CN110275026A - A kind of molecular marker and its application for diagnosing idiopathic inflammatory myopathies - Google Patents
A kind of molecular marker and its application for diagnosing idiopathic inflammatory myopathies Download PDFInfo
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Abstract
The molecular marker and its application that the present invention relates to a kind of for diagnosing idiopathic inflammatory myopathies.The molecular marker is self anti-Features of The Heat Shock Transcription Factor 1 (heat shock factor 1, the HSF1) antibody in serum.The antibody is IgG；The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis (dermatomyositis, DM)；The disease process for being diagnosed as distinguishing idiopathic inflammatory myopathies and healthy population or Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory myopathy, IIM).
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, specifically, being related to a kind of for diagnosing point of idiopathic inflammatory myopathies
Sub- marker and its application.
Background technique
Idiopathic inflammatory myopathies (idiopathic inflammatory myopathy, IIM) be one group with proximal end flesh without
It is involved outside power, muscle enzymes exception, skeletal muscle inflammatory infiltration and muscle and itself exempts from for the rare heterogeneity of main clinical manifestation
Epidemic disease mainly includes polymyositis (polymyositis, PM), dermatomyositis (dermatomyositis, DM) and inclusion body flesh
Scorching (inclusion body myositis, IBM) [1].The autoantibody of IIM includes myositis-specific antibodies (myositis
Specific autoantibodies, MSAs) and myositis correlation antibody (myositis associated
Autoantibodies, MAAs), it is seen that in the IIM patient of 60-80%, to the diagnosis of disease, guiding treatment and the pre- rear of prompt
Face have important value [2,3].MSAs is related to special clinical syndrome, and is detected in IIM；And MAAs is then found in other
Autoimmunity disease, such as systemic loupus erythematosus (systemic lupus erythematosus, SLE) and chorionitis
(systemic scleroderma,SSc)[4].In addition, some researches show that anti-SRP antibody and anti-HMGCR antibody can be direct
Participate in myocyte damage, prompt myositis autoantibody may have it is pathogenic [5,6].In addition, in the musculature of IIM patient
It can be seen that a variety of MSAs target antigens high expression [7,8].Thus, it is found that new myositis autoantibody, can not only carry out IIM
Finer parting, and the research of autoantibody has important prompt meaning to IIM pathogenesis
The MSAs majority being currently known is to be screened by immunoprecipitation, and carry out target antigen confirmatory experiment discovery.In addition,
The serological screening method of recombinant cDNA expression library is also some effective for finding unknown antibody, and this method is in identification tumour
It has been widely used in the research of related antigen.However, not only time-consuming, technical difficulty is big for construction cDNA expression library, but also
The library is to rely on bacterial expression recombinant protein, such protein expression system can not carry out posttranslational modification to albumen, for one
It can identify that the autoantibody of posttranslational modification albumen is fubaritic a bit to come out.Arrays of immobilized protein is a kind of autoantibody screening
Thousands of kinds of eukaryon system expression and purifications are obtained albumen and are coated in miniature array hole, then therewith by patients serum by new method
Reaction, being capable of efficient autoantibody of the screening into serum.At present we recognize that myositis autoantibody, target antigen is all
It is the nonspecific proteins of wide expression in human body, the albumen not only expressed and worked in IIM, therefore pass through screening
Include the very more microarray of protein classes, it may be possible to find new myositis autoantibody.
This research screens the unknown antibody in IIM using protein group chip technology (including 9374 kinds of people's full-length proteins),
Anti- Features of The Heat Shock Transcription Factor 1 (heat shock factor 1, HSF1) antibody is had found in DM, and is combined ELISA, is immunized
Precipitate (immunoprecipitation, IP), immunoblotting (immunoblot, IB) and Dot blot (dot blot, DB) method
Demonstrate the presence of the antibody.
Summary of the invention
Present invention firstly relates to one kind for diagnosing idiopathic inflammatory myopathies (idiopathic inflammatory
Myopathy, IIM) molecular marker, the molecular marker be serum in self anti-Features of The Heat Shock Transcription Factor 1
(heat shock factor 1, HSF1) antibody.
The antibody is IgG；
The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis
(dermatomyositis,DM)；
Described is diagnosed as differentiation idiopathic inflammatory myopathies and healthy population.
Specifically, the diagnosis are as follows: the IgG antibody of the anti-HSF1 in detection target patient serum is horizontal, with Healthy People
The anti-HSF1 antibody level average value of serum of group adds 3 times of standard deviations for positive critical value, such as the anti-HSF1 in target patient serum
IgG antibody level be higher than critical value, then be diagnosed as idiopathic inflammatory myopathies.
Further, the diagnosis are as follows: the IgG antibody of the anti-HSF1 in detection target patient serum is horizontal, with health
The anti-HSF1 antibody level average value of the serum of crowd adds 3 times of standard deviations for positive critical value, such as resisting in target patient serum
The IgG antibody level of HSF1 is higher than critical value, and the autoantibody of the known IIM of other in target patient increases, then is diagnosed as spy
Hair property inflammatory myopathy, other described autoantibodies are preferably anti-TIF1 gamma antibodies.
