CN109320598A - Detect neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3 and its application - Google Patents
Detect neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3 and its application Download PDFInfo
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Abstract
The present invention provides a kind of detection neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3 and its application, and the inflammatory biomarker TFF3 includes having the amino acid sequence as shown in SEQ ID:1.Present invention determine that TFF3 and the index of neonatal necrotizing enterocolitis peripheral blood sample are closely related, it is by stages in strong positive correlation with Bell, it can be detected as marker for neonatal necrotizing enterocolitis peripheral blood, provide effective evidence for the prediction, diagnosis and screening of neonatal necrotizing enterocolitis.The present invention also provides a kind of kit for detecting newborn's peripheral blood sample, the reagent comprising specific recognition inflammatory biomarker TFF3, this method is easy to be quick and economical and practical.
Description
Technical field
The present invention relates to neonatal necrotizing enterocolitis peripheral blood inflammatory biomarkers, and in particular to inflammatory biology
Marker TFF3 and its application.
Background technique
Intestinal flora field planting, formula milk are fed and other factors collaboration causes the immature intestinal mucosa of premature to be in
Inflammatory reaction state is spent, making enterocyte damage, even necrosis is neonatal necrotizing enterocolitis (necrotizing
Enterocolitis of newborn, NEC) basic pathology physiological characteristic.Therefore, intestinal inflammation is not only in regulation
Play a significant role in epithelial cell proliferation, differentiation and transition process, in intestinal epithelial cell damage, apoptosis, necrosis progression
Play a significant role.Therefore, important link of the inflammatory reaction as NEC occurrence and development, more and more researchs go to seek at present
Inflammatory biomarker relevant to NEC early diagnosis and progression of disease.
Inflammatory biomarker and pathogenesis, disease progression and severity are closely related, can be used for evaluating the state of an illness, treat
Effect has clinical indicative function well.Inflammatory biomarker relevant to NEC can be divided into nonspecific inflammatory biology mark
Remember object and specific inflammatory biomarker.The former study it is more have c reactive protein (C-reaction protein, CRP),
Calcitonin member (procalcitonin, PCT) and serum amyloid A protein (serum amyloid A, SAA), above-mentioned biology mark
Remember in object due to clinical criteria and the difference of sample size, causes the evaluation to NEC not quite identical.But it is generally considered,
CRP is worth limited but significant to the monitoring and Index for diagnosis of the state of an illness in early diagnosis；And SAA is not only in the state of an illness
Monitoring and Index for diagnosis are significant, and are worth in early diagnosis also higher；PCT is only the mark of acute inflammatory reaction
Will, with coincident with severity degree of condition without significant correlation.
Enteron aisle trefoil factor (trefoil factor 3, TFF3) is that most important small molecule is more in trefoil factor family
Peptide is distributed mainly on enteron aisle.Play a significant role in being repaired after the self-protection, damage of gastrointestinal mucosa.Currently, there is not yet
Close the relevant report of TFF3 and neonatal necrotizing enterocolitis medical diagnosis on disease and prediction.
Summary of the invention
The purpose of the present invention is to provide a kind of high sensitivities, the detection neonatal necrotizing enterocolitis of high specificity
Peripheral blood inflammatory biomarker TFF3 and its application.
To achieve the above object, the present invention provides a kind of detection neonatal necrotizing enterocolitis peripheral blood inflammatory biology
Marker TFF3, the inflammatory biomarker TFF3 include having the amino acid sequence as shown in SEQ ID:1.
It is described for detecting the present invention also provides a kind of kit for detecting neonatal necrotizing enterocolitis peripheral blood
Inflammatory biomarker TFF3 in neonatal necrotizing enterocolitis peripheral blood, the inflammatory biomarker TFF3 include tool
Just like amino acid sequence shown in SEQ ID:1.
Specifically, the kit includes the reagent of inflammatory biomarker TFF3 described in specific recognition.
Preferably, the reagent of inflammatory biomarker TFF3 described in the specific recognition, which is selected from, specifically binds the inflammation
The antibody or ligand of property biomarker TFF3.
The present invention also provides above-mentioned detection neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3 to exist
Application in preparation prediction, diagnosis and screening neonatal necrotizing enterocolitis reagent.
