 3 times, each 5 minutes.Finally with the DAPI transfect cell core of 1:10000 dilution, add anti-fluorescence quenching mounting after having washed, at fluorescence microscopy Microscopic observation.The results are shown in Figure 1, result confirms that the cell obtained after synovial membrane separation cultivation is fibroblast-like synoviocyte.
Two, there is anti-fibroblast-like synoviocyte antibody in confirmer's serum
1, fibroblast-like synoviocyte protein extraction
(1) outwell nutrient solution, culture flask slant setting 1 ~ 2 minute, residual culture transfer pipet siphons away.
(2) every bottle of cell adds the 0.01M PBS of 3mL 4 DEG C of precoolings.Keep flat and shake 1 minute gently with washed cell, then discard washing lotion.Wash cell altogether three times, PBS is abandoned only and culture flask is placed on ice.
(3) add 200uL Pierce lysate (adding protease inhibitors PMSF in advance) in every bottle of cell, leave standstill 20 minutes; 10000g, at 4 DEG C centrifugal 20 minutes, retain supernatant.After supernatant after centrifugal is measured concentration, packing, is put in-80 DEG C of preservations.
2, loading and electrophoresis
(1) in fibroblast-like synoviocyte protein sample, add 2 × SDS-PAGE albumen sample-loading buffer, mix rear 100 DEG C of boiling water baths and heat 35 minutes, make the abundant sex change of albumen.
(2) after the albumen cool to room temperature after sex change, protein sample is directly loaded in SDS-PAGE glue well, use the electrophoretic apparatus of Bio-Rad, constant voltage arranges 100V, and bromophenol blue arrives the bottom end neighbouring stopping electrophoresis of glue.
3, transferring film
Adopt pvdf membrane, pvdf membrane is dipped in respectively methyl alcohol 15 seconds, pure water 3 minutes, transfering buffering liquid pre-service in 15 minutes; Soak was in electrophoretic buffer 5 minutes; At the standard wet type membrane-transferring device of Bio-Rad, setting transferring film electric current is 300mA, and the transferring film time is 60 minutes.After having turned film, with Ponceaux dyeing liquor, film is dyeed, to observe actual transferring film effect, every part of protein sample is cut off, so that hatch different serum by its display band simultaneously.
4, close
After transferring film, with cleansing solution, film is washed 3 times, each 3 minutes; After having washed, film is added in the confining liquid that skimmed milk power is made into, close for 4 DEG C and spend the night.
5, sera incubation
RA patient, osteoarthritis (osteoarthritis, be called for short OA) patient, systemic loupus erythematosus (systemic lupus erythematosus, being called for short SLE) serum of patient and normal person (NC) is according to the pvdf membrane joined after 1:200 concentration dilution after wet turning, room temperature, on horizontal shaker, slight concussion hatches 1 hour.After hatching end, wash 6 times with cleansing solution, each 10 minutes, to alleviate background.
6, two anti-to hatch
The anti-human igg two adopting horseradish peroxidase (HRP) to mark resists, and after 1:10000 concentration dilution, room temperature, on horizontal shaker, slight concussion hatches 30 minutes.After hatching end, wash 6 times with cleansing solution, each 10 minutes, to alleviate background.
7, chemiluminescence and X film developing
Expose in magazine after pvdf membrane adds chemical luminous substrate (Thermo), carry out craft at the developer solution prepared voluntarily and stop bath and develop a film.The results are shown in Figure 2, the pvdf membrane of hatching with RA patients serum can show many and react band, and with the pvdf membrane of SLE, OA and normal person (NC) sera incubation on can not show respective strap under the same conditions, this result illustrates in RA patients serum that exist can the antibody of specificity and fibroblast-like synoviocyte albumino reaction.
Three, foundation---the indirect immunofluorescence of anti-fibroblast-like synoviocyte antibody detection method
1, fibroblast-like synoviocyte is fixing
By obtain the 3rd generation fibroblast-like synoviocyte after trypsinization, obtained single cell suspension, is laid on 96 orifice plates, every hole 100uL, 37 DEG C of overnight incubation, suck nutrient solution, after PBS washs 3 times, every hole adds 95% ethanol (or methyl alcohol, 5% glacial acetic acid) 200uL, fixes 20 minutes for 4 DEG C.After cell after fixing removes immobile liquid again after PBS washs 3 times, pat dry, be placed in-20 DEG C and save backup.