The invention further relates to a kind of Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory myopathy,
IIM) the molecular marker of disease process, the molecular marker are the self anti-1 (heat of Features of The Heat Shock Transcription Factor in serum
Shock factor 1, HSF1) antibody.
The antibody is IgG；
The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis
(dermatomyositis,DM)；
Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory myopathy, the IIM) disease into
Journey are as follows:
(1) risk of IIM secondary tumor correlation myositis (cancer associated myositis, CAM) is predicted；
(2) the treatment prognosis situation of the IIM patient of non-CAM is predicted；
Prediction prediction IIM secondary tumor correlation myositis (cancer associated myositis, CAM)
Risk is to detect the IgG antibody level of the anti-HSF1 in target patient serum, and the anti-HSF1 antibody level of the serum of healthy population is flat
Mean value adds 3 times of standard deviations to be higher than critical value for positive critical value, such as the IgG antibody level of the anti-HSF1 in target patient serum,
And the autoantibody of the known IIM of other in target patient increases, then is CAM high risk, the autoantibody of the IIM includes anti-
1 γ of transcriptional intermediary factor (TIF1 γ) antibody, anti-nuclear matrix albumen 2 (NXP2) antibody resist small ubiquitin sample modification activating enzymes (SAE)
Antibody and antihistamine acyl tRNA synzyme (Jo-1) antibody.
The treatment prognosis situation of the IIM patient of the prediction non-CAM is to detect the anti-HSF1 in target patient serum
IgG antibody it is horizontal, such as the IgG antibody level decline of the anti-HSF1 in the target patient serum after treating, then prognosis bona.
The invention further relates to one kind for diagnosis or Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory
Myopathy, IIM) disease process diagnostic kit, the kit includes: a effective amount of Features of The Heat Shock Transcription Factor of detection
1 (HSF1) and necessary related reagent, the related reagent include but is not limited to albumen preservation liquid, dilution, developing solution,
Contrast agents etc., the kit are the detection kit based on antigen-antibody interaction principle.
Preferably, the kit is ELISA kit or HP immunoblotting kit.
The invention further relates to one kind for diagnosing idiopathic inflammatory myopathies (idiopathic inflammatory
Myopathy, IIM) application of the molecular marker in reagent preparation box, the molecular marker is self in serum
Anti- Features of The Heat Shock Transcription Factor 1 (heat shock factor 1, HSF1) antibody.
The antibody is IgG；
The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis
(dermatomyositis,DM)；
Described is diagnosed as differentiation idiopathic inflammatory myopathies and healthy population.
Specifically, the diagnosis are as follows: the IgG antibody of the anti-HSF1 in detection target patient serum is horizontal, with Healthy People
3 times of the anti-HSF1 antibody level of average serum of group are positive critical value, such as the IgG antibody of the anti-HSF1 in target patient serum
Level is higher than critical value, then is diagnosed as idiopathic inflammatory myopathies.
Further, the diagnosis are as follows: the IgG antibody of the anti-HSF1 in detection target patient serum is horizontal, with health
3 times of the anti-HSF1 antibody level of the average serum of crowd are positive critical value, and the IgG such as the anti-HSF1 in target patient serum is anti-
Body level is higher than critical value, and the autoantibody of the known IIM of other in target patient increases, then is diagnosed as idiopathic inflammatory flesh
Disease, other described autoantibodies are preferably anti-TIF1 gamma antibodies.
The invention further relates to a kind of Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory myopathy,
IIM) application of the molecular marker of disease process in reagent preparation box, the molecular marker are self anti-in serum
Features of The Heat Shock Transcription Factor 1 (heat shock factor 1, HSF1) antibody.
The antibody is IgG；
The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis
(dermatomyositis,DM)；
Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory myopathy, the IIM) disease into
Journey are as follows:
(1) risk of IIM secondary tumor correlation myositis (cancer associated myositis, CAM) is predicted；
(2) the treatment prognosis situation of the IIM patient of non-CAM is predicted；
Prediction prediction IIM secondary tumor correlation myositis (cancer associated myositis, CAM)
Risk is to detect the IgG antibody level of the anti-HSF1 in target patient serum, with the anti-HSF1 antibody of the average serum of healthy population
Horizontal 3 times are positive critical value, as the IgG antibody level of the anti-HSF1 in target patient serum is higher than critical value, and target
The autoantibody of the known IIM of other in patient increases, then is CAM high risk, and the autoantibody of the IIM includes in anti-rotation record
1 γ of Jie's factor (TIF1 γ) antibody, anti-nuclear matrix albumen 2 (NXP2) antibody, resist small ubiquitin sample modification activating enzymes (SAE) antibody and
Antihistamine acyl tRNA synzyme (Jo-1) antibody.
The treatment prognosis situation of the IIM patient of the prediction non-CAM is to detect the anti-HSF1 in target patient serum
IgG antibody it is horizontal, such as the IgG antibody level decline of the anti-HSF1 in the target patient serum after treating, then prognosis bona.
