CN110174513A - Application of the TGM2 in the biomarker of preparation monitoring Crohn disease disease activity - Google Patents
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Abstract
The present invention relates to application of the plasma TG M2 in the biomarker of preparation monitoring Crohn disease disease activity, belong to Medical Biology technical field.The present invention is for the first time using plasma TG M2 as the biomarker of assessment Crohn disease disease activity.Patient plasma TGM2 concentration is positively correlated with Crohn disease disease activity index, and patient plasma TGM2 concentration is also positively correlated with severity index under Crohn disease scope, and correlation is superior to standard biologic marker.Sensibility of the present invention is 76.32%, and specificity is 93.75%.
Description
Technical field
The invention belongs to Medical Biology technical fields, and in particular to TGM2 is in preparation monitoring Crohn disease disease activity
Application in biomarker.
Background technique
Crohn disease is the gastrointestinal tract inflammation disease that a kind of cause of disease is complicated, pathogenesis is unknown.It occurs in gastrointestinal tract
Position and occurrence degree differ greatly, while lacking characteristic pathological diagnosis, at this stage to the diagnosis of Crohn disease and in advance
Assessment is mainly according to mobility under Disease Clinical mobility and scope afterwards.Due to clinically to the therapeutic purpose of Crohn disease from
It maintains remission to turn to mucous membrane healing, therefore Crohn disease pipe is become to the monitoring of mobility under cd patient scope
The most important thing of reason.
Glutamine transaminage 2 (transglutaminase 2, TGM2) is a kind of multifunctional enzyme of secreting type.It has sent out
In the blood plasma of the gum chronic inflammation patient of present ciclosporin A induction, IgA nephrosis and anaphylactoid purpura type ephritis patients with active
Urine in, TGM2 concentration rise.TGM2 concentration can be used as biomarker monitoring IgA nephrosis in urine and Lupus nephritis is living
Dynamic (referring to 1), but effect of the TGM2 in the detection or diagnosis of Crohn disease there is no research.
Enteroscopy is the goldstandard for assessing mobility under cd patient scope, but since colonoscopy is invasive inspection
It looks into, patient compliance is poor, and needs the scope doctor of profession to assess the scoring of mobility under cd patient scope,
It is more limited in the application of cd patient follow-up.
The major therapeutic goals of Crohn disease (Crohn ' s disease, CD) from induce and maintain alleviate turn to scope
Lower alleviation and mucous membrane healing (referring to 2).Compared with patient movable under disease scope, alleviation or mucous membrane heal under disease scope
Patient's prognosis is more preferable, be hospitalized and operability is decreased obviously (with reference to 3).Therefore under scope the monitoring of disease activity degree in CD patient
It is particularly important in disease control.
In recent years, it has been found that a variety of biomarkers are for monitoring mobility under cd patient scope.For example,
Vilela et al. has found that clinically used marker of inflammation c reactive protein (C-reactive protein, CRP) and red blood cell are heavy
Drop rate (erythrocyte sedimentation rate, ESR) has the monitoring of mobility under cd patient scope
Certain values (refer to 4)；But use CRP and ESR detection Crohn disease mobility specificity poor.For another example, the human hairs such as Mosli
Existing excrement calprotectin has very high correlation (referring to 5) with active level under scope；But the concentration of calprotectin is by excrement
Just it is influenced in the remaining time of enteron aisle, patient age, test method etc., it is more difficult to obtain accurate diagnostic threshold.
As it can be seen that the biomarker that uses in the prior art exists, specificity is bad, testing result is unstable precisely etc. asks
Topic.
Therefore, need it is a kind of can it is special, accurate and stablize reflection cd patient disease activity biomarker.
1:Moresco RN, Speeckaert MM, Zmonarski SC, et al.Urinary myeloid IgA
Fcalpha receptor(CD89)and transglutaminase-2as new biomarkers for active
IgAnephropathy and henoch-purpura nephritis[J].BBA Clin,2016,5:79-
84.DOI:10.1016/j.bbacli.2016.02.002.