2, close
Every hole, with containing the 0.01M PBS confining liquid 100uL of 3% BSA at room temperature, closes 1 hour.After completing, discard confining liquid, cleansing solution rinsing 3 times, each 5 minutes.
3, sera incubation
Get RA patient, SLE patient, OA patient and normal human serum respectively doubly to dilute according to 1:100, in 96 orifice plates securing fibroblast-like synoviocyte, every hole adds serum 100uL after above-mentioned dilution, incubated at room 1 hour.After having hatched, suck serum, pat dry, cleansing solution washs 6 times, each 3 minutes.
4, the goat anti-human igg two of Cy3 mark is anti-hatches
Every hole adds 100uL, the goat anti-human igg of the Cy3 mark of 1:1000 dilute concentration, incubated at room 30 minutes.After completing, discard confining liquid, cleansing solution rinsing 6 times, each 5 minutes.
5, core is contaminated
The DAPI solution 100uL that 1:10000 dilutes is added in each hole, normal-temperature reaction 30 minutes.Reaction terminates rear cleansing solution rinsing 3 times, each 5 minutes.Add a cover fluorescence microscopy Microscopic observation after cover glass mounting.
6, fluorescence microscope
Use chromium optical filter, under excitation wavelength 554nm, emission wavelength 570nm condition, observe Cy3 fluorescence intensity.The results are shown in Figure 3, the fibroblast-like synoviocyte endochylema uniform coloring (redness) of hatching with RA patients serum, and the fibroblast-like synoviocyte endochylema of hatching with SLE patient, OA patient and normal human serum is all not painted.
Four, the foundation of anti-fibroblast-like synoviocyte antibody cell chip detecting method
1, the preparation of cell chip
(1) matrix design:
Application point sample instrument ProSys 5510 Software for Design matrix, determine dot spacing, point sample order, the indexs such as point sample coordinate, each sample arranges 3 multiple holes.
(2) point sample:
Selecting pretreated slide as substrate, is 1 × 10 by concentration
3the fibroblast-like synoviocyte suspension application point sample instrument of/mL is added in substrate, dried overnight.
(3) fixing:
Be placed into by substrate after point sample in wet box, 37 DEG C of waters bath with thermostatic control fix 2 hours.
(4) close:
Adopt 1%BSA solution, every matrix adds 50uL, is placed in wet box, and half an hour is closed in 37 DEG C of waters bath with thermostatic control; 0.1%TBST washs 3 times, each 15 minutes; The substrate closed after washing is placed in 4 DEG C and saves backup.
2, the foundation of typical curve
(1) standard items point sample: join matrix relevant position according to the purifying human IgG of 50ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL five concentration gradients; 37 DEG C are reacted 30 minutes;
(2) wash: PBST washs 3 times, each 10 minutes;
(3) fluorescence two resists: select concentration to be that the anti-human igg two that 0.2ng/mL Cy3 marks resists, 37 DEG C are reacted 30 minutes;
(4) wash: PBST washs 3 times, each 10 minutes;
(5) scanner uni drawing standard curve: application American I nnopsys company Innoscan 710 laser confocal scanner scans, the result obtained after scanning is analyzed each standard items fluorescence intensity through Quant Anlysis, input Excel software, carry out linear regression analysis, obtain typical curve, the results are shown in Figure 4, linear equation is y=1.692x+11.804, R
2=0.9925.
3, the anti-fibroblast-like synoviocyte antibody concentration of test serum detects
(1) test serum is with after 1:200 dilution, and every part of serum 50uL adds matrix, and 37 DEG C are reacted 30 minutes;
(2) wash: PBST washs 3 times, each 10 minutes;
(3) fluorescence two resists: select concentration to be that the anti-human igg two that 0.2ng/mL Cy3 marks resists, 37 DEG C are reacted 30 minutes;
(4) wash: PBST washs 3 times, each 10 minutes;
(5) scanner uni calculates sample to be tested antibody content according to typical curve: application American I nnopsys company Innoscan 710 laser confocal scanner scans, the result obtained after scanning is analyzed each test serum sample fluorescence intensity through Quant Anlysis, calculates the content of antibody in test serum sample according to typical curve.24 routine RA patients (RA1-RA24) and 24 routine normal person (NC1-NC24) serum fluorescence intensity testing results are in table 1, the typical curve formula obtained according to Fig. 4 calculates, quantitatively obtain the anti-fibroblast-like synoviocyte antibody concentration of test serum, the results are shown in Table 2.