The beneficial effects of the present invention are:
This research screens discovery novel I IM autoantibody-anti-HSF1 antibody with human protein group chip technology, and further
The distribution of the antibody is had detected in the IIM and other Serum of Patients With Autoimmune Diseases of large sample.It was found thatAnti- HSF1 antibody and CAM phase It closes, but the presence of the antibody is but not detected in simple tumour.The anti-HSF1 antibody level of the serum of non-CAM patient and disease Sick mobility is positively correlated in moderate, and anti-HSF1 antibody can treat with patient and alleviate and switch to feminine gender。
The constructive expression of the visible HSF1 of various kinds of cell in human body, under non-stress situation, HSF1 is mostly with inactive list
Body form is free to be present in cytoplasm and nucleus.When cell encounters destructive stimulus, such as heat stress, ischemic, infection,
HSF1 assembles into nucleus, and phosphorylation and oligomerization occurs, and ultimately forms active tripolymer.Active HSF1 tripolymer
It can be by combining the Binding characteristic (heat shock element, HSE) in target gene promoters, regulating heat shock protein base
Cause expression [15,20].It is different from normal tissue cell, kinds of tumors tissue such as oophoroma, breast cancer, carcinoma of endometrium and liver
The visible HSF1 of cancer high expression [21-24].Moreover, research discovery HSF1 is in the active form of tripolymer in cancer cell more,
And participate in cancer cell vicious transformation [25].Wilson et al. is in early stage high-level oophoroma (high grade serious
Ovarian cancer, HGSOC) IgA of anti-phosphorylation HSF1 is detected in patients serum, but do not find anti-for this
Former other Ig types [26]。
Although the IgG of anti-non-phosphorylating HSF1 is not detected in this research in ovarian cancer alone patient, in 3 CAM
Merge in the patient of oophoroma and detects the antibody.It is worth noting that, we also merge other types tumour in 8 CAM
Patient in detect the IgG of anti-non-phosphorylating HSF1, including 2 cancer of the esophagus, 2 thyroid cancers, 1 gastric cancer, 1
Breast cancer, 1 cervical carcinoma and 1 nasopharyngeal carcinoma, but equally depositing for the antibody is not detected in the simple tumor patient to match
?.Therefore, the related immune response of HSF1 is different in CAM and simple tumour,The IgG of anti-non-phosphorylating HSF1 can be used as The serologic marker object of CAM。
Multinomial epidemiological survey shows the normal accompanying tumors of IIM patient, the incidence of tumour be about 20%~25% [27]。
For example anti-TIF1 γ of certain MSAs in IIM, anti-NXP2, anti-HMGCR and anti-SAE antibody it is related to the generation of CAM [28-31].It is interesting
, the relevant MSAs of these tumours can not predict simple tumour occurrence risk [32,33].In addition, studies have reported that
The tumour occurrence risk of MSAs feminine gender IIM patient equally increases, and there may be other unknown autoantigens in prompt CAM
[34].Therefore, there is still to further explore for new CAM related auto-antibodies.The study find that 16.9% anti-HSF1 is positive
IIM patient is CAM, and MSAs, including anti-TIF1 γ, anti-NXP2, anti-SAE and anti-Jo-1 can be detected in this group of patients serum
Antibody.Multiplicity correct the anti-HSF1 positive and anti-HSF1 feminine gender IIM patient age distribution and with after sex ratio,It was found that Anti- HSF1 antibody is still related to the generation of tumour, and prompting the antibody is the tumorigenic risk factor of IIM。
Clinical research in recent years finds that the MSAs/MAAs in IIM patients serum can disappear with the alleviation of disease, mentions
Show serum myositis antibody level can be used for monitoring patient disease mobility [3].It is anti-SRP, anti-HMGCR, anti-MDA5 in MSAs, anti-
Jo-1 and anti-NXP2 antibody have been found it is related to myositis disease activity [35-39].This research is positive by confrontation HSF1
The transverse direction and longitudinal direction research of IIM patient, discoveryThe anti-HSF1 antibody level of non-CAM IIM patients serum and myositis disease activity It is positively correlated in moderate。
Detailed description of the invention
The Immunoprecipitation studies of Fig. 1, DM patient
Patient 1 and Patient 2 is 2 anti-HSF1 positive DM patients that human protein group cDNA microarray goes out.With dilute
The recombination HSF1 (Abcam) for being interpreted as the source E.coli of 0.6ng/ μ l is IP reaction substrate, verifies anti-HSF1 antibody in serum
In the presence of.PC, positive control；HC, normal healthy controls；HSF1, Features of The Heat Shock Transcription Factor 1；DM, dermatomyositis；IP, immunoprecipitation.
The characteristic distributions of Fig. 2, anti-HSF1 antibody in IIM and other autoimmunity diseases
Characteristic distributions of (2A) the anti-HSF1 antibody in IIM (612), normal healthy controls (HCs) (65).
(2B) is coated with ELISA testing result [the anti-HSF1 positive IIM (62 of the recombination HSF1 (Abcam) in the source E.coli
Example), the anti-HSF1 feminine gender IIM (81) that randomly selects and HCs (31)] further application be coated with the recombination in the source HEK293
The ELISA of HSF1 (OriGene) is verified.