2:Allen P B, Olivera P, Emery P, et al.Review article:moving towards
common therapeutic goals in Crohn's disease and rheumatoid arthritis[J]
.Aliment Pharmacol Ther,2017,45(8):1058-1072.DOI:10.1111/apt.13995.
3:Neurath M F, Travis S P.Mucosal healing in inflammatory bowel
diseases:a systematic review[J].Gut,2012,61(11):1619-1635.DOI:10.1136/gutjnl-
2012-302830.
4:Vilela EG, Torres HO, Martins FP, et al.Evaluation of inflammatory
activity in Crohn's disease and ulcerative colitis[J].World J Gastroenterol,
2012,18 (9): 872-881.5:Mosli MH, Zou G, Garg SK, et al.C-Reactive protein, fecal
calprotectin,and stool lactoferrin for detection of endoscopic activity in
symptomatic inflammatory bowel disease patients:a systematic review and meta-
analysis[J].Am J Gastroenterol,2015,110(6):802-819.
5:D'Angelo F, Felley C, Frossard J L.Calprotectin in daily practice:
where do we stand in 2017[J].Digestion,2017,95(4):293-301.DOI:10.1159/
000476062.
Summary of the invention
For the unstable essence of biomarker poor specificity, testing result of Crohn disease disease activity in the prior art
Quasi- problem, the present invention provides a kind of biomarkers of novel Crohn disease.
In a first aspect, the biomarker is TGM2 the present invention relates to a kind of biomarker of Crohn disease；Tool
Body, the biomarker is serum TG M2, plasma TG M2, urine TGM2, Pleural effusions TGM2 or cerebrospinal fluid TGM2；It is preferred that
For plasma TG M2.
Second aspect, the present invention relates to a kind of facing for biomarker internal Crohn disease (CD) patient described in text
The monitoring method of activity condition under bed/scope.The present invention can be used for providing suitable therapeutic scheme for cd patient, such as
Identification is by receiving treatment or assisting in the treatment of the patient of income.
Specifically, the monitoring method includes obtaining the test sample of cd patient；It measures in test sample
TGM2 concentration assesses activity condition under clinic/scope of Crohn disease according to measurement result.
According to assessment result, treatment or adjuvant treatment are given for the patient of disease activity under clinic/scope, for facing
The patient of remission does not give treatment or adjuvant treatment under bed/scope.
In some specific embodiments, the monitoring is monitoring serum TG M2 concentration, plasma TG M2 concentration, urine
TGM2 concentration, Pleural effusions TGM2 concentration or cerebrospinal fluid TGM2 concentration；Preferably monitor plasma TG M2 concentration.
In some more specific embodiments, assessment result are as follows: the blood plasma of the cd patient of disease activity under scope
The concentration of TGM2 is 12.70ng/mL；The concentration of the plasma TG M2 of the cd patient of remission is 0.37ng/ under scope
mL。
In other more specific embodiments, assessment result are as follows: the blood plasma of the cd patient of clinical disease activity
The concentration of TGM2 is 16.49ng/mL；The concentration of the plasma TG M2 for the cd patient that clinical disease is alleviated is 0.55ng/mL.
More specifically, the test sample is serum TG M2, plasma TG M2, urine TGM2, Pleural effusions TGM2 or brain ridge
Liquid TGM2；Preferably plasma TG M2.
More specifically, the measuring method of TGM2 concentration is enzyme-linked immunosorbent assay in the measurement test sample.
The third aspect, the present invention include the method that selective therapy has the object of Crohn disease, and the method includes obtaining
The test sample of cd patient；Measure test sample in TGM2 concentration, according to measurement result assess Crohn disease clinic/
Activity condition under scope；According to assessment result, treatment or adjuvant treatment are given for the patient of disease activity under clinic/scope,
Treatment or adjuvant treatment are not given for the patient of remission under clinic/scope.