Table 1 RA patient and normal human serum fluorescence intensity
Anti-fibroblast-like synoviocyte antibody concentration in table 2 serum sample
Five, the value assessment of anti-fibroblast-like synoviocyte antibody test in RA diagnosis
1, apply indirect immunofluorescene assay anti-fibroblast-like synoviocyte antibody and etiologic diagnosis is carried out to RA
(1) ultimate principle: adopt the method that positive serum and negative serum and test serum are hatched jointly, test serum fluorescence intensity and the two compare, what be more than or equal to positive serum is the positive, and what be less than or equal to negative serum is feminine gender.
(2) the defining of positive serum and negative serum: in 96 orifice plates fixing fibroblast-like synoviocyte, apply 48 clear and definite RA patient's (RA1-RA48) serum of diagnosis and 48 normal person (NC1-NC48) serum, according to indirect immunofluorescence, react with the fibroblast-like synoviocyte be fixed in 96 orifice plates, result is at fluorescence microscopy Microscopic observation, and the fluorescence intensity results that each hole obtains is as shown in table 3.Serum fluorescence intensity cutoff value is determined by receiver operating curves (ROC), as shown in table 4, on this basis, fluorescence intensity a little higher than cutoff value serum is selected to be positive serum, i.e. No. 21 RA serum (fluorescence intensity 4.9), namely test serum fluorescence intensity is then positive higher than this serum fluorescence intensity; Select fluorescence intensity to be slightly negative serum lower than cutoff value serum, i.e. No. 14 NC serum (fluorescence intensity 4.88), namely test serum fluorescence intensity is then feminine gender lower than this serum fluorescence intensity.
Table 3 RA patient and normal human serum fluorescence intensity
Table 4 indirect immunofluorescene assay fluorescence intensity ROC tracing analysis
(3) quilitative method RA diagnosis in susceptibility and specificity:
According to the result of table 4, by the difference of fluorescence intensity, susceptibility is 79.17%, specificity is 97.92%, its Sensitivity and Specificity is all respectively 70% and about 86% higher than RF(bibliographical information RF susceptibility, specificity), and specificity is about 96% higher than CCP(bibliographical information CCP), susceptibility and CCP are quite (bibliographical information CCP is about 80%).
2, the anti-fibroblast-like synoviocyte antibody of application cell chip detection carries out quantitative Diagnosis to RA
(1) ultimate principle: according to aforementioned cells chip detecting method, the typical curve obtained by anti-human igg standard items, test serum fluorescence intensity, obtained antibody concentration in serum through calculating, thus realize the quantitative detection of antibody concentration in test serum.Simultaneously through ROC tracing analysis, obtain cutoff value, thus realize the quantitative Diagnosis to RA, namely test serum antibody concentration is considered as the positive higher than cutoff value, is considered as feminine gender lower than cutoff value.
(2) the positive setting judging Cutoff value: analyzed by the result application ROC curve of table 2, is set as positive cutoff value by antibody concentration time maximum for acquisition susceptibility %+ specificity % sum, the results are shown in Table 5.
Table 5 cell chip detects antibody concentration ROC tracing analysis
(3) quantivative approach RA diagnosis in susceptibility and specificity:
According to the result of table 5, anti-fibroblast-like synoviocyte antibody test susceptibility is 87.5%, specificity is 95.83%, its Sensitivity and Specificity all higher than RF and CCP(bibliographical information RF Sensitivity Specificity be respectively 70% and about 86%, CCP susceptibility, specificity be respectively 80% and about 95%).