(2C) ELISA enclosed experiment detects to be resisted in 3 anti-HSF1 positive (PS) and negative (NS) patients serum of 2 anti-HSF1
The variation of HSF1 antibody level.
The immunoblot results of (2D) anti-HSF1 positive IIM patient (49) and HCs (9) serum.
The Dot blot result of (2E) anti-HSF1 positive IIM patient (65) and HCs (7) serum.
Horizontal dotted line in 2A and 2C is the positive critical value of anti-HSF1 antibody, and the horizontal horizontal line in B is mean value, i.e. HCs
The anti-HSF1 antibody level of average serum add 3 times of standard deviations.HSF1, Features of The Heat Shock Transcription Factor 1；IIM, idiopathic inflammatory myopathies；
HCs, normal healthy controls；ELISA, enzyme linked immunosorbent assay；PS, anti-HSF1 positive serum sample；NS, anti-HSF1 negative serum sample
This.
The characteristic distributions of Fig. 3, anti-HSF1 antibody in CAM and simple tumor patient
Anti- HSF1 antibody is found in the CAM patient of 21.6% (11/51), the simple tumor patient (x in 0% (0/27)2=5.1,
P=0.024).Horizontal dotted line is the positive dividing value (0.271) of anti-HSF1 antibody in figure.HSF1, Features of The Heat Shock Transcription Factor 1；CAM,
Cancer-related myositis.
Fig. 4, the anti-anti- HSF1 antibody level of HSF1 positive non-CAM patients serum it is related to disease activity (cross section and
Vertical analysis)
The anti-HSF1 antibody level of (4A) anti-HSF1 positive non-CAM patient (51) serum and patient PGA VAS (r=
0.325, P=0.02), ordinary circumstance VAS (r=0.312, P=0.026) and muscle VAS (r=0.399, P=0.004) is in just
It is related.
The anti-HSF1 antibody level of (4B) anti-HSF1 positive non-CAM patient (10) serum with disease treatment variation.
Horizontal dotted line in figure is the positive critical value of anti-HSF1 antibody, the i.e. anti-HSF1 antibody level of the average serum of HCs
Add 3 times of standard deviations.*P<0.05；HSF1, Features of The Heat Shock Transcription Factor 1；IIM, idiopathic inflammatory myopathies；PGA, doctor is to patient's disease
The net assessment of sick mobility；VAS, visual analogue scale.
The anti-HSF1 antibody level of Fig. 5, CAM patients serum is uncorrelated to disease activity
The correlation analysis of (5A) anti-HSF1 antibody level and PGA VAS；
The correlation analysis of (5B) anti-HSF1 antibody level and ordinary circumstance VAS；
The correlation analysis of (5C) anti-HSF1 antibody level and skin VAS；
The correlation analysis of (5D) anti-HSF1 antibody level and skeletal joint VAS；
The correlation analysis of (5E) anti-HSF1 antibody level and alimentary canal VAS；
The correlation analysis of (5F) anti-HSF1 antibody level and lungs VAS；
The correlation analysis of (5G) anti-HSF1 antibody level and angiocarpy VAS；
The correlation analysis of (5H) anti-HSF1 antibody level and muscle VAS.
HSF1, Features of The Heat Shock Transcription Factor 1；CAM, cancer-related myositis；IIM, idiopathic inflammatory myopathies；PGA, doctor couple
The assessment of patient's total disease mobility；VAS, visual analogue scale.
Fig. 6, anti-HSF1 positive non-CAM Serum Antibody Levels In Patients with disease treatment variation
The anti-HSF1 antibody level of (6A) non-CAM patient (10) serum and muscle VAS with disease treatment variation；
The anti-HSF1 antibody level of (6B) non-CAM patient (10) serum and lungs VAS with disease treatment variation.
Horizontal dotted line is the positive dividing value (0.271) of anti-HSF1 antibody in figure.HSF1, Features of The Heat Shock Transcription Factor 1；IIM, it is special
Hair property inflammatory myopathy；VAS, visual analogue scale.
Specific embodiment
Material and method
1, research object
This research passes through the characteristic distributions and clinical meaning of cross section and the anti-HSF1 antibody of Longitudinal Analysis in IIM.
(1) cross-sectional study
It is included in altogether from December, 2009~2017 year and is gone to a doctor in 612 IIM patients of China-Japan Friendship Hospital, wherein wrapping December
Include 499 DM and 113 PM patients.Inclusion criteria: DM and PM diagnosis meets Bohan&Peter diagnostic criteria in 1975.It excludes
Standard: patient clinical data missing；The less IIM hypotype for seeing Chinese population, such as inclusion body myositis (inclusion body
myositis,IBM)。
The diagnostic criteria of cancer-related myositis (cancer associated myositis, CAM) be diagnosing tumor in
In the front and back 3 years that myositis occurs [9].Collect the following clinical data of patient, including age, gender, medical history, ordinary circumstance, physique
Inspection, laboratory inspection and medicining condition etc..
