CN104502602A - Application of ARHGAP26 used as marker for diagnosing closing or opening of arterial ducts - Google Patents
Application of ARHGAP26 used as marker for diagnosing closing or opening of arterial ducts Download PDFInfo
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- CN104502602A CN104502602A CN201410723729.1A CN201410723729A CN104502602A CN 104502602 A CN104502602 A CN 104502602A CN 201410723729 A CN201410723729 A CN 201410723729A CN 104502602 A CN104502602 A CN 104502602A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to an application of ARHGAP26 used as a marker for diagnosing the closing or opening of arterial ducts. According to the application disclosed by the invention, a method of ITRAQ proteomics is used for carrying out authentication and analysis on the tissues of the opened and closed human arterial ducts, 132 different proteins are screened out and a signal channel analysis is carried out, then ARHGAP26 proteins are selected, and authentication is carried out by the three methods of Western Blot, Qrt-PCR and immunohistochemistry, which proves for the first time that the proteins are expressed in the tissues of the human arterial ducts; moreover, the proteins have obvious difference in the tissues of the two groups of closed and opened arterial ducts, are possibly a key factor for regulating the closing or opening of the arterial ducts, can be used for clinical diagnosis on the patency of the arterial ducts, and have a high diagnosis accuracy.
Description
[technical field]
The present invention relates to the clinical diagnosis technology field of molecular biology and medical science, specifically, relate to ARHGAP26 and close or open mark as diagnosis arterial duct.
[background technology]
Arterial duct (ductus arteriosus, DA) is the normal channel between fetal period descending aorta and pulmonary artery, and after normal newborn is raw, 10-15h can reach functional closedown, within 3 months, reaches closedown anatomically.If DA shutdown mechanism is abnormal, be patent ductus arteriosus (patent ductus arteriosus, PDA).PDA is common congenital heart disease, and the incidence of disease accounts for the 2nd in CMH.Premature because of oxygen induction DA contractile mechanism after making a living immature, and DA declines to the reaction sensibility of vaso-active substance, and the incidence of disease of PDA is higher, and body weight is lower than 45% having PDA in the premature of 1750g.Timely closedown arterial duct contributes to improving anoxic, controls heart failure, reduces premature death rate.
The clinical manifestation of patent ductus arteriosus depend on sustainer to pulmonary artery point bleeding capacity number and whether produce Pulmonary Hypertension Secondary and its degree.The lighter can non-evident sympton, and severe one heart failure can occur.Common symptom have tired after palpitaition, out of breath, weak, easily suffer from respiratory tract infection and growth retardation.Late period, pulmonary hypertension was serious, can occur the lower part of the body cyanosis when producing reverse shunting.During patent ductus arteriosus health check-up, typical sign is that left border of sternum the 2nd intercostal hears loud continuity machinery murmur, and with trembling, pulmonary artery the 2nd sound is hyperfunction, but often cover by loud noise.Close patient for patent ductus arteriosus in infants, only can hear systolic murmur, or systolic murmur also disappears and replaces the diastolic murmur of pulmonary incompetence, diagnosis difficulty is larger.Therefore the mark providing diagnosis arterial duct to close or open is very necessary.
ARHGAP26 (Rho GTPase activating protein 26), having another name called GRAF (GTPaseRegulator Associated with Focal Adhesion Kinase), is a kind of GTP enzyme activation albumen.ARHGAP26 energy negative regulation RhoA and Cdc42, affects the performance that Rho albumen promotes cell migration and proliferation function; In addition this albumen and FAK interact and participate in integrin signaling path, also critical function (Lundmark R is played in CLIC/GEEC (clathrin-independent carrier/GPI-anchored protein-enriched earlyendocytic compartment) endocytic pathway, Doherty GJ, etal.The GTPase-Activating Protein GRAF1 Regulates the CLIC/GEEC EndocyticPathwayp [J] .Curr Biol, 2008,18 (22): 1802-1808).Generally believe that ARHGAP26 has tumor suppression function, ARHGAP26 gene defect can cause granulocytic leukemia, in the patients such as the alpha Thalassemia feeblemindedness syndrome that medullary system malignant tumour, metastatic brain tumor, X-are chain, ARHGAP26 expression obviously declines.At present, the mark closing about ARHGAP26 as diagnosis arterial duct or open have not been reported.
[summary of the invention]
The object of the invention is, for deficiency of the prior art, to provide the purposes of ARHGAP26 albumen or its specific antibody.
Of the present invention again one object be, provide a kind of for Diagnosis of Patent Ductus Arteriosus close kit.
Another object of the present invention provides a kind of chip or array.
For achieving the above object, the technical scheme that the present invention takes is:
The application of ARHGAP26 albumen in the mark open or closed as diagnosis arterial duct.
The purposes of ARHGAP26 albumen or its specific antibody, the reagent closed for the preparation of Diagnosis of Patent Ductus Arteriosus or kit.
For realizing above-mentioned second object, the technical scheme that the present invention takes is:
For the kit that Diagnosis of Patent Ductus Arteriosus closes, described kit comprises the reagent detecting ARHGAP26 protein content and the reagent detecting β-actin protein content.