The present invention analyzes inflammatory biomarker TFF3 in neonates group (77 patients NEC) and control group (80
The non-NEC newborn of example) otherness expressed in peripheral blood sample, the results showed that, inflammatory biomarker TFF3 is in case group
Expression be apparently higher than control group, illustrate that expression of the inflammatory biomarker TFF3 in peripheral blood can reflect new life
The pathological characteristic of youngster's necrotizing enterocolitis, and then determine that TFF3 can be used as detection neonatal necrotizing enterocolitis periphery
The biomarker of blood.
Further, the present invention using Spearman grade to TFF3 and Bell by stages (referring to " practical neonatology " (the
Four editions) the modified Bell staging scale of NEC) do correlation analysis, the results showed that, TFF3 and Bell is positively correlated by stages, and closes
Connection property is very strong.Further the results show that peripheral blood T FF3 expression and neonatal necrotizing enterocolitis it is serious
Degree is related, clinically can marker using TFF3 as neonatal necrotizing enterocolitis Bell by stages.
Compared with prior art, the present invention has the advantage that
(1) present invention determine that TFF3 and the index of neonatal necrotizing enterocolitis peripheral blood sample are closely related, with
Bell is in strong positive correlation by stages, can be detected as marker for neonatal necrotizing enterocolitis peripheral blood, for new life
Prediction, diagnosis and the screening of youngster's necrotizing enterocolitis provide effective evidence；
(2) peripheral blood sample materials are convenient in the present invention, non-invasive, and can continuous vitro detection, therefore detect periphery
Inflammatory biomarker TFF3 in blood can improve the screening of neonatal necrotizing enterocolitis and diagnosis to one newly
Level, and then improve cure rate and reduce the death rate；
(3) present invention provides the kit of detection newborn's peripheral blood sample, includes specific recognition inflammatory biomarker
The reagent of object TFF3, this method are easy to be quick and economical and practical；
(4) marker of detection peripheral blood provided by the invention, can combine with other markers, be newborn's necrosis
Property the prediction of enterocolitis, diagnosis, treatment and prognosis new comprehensive kit is provided, facilitate clinical application.
Detailed description of the invention
Fig. 1 is the working curve diagram of TFF3 standard items；
Fig. 2 is expression figure of the TFF3 in case group and control group peripheral blood sample；
Fig. 3 is TFF3 and Bell correlation analysis figure by stages.
Specific embodiment
Purpose in order to better illustrate the present invention, technical scheme and beneficial effects, below in conjunction with specific embodiment and attached
The invention will be further described for figure.It should be noted that following the methods of implementing are explained further to what the present invention was done
It is bright, it should not be taken as limitation of the present invention.If material used in the embodiment of the present invention, reagent all can be from without specified otherwise
Commercial sources obtain.
1 sample Collecting and dealing of embodiment
Case group: in March, 2016 is chosen in September, 2017 in ZhongShan University attached No.6 Hospital neonate department, Guangdong Province
Healthcare hospital for women & children's neonate department (including newborn surgery), the 5th the People's Hospital's neonate department of Dongguan City, children's health care institute of Foshan City
77 patients NEC of neonate department ward hospitalization, by stages referring to the modified Bell of " practical neonatology " (fourth edition) NEC
Standard is divided into 3 groups according to final diagnosis result: doubtful NEC (NEC I phase 28) makes a definite diagnosis NEC (NEC II phase 30), progress
NEC (NEC III phase 19), wherein feeding intolerance symptom (abdominal distension, gastric retention) occurs in doubtful NEC, all cases are equal
By imageological examination.
Control group: with the same period be hospitalized with the same gestational age of case, gender, birth weight, childbirth options, birth 5min Apgar
80 non-NEC newborns that scoring and blood sampling age in days match (it is abnormal to exclude systemic inflammatory response syndrome, pyemia, alimentary canal
Shape and Inherited Metabolic Disorders infant) it is control group.This research has passed through ZhongShan University attached No.6 Hospital Ethics Committee and has examined symbol
The ethical standard that Chinese Ethics Committee formulates is closed, while obtaining the informed consent of research object guardian.