Fourth aspect, the present invention include that the reagent of detection TGM2 concentration refers in preparation for assessing, monitoring, detect, treat
It leads, the purposes in the tool of prognosis prediction or prognosis intervention Crohn disease.The tool is kit.
5th aspect, the present invention includes a kind of kit, it is characterised in that: the kit includes for measuring blood plasma
The reagent of 2 concentration of glutamine transaminage；The kit further includes Crow grace disease activity situation under analysis clinic/scope
To predict whether patient can benefit from the tool for the treatment of or adjuvant treatment.
6th aspect, the present invention include a kind of prognosis of the object for predicting to have Crohn disease or the calculating classified to it
Machine product, the product include the hand for receiving the data of TGM2 concentration in the sample for correspond to the object for having Crohn disease
Section；And the means of risk score are generated based on expression value is inputted to database, comprising related to prognosis in the database
Control concentration table, wherein risk score predicts the survival prognosis of the object or to be divided into remission group or disease living
Dynamic group.
" biomarker " can be used as the molecule of biological condition indicant in object.About this theme, it is disclosed herein
Biomarker can be display density variation and it exists can be used for prognosis or prediction object whether to benefit from receiving special
Surely the molecule treated.
In a series of specific embodiments of above-mentioned various aspects, plasma TG M2 concentration is that 4.81ng/mL is Crow grace
Disease assessment threshold value is determined as that the object is in Crohn disease as the plasma TG M2 concentration > 4.81ng/mL of samples sources object
Clinic/scope lower active stage is determined as that the object is in as plasma TG M2 concentration≤4.81ng/mL of samples sources object
Crohn disease clinic/scope lower paracmasis.
Compared with prior art, the beneficial effects of the present invention are as follows:
1, the present invention is for the first time using plasma TG M2 as the biomarker of assessment Crohn disease disease activity, patient plasma
TGM2 concentration is positively correlated with Crohn disease disease activity index (CDAI), under patient plasma TGM2 concentration and Crohn disease scope
Severity index (CDEIS) is also positively correlated, and correlation is superior to standard biologic marker.
2, the present invention is using TGM2 concentration as the evaluation index of Crohn disease activity condition, sensibility 76.32%, specifically
Property is 93.75%.
3, in general, the present invention compared with the existing technology, the biology of the Crohn disease disease activity situation that provides assessment
Marker and its corresponding index specificity is more preferable, testing result is more stable precisely.
Detailed description of the invention
Fig. 1 is TGM2, CRP, ESR, PLT in experimental example 1 to diagnostic figure movable under Crohn disease scope.
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is furture elucidated, and however, the following embodiments are merely preferred embodiments of the present invention, and not all.
Based on the implementation example in the implementation mode, those skilled in the art's obtained other realities without making creative work
Example is applied, protection scope of the present invention is belonged to.Experimental method in following embodiments is unless otherwise specified conventional method,
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Clinical blood sample information
(1) samples sources
In October, 2017 to during in October, 2018, the Crow of enteroscopy is planned to implement in No.1 Hospital Affiliated to Zhongshan Univ.
Grace patient 70 and the normal healthy volunteer of No.1 Hospital Affiliated to Zhongshan Univ.'s enteroscopy 35.Patient and health aspiration
Person knows and signs informed consent form, this research has obtained No.1 Hospital Affiliated to Zhongshan Univ.'s clinical research and experimental animal human relations
The reason committee ratifies (human relations Shen [2019] 084).
(2) patient and healthy volunteer are included in standard
With reference to Crohn disease diagnosis and treatment Europe evidence-based National Consensus in 2016 (bibliography Gomoll ó n F, Dignass A,
Annese V,et al.3rd European evidence-based consensus on the diagnosis and
management of Crohn's disease 2016:part 1:diagnosis and medical management
[J] .J Crohns Colitis, 2017,11 (1): 3-25.), cd patient is specifically included in standard are as follows: 1. clarifies a diagnosis
For Crohn disease person；2. less than 70 years old age person；3. complete clinical data has c reactive protein in 3 days before and after colonoscopy
(C-reactive protein, CRP), erythrocyte sedimentation rate (ESR) (erythrocyte sedimentation rate, ESR) and blood
Platelet number (platelets, PLT) testing result person.