The reagent of described detection ARHGAP26 protein content is the specific antibody of ARHGAP26 albumen, and the reagent of described detection β-actin protein content is the specific antibody of β-actin albumen.
For realizing above-mentioned 3rd object, the technical scheme that the present invention takes is:
A kind of chip or array, described chip or array have the reagent detecting ARHGAP26 protein content and the reagent detecting β-actin protein content.
The reagent of described detection ARHGAP26 protein content is the specific antibody of ARHGAP26 albumen, and the reagent of described detection β-actin protein content is the specific antibody of β-actin albumen.
The invention has the advantages that:
The present invention uses the method for ITRAQ proteomics to carry out identification and analysis to open and closed human artery tracheal tissue, filter out 132 discrepant albumen, analyzed by signal path, pick out ARHGAP26 albumen, use western blot, Qrt-PCR and SABC three kinds of methods are identified, prove that this albumen is shown in human artery tracheal tissue first, and these albumen have obvious difference at two groups of closed and open arterial duct tissues, may be that regulation and control arterial duct closes or open key factor.The invention provides a protein marker ARHGAP26 for distinguishing closed arterial duct and not closed arterial duct, the reagent, kit, chip and the array that utilize this mark to prepare Diagnosis of Patent Ductus Arteriosus to close.The present invention provides reliable detection means for effectively judging arterial duct opening or closing.
[accompanying drawing explanation]
Accompanying drawing 1 is the comparison of closed (Constricted) and open (Patent) arterial duct techtology.
Accompanying drawing 2 is western-blot results of ARHGAP26 albumen.
Accompanying drawing 3 is RT-PCR results of ARHGAP26 albumen.
Accompanying drawing 4 is ImmunohistochemistryResults Results of ARHGAP26 albumen.
[embodiment]
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
Embodiment 1
One, materials and methods
1.1 are separated human artery tracheal tissue
According to health ministry related medical Ethic review method, this experiment all obtains the examination and approval of Ethics Committee of Medical College, Shanghai Communication Univ. with process in steps and signs Informed Consent Form with patient.
Open (n=10, age=30.2 ± 6.3days) or the arterial duct tissue of closed (n=10, age=25.3 ± 7.9days) be taken from the children with congenital heart disease (infant details are in table 1) of the Ductal dependence in Shanghai Jiaotong University Medical College subsidiary Shanghai Children's Medi's surgical procedure.All patients all use PGE1 before surgery, but the arterial duct of closed group is still closed before surgery.After all samples are in vitro, with normal saline flushing number time, be divided into four parts, then frozen in liquid nitrogen rapidly.The state (open or closed) of all samples all confirms through cardiac ultrasonic and CT, and is confirmed at surgical procedure.
Table 1
Note: TGA/IVS, complete transposition of great arteries/complete ventricular septum; PA/IVS; Pulmonary atresia; IAA, medium sized artery bow interrupts; PS, pulmonary artery blocks; NA, cannot obtain.* two groups of difference are pointed out to have significant difference.
The extraction of 1.2 protein and ITRAQ reagent mark
Get tissue samples, through lysate (7M urea, 2M thiourea, 65mM dithiothreitol, 0.1mMphenylmethylsulfonyl fluoride) homogenate cracking 1 hour under 4 DEG C of conditions, by centrifugal 30 minutes of lysate 12000rpm at 4 DEG C.Collect supernatant, by 2D quantification kit (Amersham Biosciences company) Quantitative Western concentration.For reducing the sample error that causes because of biomutation by common for 5 different sample mixing acquisitions one sample (closed group and open organize each one), then carry out proteome analysis.Therefore, acquisition 4 common samples (tissue that correspondence 2 kinds is different) carry out ITRAQ mark altogether.Trypsinization and ITRAQ mark carry out according to kit instructions.In brief, 100 μ g albumen first reductive alkylation, then 37 degree of pancreatin (mass spectrometry grade of the common sample acquisition of each mixing; Promega) digestion is spent the night.Sample iTRAQ
tMreagent (Applied Biosystems company) marks, and marks as follows: closed arterial duct tissue, iTRAQ reagent 114; Open arterial duct tissue, iTRAQ reagent 115.Two groups of different Isobaric Tags apply to four kinds of common albumen samples altogether.