The diagnostic criteria of interstitial lung lesion (interstitial lung disease, ILD) is high-resolution ct (HRCT)
Prompting ILD sign and/or restrictive ventilatory functional disturbance, (total lung capacity, lung capacity, carbon monoxide transport factor value are less than predicted value
80%) [10]。
The diagnostic criteria of weight loss be mitigate suddenly in patient's weight 6 months reach 5% or more original weight [11]。
Visual analogue scales (Myositis disease activity assessment is assessed according to myositis disease activity
Visual analog scales, MYOACT) 6 muscle external system mobilities of assessment, including ordinary circumstance, skin, bone pass
Section, gastrointestinal tract, lungs and cardiac system mobility.
Patient's overall disease mobility is assessed using 10cm visual analogue scales (visual analog scale, VAS)
Outside (physician global assessment, PGA), muscle involvement situation and muscle mass activity degree [12]。
(2) longitudinal research
It is included in 10 anti-HSF1 positive IIM patients, follow-up 16m (6~34m) altogether, and collects patient per and is admitted to hospital serum (1
~20m).Situation is treated according to 3 core index (MYOACT, PGA and muscle scoring) assessment patient disease.Judge patient disease
The standard whether improved be in 3 core index at least 2 improve >=20%, and remaining 1 must not deteriorate >=25% [13]。
(3) control group
Including 65 normal healthy controls (HCs), 27 simple tumor patients.
The diagnosis of tumour has obtained proved by pathology in CAM and simple tumor patient (see Table 1 for details).The sex ratio of HCs
Match with IIM patient with age distribution.All serum samples are acquired before patient's hospitalization, and are saved at -80 DEG C.
Tumor type and the demographic characteristics of 1 CAM of table and simple tumor patient
aCAM (cancer-related myositis): patient tumors diagnosis is before and after IIM generation in 3 years.
This research has passed through the approval of Ethics Committee, China-Japan Friendship Hospital (number: 2013-6), and all patients in group are equal
It endorsed informed consent form.
2, statistical method:
Continuous variable meets normal distribution person and is described with mean ± standard deviation, and Non-Gaussian Distribution person is with median and quartile
Spacing (IQR) description.Classified variable is indicated with percentage and absolute frequency.The inspection of measurement data comparison among groups application independent sample t
It tests or Mann-Whitney U is examined.Enumeration data then applies x2It examines or Fisher is accurately examined.Logistic regression analysis
Assess the tumour occurrence risk of anti-HSF1 positive IIM patient.Spearman correlation analysis and Generalized estimating equation
(generalized estimating equation model, GEE) analyzes the anti-HSF1 antibody level of IIM patients serum and disease
The correlation of sick mobility.Using SPSS 23.0 (SPSS institute, USA) and GraphPad Prism 5.0
(GraphPad Software, USA) carries out data management and statistical analysis.
Embodiment 1, the antibody with high specificity in screening IIM and the presence for determining HSF1 antibody
UsingHuman Protein Microarray v5.1 (Life Technologies, USA) detection
Antibody distribution in 10 DM patients and 10 HCs serum, and it is for statistical analysis.Method particularly includes:
1. it closes, and configuration confining liquid (50mM HEPES, 200mM NaCl, 0.01%Triton X-100,25%
Glycerol, 20mM reduced glutathione, 1.0mM DTT, 1X Synthetic Block), chip is placed in core
Piece is incubated in box in 4 DEG C of closing 1h；
2. serum sample is incubated for, by ready Sample dilution (1:500；DM patient 10, HCs10) add to chip
It is incubated in box, 4 DEG C of incubation 1.5h；
3. secondary antibody is incubated for, by Alexa647-conjugated goat anti-human IgG fluorescence secondary antibody adds to
Chip is incubated in box, and 4 DEG C are protected from light incubation 1.5h；
4. drying, using chip centrifuge by chip centrifugal drying (200g, 2min)；
4. scanning and data collection, Tecan fluorescent microarray PowerScanner scans chip, and
Data are collected using 6.0 software of GenePix Pro (Molecular Devices, Sunnyvale, CA)；
5. data are analyzed, using Life Technologies ' proprietaryProspector
Software analyzes data, screens the unknown antibody in DM patients serum.
By anti-in 10 DM patients of detection (2 anti-MDA5 are positive, 8 myositis negative antibodies) and 10 HCs serum
Body is distributed to screen the unknown antibody in IIM.2 anti-MDA5 positive DM patients serums detect MDA5 antigen (anti-black
Plain tumor correlation differentiation gene -5, melanoma differentiation-associated gene-5) high RST, it was demonstrated that egg
White matter chip is used for the feasibility of Large-scale Screening autoantibody.2/8 DM patients serum detects the high letter of HSF1 antigen
Number, IP (immunoprecipitation) it is again seen that in patients serum anti-HSF1 antibody presence (Fig. 1).Therefore, we have selected anti-HSF1
Antibody does further expand and studies.