Disease parting, exclusion criteria are as follows: 1. simple upper digestive tract are carried out to Crohn disease according to Montreal parting standard
Type Crohn disease；2. autoimmune disease is suffered from, such as systemic loupus erythematosus, rheumatoid arthritis, ankylosing spondylitis
Deng；3. with anaphylactias such as asthma, allergic urticarias；4. nearly one month once had respiratory tract, urethral infection etc. acute
Infect history；5. being in the gestational period or nursing period, virus hepatitis active stage etc..
Healthy volunteer is included in standard are as follows:
1. the past physical examination is without enterogastric diseases, autoimmune, chronic, tumour medical history and acute infection history in January；②
Enteroscopy is normal.
It is included in 70 cd patients and 35 healthy volunteers altogether according to the above standard.Wherein, cd patient
In, male 48, women 22, the median age is 28 years old；In healthy volunteer, male 20, women 15, the median age is
25 years old.
(3) 70 cd patient data
Collect all cd patient venous samples for being included in research and clinical data, comprehensive assessment.
Patient is judged according to Crohn disease disease activity index (Crohn ' s disease activity index, CDAI)
Disease activity.70 cd patients can be divided into two subgroups, clinical event group 33 (CDAI >=150) and clinic are delayed
The system of solutions 37 (CDAI < 150).
According to (the Crohn's disease endoscopic index of of severity index under Crohn disease scope
Severity, CDEIS) assess mobility under scope.70 cd patients can be divided into two subgroups, lived under scope
Alleviation group 32 (CDEIS≤3) under dynamic group 38 (CDEIS > 3) and scope.
Specific cd patient data such as table 1.
1 70 cd patient data of table
The measuring method of clinical indices
(1) plasma TG M2 concentration detection method
1. the acquisition of blood specimen, processing and preservation: in 3 days, using adopting for the anti-coagulants containing EDTA-K2 before and after enteroscopy
The cubital venous blood 4mL of blood vessel acquisition research object.1900g is centrifuged under the conditions of 4 DEG C in blood sample collection half an hour after
10min, transfer supernatant to 1.5mL centrifuge tube.Then 16000g is centrifuged 10min and removes remaining cell ingredient under the conditions of 4 DEG C, will
Upper plasma is transferred to new 1.5mL centrifuge tube.The blood plasma of acquisition is transferred to -80 DEG C of refrigerators and saves, and TGM2 is completed in two weeks
Concentration mensuration.
2. key instrument and reagent
TGM2 enzyme-linked immunosorbent assay kit (is purchased from Britain RayBiotech Life company), and microplate reader is (purchased from auspicious
Scholar Tecan company, model F50).
3. plasma TG M2 concentration mensuration
A. it configures reagent: seminal plasma fructose detection kit being answered and is warmed to room temperature.Using Sample dilution in kit by plasma sample
3 times of dilution；Respectively different concentration configuration TGM2 standard items (200ng/mL, 80ng/mL, 32ng/mL, 12.80ng/mL,
5.120ng/mL,2.048ng/mL,0.819ng/mL,0ng/mL).It is clear that 20X cleaning buffer solution is diluted to 1X using distilled water
Wash buffer.Use antibody and biotin dilution configuration biotin antibody working solution and horseradish peroxidase Avidin
Working solution.