In sum, fibroblast-like synoviocyte as the diagnostic reagent of rheumatoid arthritis, can have higher Sensitivity and Specificity in the diagnosis of RA.
embodiment 2 is applied cartilage cell and is not suitable for RA clinical assistant diagnosis as antigen substrate
1, the separation of RA cartilage cell is cultivated
(1) draw materials: the RA patient articular cartilage getting row knee replacements, peel off manadesma and the perichondrium of parcel cartilaginous tissue, put into the double dish filling PBS liquid;
(2) cartilaginous tissue is shredded into the thin block of 0.5 ~ 0.8mm, rinse by the PBS solution containing penicillin and streptomysin;
(3) 0.25% trypsase stop after 2 hours 37 DEG C of digestion;
(4) 0.02%II Collagenase Type spends the night 37 DEG C of digestion;
(5) observe under inverted microscope, when after the individual cells that dissociates, add DMEM nutrient solution and the dilution of II Collagenase Type is stopped digestion;
(6) filter with 200 mesh filter screens after cell mass being blown and beaten into individual cells, collect filtrate, the centrifugal 5min of 1500r/min;
(7) abandon supernatant, add nutrient solution re-suspended cell, 0.2% Trypan Blue, living cell rate is greater than 90%, CO
2incubator is hatched.
2, cartilage cell's qualification
The cartilage cell of chondrocyte marker II Collagen Type VI (CII) antibody to original cuiture is adopted to dye to determine cell category.
Double dish sucks supernatant, fixes 10 minutes with methyl alcohol at-20 DEG C.Wash 3 times with 0.01M PBS, sheep blood serum closes 30 minutes, and 0.01M PBS washs 3 times, each 5 minutes.Incubated at room 1 hour after diluting according to 1:1000 by chondrocyte-specific markers CII monoclonal antibody (Abcam), 0.01M PBS washs 3 times, each 5 minutes.Anti-according to 1:2000 dilution with the goat anti-mouse igg two of Cy3 mark, incubated at room 30 minutes, 0.01M PBS washs 3 times, each 5 minutes.Finally with the DAPI transfect cell core of 1:10000 dilution, add anti-fluorescence quenching mounting after having washed, at fluorescence microscopy Microscopic observation, CII is mainly distributed in cartilage cell's endochylema, confirms that the cell of original cuiture is cartilage cell, the results are shown in Figure 5.
3, the method for Western-blot detects anti-cartilage cell's antibody in RA patient and normal human serum
(1) chondrocyte proteins extracts:
A, outwell nutrient solution, culture flask slant setting 1 ~ 2 minute, residual culture transfer pipet siphons away.
B, every bottle of cell add the 0.01M PBS of 3mL 4 DEG C of precoolings; Keep flat and shake 1 minute gently with washed cell, then discard washing lotion; Wash cell altogether three times, PBS is abandoned only and culture flask is placed on ice.
Add 200uL Pierce lysate (adding protease inhibitors PMSF in advance) in C, every bottle of cell, leave standstill 20 minutes; 10000g, at 4 DEG C centrifugal 20 minutes, retain supernatant; After supernatant after centrifugal is measured concentration, packing, is put in-80 DEG C of preservations.
(2) loading and electrophoresis:
A, in chondrocyte proteins sample, add 2 × SDS-PAGE albumen sample-loading buffer, mix rear 100 DEG C of boiling water baths and heat 35 minutes, make the abundant sex change of albumen.
After albumen cool to room temperature after B, sex change, protein sample is directly loaded in SDS-PAGE glue well, use the electrophoretic apparatus of Bio-Rad, constant voltage arranges 100V, and bromophenol blue arrives the bottom end neighbouring stopping electrophoresis of glue.
(3) transferring film:
Adopt pvdf membrane, pvdf membrane is dipped in respectively methyl alcohol 15 seconds, pure water 3 minutes, transfering buffering liquid pre-service in 15 minutes; Soak was in electrophoretic buffer 5 minutes; At the standard wet type membrane-transferring device of Bio-Rad, setting transferring film electric current is 300mA, and the transferring film time is 60 minutes.After having turned film, with Ponceaux dyeing liquor, film is dyeed, to observe actual transferring film effect, every part of protein sample is cut off, so that hatch different serum by its display band simultaneously.
(4) close:
After transferring film, with cleansing solution, film is washed 3 times, each 3 minutes; After having washed, film is added in the confining liquid that skimmed milk power is made into, close for 4 DEG C and spend the night.