Embodiment 2, ELISA detect anti-HSF1 antibody
The recombination HSF1 albumen (Abcam, Cambridge, UK) of commercialization is diluted and is coated in polystyrene board
In the reacting hole of (Thermo Scientific, Roskilde, Denmark) (hole 200ng/), ELISA, which is detected in serum, to be resisted
HSF1 antibody level.Simultaneously using another commercialization recombination HSF1 albumen (OriGene, Maryland, USA) ELISA inspection
Survey anti-HSF1 antibody level in serum.Gradient dilution ELISA (1:2) measures the best dilution ratio of serum, ELISA enclosed experiment
Measurement ELISA detects the specificity of anti-HSF1 antibody.All serum samples detect 2 times.ELISA detects the anti-HSF1 antibody of serum
Before, first by the serum of the anti-HSF1 positive IIM patient (3) and HCs (2) that have diluted and recombination HSF1 albumen (Abcam,
Cambridge, UK) 4 DEG C be incubated overnight, then detect anti-HSF1 antibody level again.
As the result is shown: being used as coating protein, gradient dilution patient's blood using the commercialization HSF1 (Abcam) in the source E.coli
Measuring its optimum diluting multiple clearly is 1:200.3 times of standard deviations are added to face as the positive using the anti-HSF1 antibody level of the average serum of HCs
Dividing value (0.271), calculating antibody positive rate.Anti- HSF1 antibody is found in 10.6% (65/612) IIM and 3.1% (2/65)
HCs (Fig. 2A).The HSF1 antigen testing result and ELISA testing result one of 10 DM patients in human protein group chip research
It causes, it was confirmed that the feasibility of two kinds of experimental methods.Simultaneously using the commercialization HSF1 in another source HEK293 as coating
Albumen, ELISA detect the anti-HSF1 antibody (Fig. 2 B) in IIM patients serum, as a result, it has been found that the ELISA detection of two kinds of HSF1 albumen
As a result there is preferable consistency and correlation.
ELISA enclosed experiment discovery, the serum sample of anti-HSF1 positive after being closed by the HSF1 in the source E.coli its resist
HSF1 antibody level is decreased obviously, it was confirmed that the specificity of the antibody has (Fig. 2 C) in serum.In addition, we fight HSF1 sun
The IIM (Fig. 2 D, E) of property, has found that it all has preferable consistency between ELISA result.
Embodiment 3, the clinical characters of anti-HSF1 positive IIM patient
The CAM ratio of anti-HSF1 positive IIM patient is higher (16.9%vs 7.3%, P=0.008), and common weight subtracts
Gently (P=0.046) and pruitus (P=0.039).Meanwhile anti-HSF1 antibody and anti-TIF1 gamma antibodies (P=0.046), high ball
Proteinemia (P < 0.001), c reactive protein increase (P=0.039) and erythrocyte sedimentation rate increases (ESR, P=0.006) correlation.We are simultaneously
Do not find that anti-HSF1 antibody is related to other typical IIM clinical characters, as skin involvement, interstitial lung lesion and creatase increase
(table 2).In addition, the ANA caryogram of anti-HSF1 positive IIM patients serum is mainly shown as spotted type or cytoplasmic granule type.
The clinical characters of the anti-HSF1 antibody positive IIM patient of table 2
HSF1, Features of The Heat Shock Transcription Factor 1；IIM, idiopathic inflammatory myopathies；PM, polymyositis；DM, dermatomyositis；ANA,
Antinuclear antibodies；CK, creatine kinase；LDH, lactic dehydrogenase；aWeight loss: weight surprisingly mitigates >=5%；b(tumour is related by CAM
Property myositis): the diagnosis of patient tumors IIM generation before and after in 3 years；Myositis antibody, including anti-Mi-2, anti-TIF1 γ resist
MDA5, anti-NXP2, anti-Jo-1, anti-PL-7, anti-EJ, anti-OJ, anti-SRP, anti-HMGCR, anti-SAE, anti-Ku, anti-Scl-100, anti-Scl-
75 antibody.cData are found in 611 patients,dData are found in 594 patients,eData are found in 601 patients,fData can
551 patients are seen,gData are found in 600 patients,hData are found in 541 patients,iVisible 588 patients of data,jNumber
According to being found in 590 patients,kData are found in 382 patients.
Embodiment 4, anti-HSF1 antibody and IIM tumour occur
In this research, 8.3% (51/612) IIM patient generation tumour, including urogenital neoplasm (23/51,
45.1%), digestive system tumor (9/51,17.6%), tumor in respiratory system (7/51,13.7%), hematological system tumor (3/
And endocrine system carcinoma (9/51,17.6%) 51,5.9%).Wherein, the anti-HSF1 of 21.6% (11/51) CAM patient is positive, so
And the presence (Fig. 3) of the antibody is not detected in its corresponding simple tumor patient.11 anti-HSF1 positive CAM patients
It is the MSAs positive, including anti-TIF1 γ 7 (63.6%) example, anti-NXP2 1 (9.1%) example, anti-SAE 1 (9.1%) example and anti-
Jo-1 2 (18.2%) example.Univariate analysis discovery, age increase (OR 3.6 [95%CI 2.0-6.4], P < 0.001), resist
The HSF1 positive (OR 2.6 [95%CI 1.3-5.3], P=0.008), anti-HSF1 or the anti-TIF1 γ positive (10.4 [95%CI of OR
5.4-20.0], P < 0.001) it is related to the generation of tumour.Multi-variables analysis is still found after the influence of correction age distribution
Anti- HSF1 positive (OR 2.4 [95%CI 1.1-5.0], P=0.022), anti-HSF1 or the anti-TIF1 γ positive (OR 9.5 [95%
CI 4.9-18.4], P < 0.001) the tumorigenic risk increase (table 3) of patient.