B.TGM2 Specification Curve of Increasing: it is each that various concentration standard solution is added in every hole in 96 holes of pre-coated antibody
A secondary orifices is arranged in 100uL, each standard items gradient solution, is incubated at room temperature 2.5h on circumference concussion shaking table.Get rid of liquid in hole
Body, every hole are added 300uL 1X cleaning buffer solution and clean four times, pat dry on dry paper handkerchief after last time is cleaned remaining in hole
Liquid.100uL biotin antibody working solution is added in every hole after cleaning, and circumference, which shakes, dries liquid in hole after incubation at room temperature 1h on shaking table
Body.1X cleaning buffer solution cleans 4 times.100uL Avidin working solution is added in every hole after cleaning, is incubated at room temperature on circumference concussion shaking table
Liquid in hole is dried after 45min.1X cleaning buffer solution cleans 4 times.The colour developing of 100uL tetramethyl benzidine is added in every hole after cleaning
Liquid, circumference, which shakes, is added 50uL terminate liquid after incubation at room temperature 30min on shaking table, and microplate reader is read under 450nm wavelength immediately.Make
With 1.4 Software on Drawing TGM2 concentration of CurveExpert-absorbance standard curve, calibration curve equation is obtained.
C. it measures TGM2 concentration: according to step b the method, replacing standard items with plasma sample, obtain absorbance value, it will
Absorbance value calculates each sample TGM2 concentration after substituting into calibration curve equation.
(2) measurement of other clinical indices: using conventional method measurement c reactive protein (C-reactive protein,
CRP), erythrocyte sedimentation rate (ESR) (erythrocyte sedimentation rate, ESR) and platelet count (platelets,
PLT)
Embodiment 1 carries out clinical indices measurement to sample
Healthy volunteer and cd patient plasma TG M2 concentration, CRP, ESR, PLT result such as table 2.Using IBM
24.0 software of SPSS analyzes data, meets the quantitative data of partial velocities with M (P25, P75) expression；Mean between two groups
Compare using Mann-Whitney rank sum test, multisample mean compares using after Kruskal-Wallis rank sum test
Bonferroni method compares averagely sum of ranks two-by-two；Correlation analysis uses Spearman correlation analysis.It is poor with P < 0.05
It is different statistically significant.
The healthy group of table 2 and sufferer group plasma TG M2 concentration, CRP, ESR, PLT result
(1) CRP, ESR and PLT compare:
Compared with clinical remission group, CPR (U=3.971, P < 0.01), ESR (U=3.266, the P < of clinical event group
0.001) it increases, PLT no significant difference (P=0.120).
Compared with alleviation group under scope, CPR (U=2.912, P < 0.01), ESR (U=2.902, the P of activity group under scope
< 0.01) it increases, PLT no significant difference (P=0.088).
(2) TGM2 concentration compares:
The TGM2 concentration of clinical event group is higher than clinical remission group (Z=5.565, P < 0.01) and healthy group (Z=
5.074, P < 0.01), the no significant difference (P=of plasma TG M2 concentration between clinical remission group and healthy group
0.566)。
Activity group plasma TG M2 concentration is higher than alleviation group (Z=5.447, P < 0.01) and normal control under scope under scope
Group (Z=5.572, P < 0.01), under scope between alleviation group and healthy group plasma TG M2 concentration no significant difference (P
=0.995).
(3) correlation of each index and CDAI, CDEIS
The correlation of table 3 TGM2, CRP, ESR, PLT and CDAI, CDEIS
| CDAI | CDEIS | |
| TGM2 | ρ=0.647 | ρ=0.727 |
| CRP | ρ=0.483 | ρ=0.505 |
| ESR | ρ=0.465 | ρ=0.448 |
| PLT | ρ=0.182, P=0.131 | ρ=0.312 |
According to table 3, plasma TG M2 concentration, CRP and ESR and CDAI are positively correlated, and PLT and CDAI correlation is without statistics
It learns meaning (P=0.131)；Plasma TG M2 concentration, CRP, ESR and PLT are positively correlated with CDEIS.
In conclusion cd patient plasma TG M2 concentration and CDAI (ρ=0.647, P < 0.01) and CDEIS (ρ=
0.727, P < 0.01) there is correlation, and it is substantially better than CRP, ESR, PLT.