Case group sample collection is after clinical diagnosis NEC in 4h, and application is sterile, the anticoagulant heparin pipe acquisition periphery 1ml is quiet
Arteries and veins blood 4 DEG C, is centrifuged 10min, takes upper plasma in sterile EP tube, be subsequently placed in -80 DEG C through 3000r/min after mixing well
Refrigerator saves, and concentrates with enzyme-linked immunosorbent assay kit (ELISA) to relevant acute phase reactive protein and Gut barrie r function
It can Protein Detection.Group selection blood sampling selection is compareed to take a blood sample with the consistent blood sampling time point of case group.
2 ELISA of embodiment detects blood plasma inflammatory biomarker
Concentration application enzyme-linked immunosorbent assay kit (the Wuhan China of blood plasma TFF3 acute stage and intestinal barrier function albumen
U.S. bioengineering Co., Ltd), operation requires to be detected to specifications, specific as follows:
1) kit basic parameter:
TFF3ELISA detection kit (detection range: 1.56ng/ml-100ng/ml；Sensitivity: 0.39ng/ml；It is accurate
Degree: batch internal difference CV% < 8%, difference between batch CV% < 10%；Specificity: this kit specific detection human TFF 3, and with other phases
Close albumen no cross reaction).
2) inflammatory biomarker testing principle: using double-antibody sandwich elisa (Sandwich ELISA), and this method is suitable
Inspection for the soluble antigen in the various liquid phases such as serum, cerebrospinal fluid, Pleural effusions.In simple terms, first by known antibodies packet
In solid phase, unadsorbed antibody is washed away；Sample to be examined is added, sufficiently after effect, in sample in corresponding antigens and solid phase
Know that antibody combines, washes away unbonded antigenic component；Known enzyme labelled antibody is added, then washes away unbonded enzyme labelled antibody；Add
After substrate, enzyme bottom exploded object generates color reaction, determines antigenic content in sample to be examined according to shade.Coated antibody and enzyme
Antibody of the labeling antibody generally be directed to different epitopes in antigen molecule.
3) preparation of sample and reagent:
A. various reagents the rewarming of reagent, sample: are moved into (18-25 DEG C) 30~40min of balance of room temperature.
B. the dilution of standard items: taking the standard items restored to room temperature, and 10000rpm is centrifuged 30s, its whole is made to be sunken to bottom of bottle.
Then it gently opens and bottle cap and 1ml Sample dilution is added, blow and beat 5~8 times repeatedly with sample loading gun, after sufficiently dissolving and mix i.e.
The dilution mother liquor S7 of standard items can be obtained.
Standard items mother liquor is diluted using gradient dilution method.Take 7 1.5ml EP pipes according to number consecutively S0~S6,
Every pipe is separately added into 250 μ l Sample dilutions, and 250 μ l standard items are drawn out of number S7 to S6, mixes, then draws from S6
In 250 μ l to S5, mix, then and so on, the doubling dilution of standard items can be completed.Sample dilution is as blank control
(S0), the standard items of TFF3 (table 1) various concentration are obtained, standard items need to be prepared in 15min before use.
1 TFF3 standard concentration of table
C. thickening and washing buffer dilutes process: 25 times of dilution is carried out to thickening and washing buffer, it is specific as follows: to measure
The deionized water of 480ml is in beaker, then measures the dense washing buffer of 20ml, and be slowly added in deionized water, to be mixed equal
It can be used after even, generally in prepared before use.When cryo-conservation, dense washing buffer, which has, to be salted out, can be in temperature before dilution
Heating helps its dissolution in water-bath.
D. it prepares biotin labelled antibodies working solution: the biotin labelled antibodies bottle 10000rpm of rewarming being centrifuged 30s, is made
Bottle interior antibody all focuses on the dilution that pipe is low, then antibody diluent is applied to carry out 100 times to it.After being mixed gently after dilution
It can be used, generally in prepared before use.
E. Horseradish peroxidase-conjugated avidin working solution is prepared: by the Horseradish peroxidase-conjugated avidin of rewarming
Bottle 10000rpm is centrifuged 30s, so that bottle content is all focused on pipe low, then dilute using Horseradish peroxidase-conjugated avidin
Release the dilution that liquid carries out 100 times to it.It can be used after being mixed gently after dilution, generally in prepared before use.
4) ELISA operating procedure:
A. be loaded: setting standard sample wells, sample to be tested hole are respectively provided with 1 multiple holes, and every hole is loaded 100 μ l.