Table 3, anti-HSF1 positive IIM patient tumour occurrence risk
OR, odds ratio；95%CI, 95% confidence interval；IIM, idiopathic inflammatory myopathies；HSF1, Features of The Heat Shock Transcription Factor
1；TIF1 γ, 1 γ of transcriptional intermediary factor；* ROC curve, which is analyzed, determines that the best age critical value that IIM merges tumor patient is 56
Year.
Embodiment 5, the anti-HSF1 antibody level of IIM patients serum are related to disease activity
Cross-sectional study finds that the PGA VAS of anti-HSF1 positive non-CAM patient is apparently higher than anti-HSF1 negative patient (P
=0.014).Spearman correlation analysis discovery, the antibody level of serum and PGA VAS (r of anti-HSF1 positive non-CAM patient
=0.325, P=0.02), ordinary circumstance VAS (r=0.312, P=0.026) and muscle VAS (r=0.399, P=0.004) be in
Be positively correlated (Fig. 4 A), and anti-HSF1 positive CAM Serum Antibody Levels In Patients do not find it is related to disease activity (Fig. 5, P >
0.05).10 anti-anti- HSF1 antibody levels of HSF1 positive non-CAM patients serum of Longitudinal Analysis are in treatment of diseases
In variation.During follow-up, 9/10 (90%) example patient therapeutic response is good and has reached clinical remission, and resists in its serum
The decline of HSF1 antibody level, wherein 7 patient's lasts when medical anti-HSF1 antibody switch to feminine gender；1/10 (10%) example conditions of patients
Recurrence, and anti-HSF1 antibody level increases (Fig. 4 B, Fig. 6) in its serum.GEE modeling statistics analysis finds that the anti-HSF1 of serum is anti-
Body level is positively correlated with PGA VAS, muscle VAS and lungs VAS.
Finally, it should be noted that above embodiments are used only as helping skilled in the art to understand essence of the invention,
It is not used as the restriction to protection scope.
Claims (10)
1. a kind of molecular marker is in preparation for diagnosing idiopathic inflammatory myopathies (idiopathic inflammatory
Myopathy, IIM) diagnostic kit in application, the molecular marker be serum in self anti-heat shock transcription
The factor 1 (heat shock factor 1, HSF1) antibody.
2. application according to claim 1, which is characterized in that the antibody is IgG；
The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis
(dermatomyositis,DM)；
Described is diagnosed as differentiation idiopathic inflammatory myopathies and healthy population.
3. application according to claim 2, which is characterized in that the diagnosis are as follows: anti-in detection target patient serum
The IgG antibody of HSF1 is horizontal, is reference with the anti-HSF1 antibody level of the average serum of healthy population, in target patient serum
The IgG antibody level of anti-HSF1 is higher than positive critical value, then is diagnosed as idiopathic inflammatory myopathies.
4. application according to claim 3, which is characterized in that the positive critical value is that the serum of healthy population is anti-
HSF1 antibody level average value adds 3 times of standard deviations.
5. application according to claim 2, which is characterized in that the diagnosis are as follows: anti-in detection target patient serum
The IgG antibody of HSF1 is horizontal, is reference with the anti-HSF1 antibody level of the serum of healthy population, such as resisting in target patient serum
The IgG antibody level of HSF1 is higher than positive critical value, and the autoantibody of the known IIM of other in target patient increases, then diagnoses
For idiopathic inflammatory myopathies；
Preferably,
Other described autoantibodies are anti-TIF1 gamma antibodies；
The positive critical value is that the anti-HSF1 antibody level average value of serum of healthy population adds 3 times of standard deviations.
6. a kind of molecular marker is used for Predicting Idiopathic inflammatory myopathy (idiopathic inflammatory in preparation
Myopathy, IIM) disease process diagnostic kit in application, the molecular marker be serum in self heat resistanceheat resistant
Shock transcription factor 1 (heat shock factor 1, HSF1) antibody, the Predicting Idiopathic inflammatory myopathy
(idiopathic inflammatory myopathy, IIM) disease process are as follows:
(1) risk of IIM secondary tumor correlation myositis (cancer associated myositis, CAM) is predicted；
(2) the treatment prognosis situation of the IIM patient of non-CAM is predicted.
7. application according to claim 6, which is characterized in that the antibody is IgG；
The idiopathic inflammatory myopathies are polymyositis (polymyositis, PM) or dermatomyositis
(dermatomyositis,DM)。
8. application according to claim 7, which is characterized in that
The risk of prediction prediction IIM secondary tumor correlation myositis (cancer associated myositis, CAM)
For the IgG antibody for detecting the anti-HSF1 in target patient serum is horizontal, average with the anti-HSF1 antibody level of the serum of healthy population
Value adds 3 times of standard deviations to be higher than critical value for positive critical value, such as the IgG antibody level of the anti-HSF1 in target patient serum, and
The autoantibody of the known IIM of other in target patient increases, then is CAM high risk, the autoantibody of the IIM includes anti-rotation
Record 1 γ of intermediary's factor (TIF1 γ) antibody, anti-nuclear matrix albumen 2 (NXP2) antibody resist small ubiquitin sample modification activating enzymes (SAE) anti-
Body and antihistamine acyl tRNA synzyme (Jo-1) antibody.