Diagnostic value of the 2 plasma TG M2 concentration of embodiment to mobility under cd patient scope
Using MedCalc Software on Drawing ROC curve and calculate area under each index ROC curve, the best critical value of diagnosis, quick
Diagnostic between sensitivity, specificity and each index of comparison.It is that difference is statistically significant with P < 0.05.
Diagnostic figure is as shown in Figure 1.
ROC curve is analyzed the results show that plasma TG M2 concentration (AUC=0.871, P < 0.01), CRP (AUC=0.703, P
< 0.01), ESR (AUC=0.702, P < 0.01) have diagnostic value to activity under cd patient scope, and PLT is to CD
The activity of patient's scope is without diagnostic value (AUC=0.619, P=0.088).Plasma TG M2 concentration diagnostic value is compared with CRP (P=
0.017) and ESR (P=0.021) is high, but the no significant difference (P=0.989) of CRP and ESR diagnostic value.
Plasma TG M2 concentration, CRP, ESR, PLT are shown in Fig. 1 to movable diagnostic under CD scope.As shown in Figure 1, by blood
(as the plasma TG M2 concentration > 4.81ng/mL of samples sources object when starching TGM2 concentration > 4.81ng/mL as assessment threshold value
When, be determined as that the object is in Crohn disease clinic/scope lower active stage, when samples sources object plasma TG M2 concentration≤
When 4.81ng/mL, it is determined as that the object is in Crohn disease clinic/scope lower paracmasis.), Youden index reaches peak
0.70, sensibility 76.32%, specificity is 93.75%.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of biomarker of Crohn disease, it is characterised in that: the biomarker is glutamine transaminage 2.
2. biomarker according to claim 1, it is characterised in that: the biomarker is serum, blood plasma, urine
Glutamine transaminage 2 in liquid, Pleural effusions or cerebrospinal fluid.
3. biomarker according to claim 2, it is characterised in that: the biomarker is blood plasma glutamine
Transaminase 2.
4. the reagent for detecting 2 concentration of glutamine transaminage is being prepared for assessing, monitoring, detect, treat guidance, prognosis prediction
Or the purposes in the tool of Crohn disease is intervened in prognosis.
5. purposes according to claim 4, it is characterised in that: the tool is kit.
6. purposes according to claim 4, it is characterised in that: the glutamine transaminage 2 is serum, blood plasma, urine
Glutamine transaminage 2 in liquid, Pleural effusions or cerebrospinal fluid.
7. purposes according to claim 6, it is characterised in that: the glutamine transaminage 2 is blood plasma glutamine
Transaminase 2.
8. according to purposes described in claim 4-6 any one, it is characterised in that: the assessment Crohn disease is according to blood
Starch glutamine transaminage 2 concentration evaluation Crohn disease activity condition, be by 2 concentration of blood plasma glutamine transaminage
4.81ng/mL is that Crohn disease assesses threshold value, as the 2 concentration > 4.81ng/ of blood plasma glutamine transaminage of samples sources object
When mL, it is determined as that the object is in Crohn disease clinic/scope lower active stage, when the blood plasma glutamine of samples sources object turns
When 2 concentration of amine enzyme≤4.81ng/mL, it is determined as that the object is in Crohn disease clinic/scope lower paracmasis.
9. a kind of kit, it is characterised in that: the kit includes for measuring 2 concentration of blood plasma glutamine transaminage
Reagent.
10. a kind of prognosis of object for predicting to have Crohn disease or the computer product classified to it, it is characterised in that: institute
Stating product includes the means for receiving the data of TGM2 concentration in the sample for correspond to the object for having Crohn disease；And it is based on
Expression value is inputted to database to generate the means of risk score.
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| CN115932272B (en) * | 2022-09-23 | 2023-12-12 | 上海市第十人民医院 | Application of serological biomarker in clinical diagnosis of Crohn disease |
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