B. it is incubated for: after sample adds, shaking gently mixing, cover plate patch, 37 DEG C of incubation 2h.
C. board-washing: carefully going board falling to paste, and discards liquid, drying, repeated washing.
D. plus biotin labelled antibodies working solution, 100 holes μ l/.
F. new plate patch, 37 DEG C of incubation 1h are covered.
G. it carefully goes board falling to paste, discards liquid, drying, board-washing 3 times on automatic plate washer, every hole adds dilution buffer
200 μ l impregnate 2min.
H. 100 hole μ l/ of Horseradish peroxidase-conjugated avidin working solution is added.
I.37 DEG C incubation 1h.
J. it discards liquid and dries, board-washing 3 times on automatic plate washer, every hole adds 200 μ l of dilution buffer, impregnates
2min。
K. TMB, 90 holes μ l/ are added.
L.37 it DEG C is protected from light and is incubated for 15min to 30min.
M. it terminates reaction: 50 hole μ l/ of terminate liquid is added.
N. data are read: being successively read each hole optical density at 450nm wavelength with microplate reader, time control is terminating reaction
In 5min.
Using the optical density of read standard items as abscissa, the concentration value of standard items is ordinate, draws working curve.
The working curve of gained standard items is as shown in Figure 1.The OD value for recording sample aperture, according to the working curve of the standard items of drafting
Calculate the concentration value of institute's test sample sheet.
The Bell of 3 NEC of embodiment is associated with Journal of Sex Research with intestinal barrier function albumen TFF3 by stages
Intestinal barrier function GAP-associated protein GAP blood plasma level is detected, statistics is all made of Mann-Whitney U inspection, with Bell
Correlation analysis uses Spearman grade related check by stages.It was found that expression explanation of the TFF3 in case group is significantly higher than pair
According to group, as shown in Fig. 2, CTRL is control group；NEC is case group.
Correlation analysis discovery done by stages (I phase, II phase, III phase) to TFF3 and Bell, TFF3 and Bell relationship degree by stages
By force, it is positively correlated (Fig. 3) in strong, related coefficient > 0.8.
The present invention provides the kit of detection neonatal necrotizing enterocolitis peripheral blood, including specific bond TFF3's
Reagent, the specific antibody comprising specific binding TFF3.The peripheral blood sample that patient is acquired according to mode in embodiment 1, with examination
The antibody contained in agent box is reacted, and the expression of TFF3 in patient peripheral's blood sample is detected, this is to detection, diagnosis patient
It is of great significance with the presence or absence of neonatal necrotizing enterocolitis.
The present invention analyzes inflammatory biomarker TFF3 in neonates group (77 patients NEC) and control group (80
The non-NEC newborn of example) otherness expressed in peripheral blood sample, the results showed that, inflammatory biomarker TFF3 is in case group
Expression be apparently higher than control group, illustrate that expression of the inflammatory biomarker TFF3 in peripheral blood can reflect new life
The pathological characteristic of youngster's necrotizing enterocolitis, and then determine that TFF3 can be used as detection neonatal necrotizing enterocolitis periphery
The biomarker of blood.
Further, the present invention does correlation analysis to TFF3 and Bell using Spearman grade by stages, the results showed that,
TFF3 and Bell is positively correlated by stages, and relevance is very strong.Further the results show that the expression of peripheral blood T FF3 and new
The severity of raw youngster's necrotizing enterocolitis is related, clinically can be using TFF3 as neonatal necrotizing enterocolitis
The marker of Bell by stages.