(5) sera incubation:
RA patient and normal human serum are according to the pvdf membrane joined after 1:200 concentration dilution after wet turning, and room temperature, on horizontal shaker, slight concussion hatches 1 hour.After hatching end, wash 6 times with cleansing solution, each 10 minutes, to alleviate background.
(6) two anti-hatch:
The anti-human igg two adopting horseradish peroxidase (HRP) to mark resists, after 1:10000 concentration dilution, and room temperature, on horizontal shaker, slight concussion hatches 30 minutes; After hatching end, wash 6 times with cleansing solution, each 10 minutes, to alleviate background.
(7) chemiluminescence and X film developing:
Expose in magazine after pvdf membrane adds chemical luminous substrate (Thermo), carry out craft at the developer solution prepared voluntarily and stop bath and develop a film.The results are shown in Figure 6, on the pvdf membrane of hatching with RA patient and normal human serum, all can show many reacts band, this result illustrates except RA patients serum, also the antibody that can react with chondrocyte proteins is there is, so anti-cartilage cell's antibody is not be present in specifically in RA patients serum in normal human serum.
4, IIF method detects anti-cartilage cell's antibody in RA patient and normal human serum
(1) cartilage cell's is fixing:
By the cartilage cell of acquisition after trypsinization, obtained single cell suspension, is laid on 96 orifice plates, every hole 100uL, 37 DEG C of overnight incubation, suck nutrient solution, after PBS washs 3 times, every hole adds 95% ethanol (or methyl alcohol, 5% glacial acetic acid) 200uL, fixes 20 minutes for 4 DEG C.After cell after fixing removes immobile liquid again after PBS washs 3 times, pat dry, be placed in-20 DEG C and save backup.
(2) close:
Every hole, with containing the 0.01M PBS confining liquid 100uL of 3% BSA at room temperature, closes 1 hour.After completing, discard confining liquid, cleansing solution rinsing 3 times, each 5 minutes.
(3) sera incubation:
Get RA patient respectively and normal human serum doubly dilutes according to 1:100, in 96 orifice plates securing cartilage cell, every hole adds serum 100uL after above-mentioned dilution, incubated at room 1 hour.After having hatched, suck serum, pat dry, cleansing solution washs 6 times, each 3 minutes.
(4) goat anti-human igg two of Cy3 mark is anti-hatches:
Every hole adds 100uL, the goat anti-human igg of the Cy3 mark of 1:1000 dilute concentration, incubated at room 30 minutes.After completing, discard confining liquid, cleansing solution rinsing 6 times, each 5 minutes.
(5) core is contaminated:
DAPI solution 100uL containing 1:10000 dilution adds in each hole, normal-temperature reaction 30 minutes.Reaction terminates rear cleansing solution rinsing 3 times, each 5 minutes.Add a cover fluorescence microscopy Microscopic observation after cover glass mounting.
(6) fluorescence microscope:
Use chromium optical filter, observe Cy3 fluorescence intensity under excitation wavelength 554nm, emission wavelength 570nm condition, the results are shown in Figure 7.Experimental result shows, have an appointment respectively 40%, 30% RA patients serum and normal human serum cartilage cell's endochylema uniform coloring (green) of hatching, the RA patients serum of about 60% and 70% and normal human serum do not react (cartilage cell's endochylema is not painted) with cartilage cell.This result confirms that anti-cartilage cell's antibody is not be present in RA patients serum specifically further, and thus cartilage cell is not suitable as the auxiliary diagnosis of cell substrate for RA.
embodiment 3 is applied Th17 positive cell and is not suitable for RA clinical assistant diagnosis as antigen substrate
1, the magnetic bead sorting of Th17 cell in RA peripheral blood in patients
(1) adopt the method for gradient centrifugation to be separated from RA peripheral blood and obtain mononuclearcell (PBMC).
(2) select the Th17 cell positive sorting post of German U.S. sky girl to carry out sorting to the PBMC in RA peripheral blood to specifications, obtain Th17 positive cell.
2, Th17 cell protein total protein extraction
Add the protease inhibitors of anti-protein degradation, by the method smudge cells of ultrasonic degradation, obtain total protein of cell.