9. application according to claim 7, which is characterized in that
The treatment prognosis situation of the IIM patient of the prediction non-CAM is to detect the anti-HSF1's in target patient serum
IgG antibody is horizontal, the IgG antibody level decline of the anti-HSF1 in target patient serum after such as treating, then prognosis bona.
10. one kind is for diagnosing idiopathic inflammatory myopathies (idiopathic inflammatory myopathy, IIM) or prediction
The diagnostic kit of idiopathic inflammatory myopathies (idiopathic inflammatory myopathy, IIM) disease process,
The kit is the detection kit based on antigen-antibody interaction principle, it is preferred that is ELISA kit
Or HP immunoblotting kit；
The kit includes: to detect a effective amount of Features of The Heat Shock Transcription Factor 1 (HSF1) and necessary related reagent, preferably
, the related reagent includes but is not limited to that albumen saves liquid, dilution, developing solution, contrast agents.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323590A (en) * | 2020-03-19 | 2020-06-23 | 福建省立医院 | Application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnostic markers in lung cancer diagnosis |
| CN116593700A (en) * | 2023-05-24 | 2023-08-15 | 中日友好医院(中日友好临床医学研究所) | Molecular marker for identifying MDA 5-resistant positive dermatomyositis patient |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1950701A (en) * | 2004-02-20 | 2007-04-18 | 维里德克斯有限责任公司 | Breast cancer prognostics |
| WO2008097840A2 (en) * | 2007-02-02 | 2008-08-14 | Dana-Farber Cancer Institute, Inc. | Methods and compositions relating to the regulation of muc1 by hsf1 and stat3 |
| CN102369014A (en) * | 2008-12-08 | 2012-03-07 | 西北大学 | Method of modulating HSF-1 |
| CN103983687A (en) * | 2014-05-12 | 2014-08-13 | 缪应雷 | Application of human derived HSF2 as specific diagnosis molecular marker of ulcerative colitis |
| US20170227553A1 (en) * | 2014-08-04 | 2017-08-10 | Duke University | Compositions and methods for identifying and treating conditions involving hsf1 activity |
-
2019
- 2019-07-03 CN CN201910592945.XA patent/CN110275026B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1950701A (en) * | 2004-02-20 | 2007-04-18 | 维里德克斯有限责任公司 | Breast cancer prognostics |
| WO2008097840A2 (en) * | 2007-02-02 | 2008-08-14 | Dana-Farber Cancer Institute, Inc. | Methods and compositions relating to the regulation of muc1 by hsf1 and stat3 |
| CN102369014A (en) * | 2008-12-08 | 2012-03-07 | 西北大学 | Method of modulating HSF-1 |
| CN103983687A (en) * | 2014-05-12 | 2014-08-13 | 缪应雷 | Application of human derived HSF2 as specific diagnosis molecular marker of ulcerative colitis |
| US20170227553A1 (en) * | 2014-08-04 | 2017-08-10 | Duke University | Compositions and methods for identifying and treating conditions involving hsf1 activity |
Non-Patent Citations (4)
| Title |
|---|
| A.MILLER, ET AL.: "Development of an in vitro system for the evaluation of new therapeutic approaches in Sporadic Inclusion Body Myositis.", 《41ST ANNUAL MEETING OF THE SOCIETY-FOR-NEUROSCIENCE》 * |
| AMY L. WILSON, ET AL.: "Autoantibodies against HSF1 and CCDC155 as Biomarkers of Early-Stage, High-Grade Serous Ovarian Cancer.", 《AMERICAN ASSOCIATION FOR CANCER RESEARCH》 * |
| HANBO YANG, ET AL.: "Identification of multiple cancer-associated myositis-specific autoantibodies in idiopathic inflammatory myopathies: a large longitudinal cohort study.", 《ARTHRITIS RESEARCH & THERAPY》 * |
| PING WANG, ET AL.: "Acetylation-induced TDP-43 pathology is suppressed by an HSF1-dependent chaperone program.", 《NATURE COMMUNICATIONS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111323590A (en) * | 2020-03-19 | 2020-06-23 | 福建省立医院 | Application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnostic markers in lung cancer diagnosis |
| CN111323590B (en) * | 2020-03-19 | 2022-04-22 | 福建省立医院 | Application of anti-TIF 1 gamma-IgA and anti-TIF 1 gamma-IgG serving as combined diagnostic markers in lung cancer diagnosis |
| CN116593700A (en) * | 2023-05-24 | 2023-08-15 | 中日友好医院(中日友好临床医学研究所) | Molecular marker for identifying MDA 5-resistant positive dermatomyositis patient |
| CN116593700B (en) * | 2023-05-24 | 2024-02-06 | 中日友好医院(中日友好临床医学研究所) | Molecular marker for identifying MDA 5-resistant positive dermatomyositis patient |
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