In conclusion the present invention has the advantage that
(1) present invention determine that TFF3 and the index of neonatal necrotizing enterocolitis peripheral blood sample are closely related, with
Bell is in strong positive correlation by stages, can be detected as marker for neonatal necrotizing enterocolitis peripheral blood, for new life
Prediction, diagnosis and the screening of youngster's necrotizing enterocolitis provide effective evidence；
(2) peripheral blood sample materials are convenient in the present invention, non-invasive, and can continuous vitro detection, therefore detect periphery
Inflammatory biomarker TFF3 in blood can improve the screening of neonatal necrotizing enterocolitis and diagnosis to one newly
Level, and then improve cure rate and reduce the death rate；
(3) present invention provides the kit of detection newborn's peripheral blood sample, includes specific recognition inflammatory biomarker
The reagent of object TFF3, this method are easy to be quick and economical and practical；
(4) marker of detection peripheral blood provided by the invention, can combine with other markers, be newborn's necrosis
Property the prediction of enterocolitis, diagnosis, treatment and prognosis new comprehensive kit is provided, facilitate clinical application.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<110>the 5th the People's Hospital of Dongguan City (Dongguan City peace the People's Hospital)
<120>neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3 and its application are detected
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<170> SIPOSequenceListing 1.0
<210> 1
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<212> PRT
<213> Homo sapiens
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Met Leu Gly Leu Val Leu Ala Leu Leu Ser Ser Ser Ser Ala Glu Glu
1 5 10 15
Tyr Val Gly Leu Ser Ala Asn Gln Cys Ala Val Pro Ala Lys Asp Arg
20 25 30
Val Asp Cys Gly Tyr Pro His Val Thr Pro Lys Glu Cys Asn Asn Arg
35 40 45
Gly Cys Cys Phe Asp Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys
50 55 60
Pro Leu Gln Glu Ala Glu Cys Thr Phe
65 70
Claims (5)
1. a kind of detection neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3, it is characterised in that: described
Inflammatory biomarker TFF3 includes having the amino acid sequence as shown in SEQ ID:1.
2. a kind of kit for detecting neonatal necrotizing enterocolitis peripheral blood, it is characterised in that: described new for detecting
Inflammatory biomarker TFF3 in raw youngster's necrotizing enterocolitis peripheral blood, the inflammatory biomarker TFF3 include having
The amino acid sequence as shown in SEQ ID:1.
3. the kit of detection neonatal necrotizing enterocolitis peripheral blood as claimed in claim 2, it is characterised in that: institute
State the reagent that kit includes inflammatory biomarker TFF3 described in specific recognition.
4. the kit of detection neonatal necrotizing enterocolitis peripheral blood as claimed in claim 3, it is characterised in that: institute
The reagent for stating inflammatory biomarker TFF3 described in specific recognition, which is selected from, specifically binds the inflammatory biomarker TFF3
Antibody or ligand.
5. detection neonatal necrotizing enterocolitis peripheral blood inflammatory biomarker TFF3 is making as described in claim 1
Application in standby prediction, diagnosis and screening neonatal necrotizing enterocolitis reagent.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014182940A1 (en) * | 2013-05-08 | 2014-11-13 | Northeastern University | Mucus strengthening formulations to alter mucus barrier properties |
-
2018
- 2018-10-29 CN CN201811268333.7A patent/CN109320598A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014182940A1 (en) * | 2013-05-08 | 2014-11-13 | Northeastern University | Mucus strengthening formulations to alter mucus barrier properties |
Non-Patent Citations (10)
| Title |
|---|
| BING-HONG ZHANG: "The therapeutic effect of recombinant human trefoil factor 3 on hypoxia-induced necrotizing enterocolitis in immature rat", 《REGULATORY PEPTIDES》 * |
| EDDY W.Y.NG: "Gut-Associated Biomarkers L-FABP,I-FABP,and TFF3 and LIT Score for Diagnosis of Surgical Necrotizing Enterocolitis in Preterm Infants", 《ANNALS OF SURGERY》 * |
| NINA A. MARTIN: "Active Transport of Bile Acids Decreases Mucin 2 in Neonatal Ileum: Implications for Development of Necrotizing Enterocolitis", 《PLOS ONE》 * |
| PAK CHEUNG NG: "An update on biomarkers of necrotizing enterocolitis", 《SEMINARS IN FETAL AND NEONATAL MEDICINE》 * |
| 于学林: "《现代急诊影像诊断手册》", 30 April 2018, 科学技术文献出版社 * |
| 佚名: "GenBank 登录号：AAH17859.1", 《NCBI》 * |
| 刘素佳: "新生儿坏死性小肠结肠炎相关炎性标志物研究进展", 《中国新生儿科杂志》 * |
| 安翠平: "《免疫***疾病临床检验与诊断》", 30 June 2017, 科学技术文献出版社 * |
| 邹鸿燕: "《免疫学检验理论与临床》", 31 January 2018, 天津科学技术出版社 * |
| 陈仁慧: "新生儿坏死性小肠结肠炎早期诊断生物标记物的研究进展", 《中国小儿急救医学》 * |
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Application publication date: 20190212 |