3, the method for Western-blot detects anti-Th17 cell antibody in RA patient and normal human serum
It is identical that this Part Methods and use Western-blot method in embodiment 2 detect anti-cartilage cell's antibody in serum.Through above-mentioned experiment, the pvdf membrane that RA patient and normal human serum are hatched all does not find react band, refer to Fig. 8, this result illustrates it is no matter the antibody that all there is not anti-Th17 cell protein in normal human serum or RA patients serum, so Th17 cell is not suitable for the auxiliary diagnosis of RA.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (10)
1. fibroblast-like synoviocyte is preparing the application in Diagnosis of Rheumatoid Arthritis reagent.
2. application according to claim 1, is characterized in that, described fibroblast-like synoviocyte is the human desmocyte sample synovial cell of in vitro culture.
3. fibroblast-like synoviocyte is preparing the application in Diagnosis of Rheumatoid Arthritis kit.
4. application according to claim 3, is characterized in that, described diagnostic kit comprises the human desmocyte sample synovial cell of in vitro culture and indirect immunofluorescene assay method other reagent used.
5. application according to claim 4, is characterized in that, the step of described indirect immunofluorescene assay method is:
(1) the human desmocyte sample synovial cell of in vitro culture is fixed;
(2) close with the PBS confining liquid containing BSA;
(3) get test serum and hatch fixing fibroblast-like synoviocyte;
(4) goat anti-human igg two of Cy3 mark is anti-hatches;
(5) DAPI solution dye core;
(6) fluorescence microscope, the fibroblast-like synoviocyte endochylema uniform coloring of rheumatoid arthritis patients sera incubation, the fibroblast-like synoviocyte endochylema that other patients and normal human serum are hatched is all not painted.
6. application according to claim 5, is characterized in that, described fluorescence microscope condition is: use chromium optical filter, excitation wavelength 554nm, emission wavelength 570nm.
7. fibroblast-like synoviocyte is preparing the application in Diagnosis of Rheumatoid Arthritis cell chip.
8. application according to claim 7, is characterized in that, is fixed with the human desmocyte sample synovial cell of in vitro culture in described cell chip.
9. the application according to claim 7 or 8, is characterized in that, the preparation method of described cell chip is:
(1) apply point sample instrument Software for Design matrix, determine dot spacing, point sample order, point sample coordinate, each sample arranges 3 multiple holes;
(2) selecting pretreated slide as substrate, is 1 × 10 by concentration
3the fibroblast-like synoviocyte suspension application point sample instrument of/mL is added in substrate, dried overnight;
(3) be placed in wet box by the substrate after point sample, 37 DEG C of waters bath with thermostatic control fix 2 hours;
(4) close and wash substrate, being placed in 4 DEG C and saving backup.
10. the application according to claim 7 or 8, it is characterized in that, the using method of described cell chip is: get test serum and add the reaction of cell chip matrix, through washing, anti-human igg two anti-reflective then marked with Cy3 is answered, again after washing, scanning of fluorescent intensity is carried out with laser confocal scanner, finally analyze anti-fibroblast-like synoviocyte antibody concentration in test serum according to typical curve Quant Anlysis, then compare with cutoff value, judge whether test serum is positive.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1218506A (en) * | 1996-03-13 | 1999-06-02 | 盐野义制药株式会社 | Human T cell clone specific for rheumatoid arthrilis |
| CN1491280A (en) * | 2000-12-22 | 2004-04-21 | ��ī�����˹��ʽ���� | Synovial membrane cell protein |
-
2013
- 2013-09-09 CN CN201310405870.2A patent/CN104422761A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1218506A (en) * | 1996-03-13 | 1999-06-02 | 盐野义制药株式会社 | Human T cell clone specific for rheumatoid arthrilis |
| CN1491280A (en) * | 2000-12-22 | 2004-04-21 | ��ī�����˹��ʽ���� | Synovial membrane cell protein |
Non-Patent Citations (1)
| Title |
|---|
| 史战国 等: "类风湿关节炎成纤维滑膜细胞的表型及其侵蚀软骨的特性", 《细胞与分子免疫学杂志》, vol. 22, no. 4, 31 December 2006 (2006-12-31) * |
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