1.32D LC-MS/MS
Hybrid peptide section is through strong cation exchange chromatography (SCX) fractionation, and fractionating system is 20AD highly effective liquid phase chromatographic system (HPLC, Shimadzu Corporation).In brief, hybrid peptide Duan Xianyong Sep-Pak Cartridge (Waters, USA) desalination, sample loading buffer (10mM KH
2pO
4in 25%acetonitrile [ACN], pH 2.8) dilution, load on separating column.Buffer A composition is identical with sample loading buffer, and buffer B is that sample loading buffer adds 350mM KCl.The separation of peptide section uses linear binary gradient to be separated, and namely 0-80% buffer B is dissolved in buffer A, and flow velocity is that 200 μ l are per minute, and the time is 60 minutes.The absorbance at monitoring 214nm and 280nm place, along separating column, collects altogether 30 sections of SCX fragments.After each segregation section drying, be dissolved in damping fluid C (5%ACN, 0.1%formic acid [FA]), and analyze on QSTARXL mass spectrometer (Applied Biosystems).In brief, peptides separation uses anti-phase (RB) separating column (ZORBAX300SB-C18 post, 5 microns, 300 dusts, the 0.1mm × 15mm of 20ADHPLC system (Shimadzu); Micromass).The gradient of HPLC is that the damping fluid D (95%CAN, 0.1%FA) of 5%-35% is dissolved in damping fluid C, and flow velocity is that 0.2 μ l is per minute, and the time is 65 minutes.The peptide section of ITRAQ mark produces report ion in 114.1 and 115.1Th under the separation condition of collision-induced.The ratio of report ion peak area reflects the relative content of peptide section, and result demonstrates the relative content of albumen in sample.
1.4 data analysis
The protein urine that iTRAQ obtains and quantitative test adopt ProteinPilot software (version 4.2, revision number:1340; Applied Biosystems).Data analysis optimum configurations is as follows: sample type: ITRAQ (labeled peptide); Halfcystine alkylation: methyl sulphur methanesulfonates; Digestion: pancreatin; Equipment: QSTARESI; Kind: the mankind; ID focus: biological regulation; Database: international protein index human data storehouse (version:3.45; 143,958entries); Query context: all; Maximum loss is sheared: 2; FDR analyzes: be; User's regulating parameter file: no; Bias Correction: automatically; Background is corrected: be.Obtain albumen through software enrichment to reduce repetition as far as possible.All peptides being used for calculating Protein ratios are all unique for given albumen.The cut-off of protein confidence threshold value is 1.3 (untapped ProtScore), has 95% confidence with at least one peptide.
1.5 histological stain
Arterial duct tissue is after 4% paraformaldehyde 4 spends night, and sample washs through PBS, after dehydration, and wax embedding.Lycra microtome (5 μm) Superfrost slide mounting, 37 degree of dried overnight.Dimethylbenzene dewaxes, and aquation, washs in distilled water.Then, tissue hematoxylin eosin staining.Endochylema dyes redness, and karyon dyes blueness.
1.6 gene ontologies (GO) and network analysis
Differentially expressed protein is analyzed by the GO of DAVID software (6.7 version) and is indicated.GO term is less than 0.05 with calculating P value and is considered to be remarkable enriching.The path analysis of protein expression form adopts IPA software (Ingenuity Systems Inc.) IPA software analysis to be based on Ingenuity Knowledge Base database, in the middle of contain known point subrelation, the association of function and disease.
1.7Western blotting analyzes
Following protein vs protein matter group result is selected to verify: Rho GTPase activating protein26 (ARHGAP26).Carry out a Western blotting analysis antibody information used as follows: ARHGAP26polyclonal antibody (Ab37618, and β-actin rabbit monoclonal antibody (4970, Cell Signaling Technology) Abcam).
By the albumen of purification with 10% separation gel be separated, 80 volts, 1 hour.Albumen carries out conventional transferring film, and 5% skimmed milk power closes 1 hour.According to antibody instructions, add primary antibodie, after hatching 2 hours under normal temperature, add the two anti-(1:5000 that corresponding HRP is coupled; Thermo Scientific Pierce) hatch 1 hour after, add ECL chemical luminescence for liquid (Merck, Milipore, USA).The optical density Chemi-Doc imaging system (Bio-Rad Laboratories) of band is analyzed.
1.8 real-time fluorescence quantitative PCR analyses
SYBR Green (Applied Biosystems) two-step approach is adopted to detect the mRNA level in-site of above-mentioned albumen.From frozen tissue, extract RNA with total RNA extraction reagent box (sky root), then with Takara Reverse Transcription box reverse transcription synthesis cDNA, then use SYBR Green Power Premix (ABI) to carry out polymerase chain reaction.Reaction system is 10 μ l, and DNA profiling is 1.0 μ l.ABI7900 real-time fluorescence quantitative PCR reaction system is as follows: 95 degree of 1 circulations in 10 seconds, 95 degree of 40 circulations in 15 seconds, 60 degree 60 seconds.Primer is synthesized by Chinese Jie Rui bio tech ltd, and sequence is in table 2.All sample standard deviations do 3 multiple holes, if the difference of Ct value is greater than 0.3 between multiple hole, then this multiple hole is not considered.
Table 2 primer sequence
1.9 immunohistochemical analysis
Arterial duct tissue is after 4% paraformaldehyde 4 spends night, and PBS washs, dehydration, wax embedding.Lycra microtome (5 μm) Superfrost slide mounting, 37 degree of dried overnight.Dimethylbenzene dewaxes, and aquation, washs in distilled water.. section is immersed containing 0.3%H
2o
2formaldehyde in 20 minutes with endogenic peroxidase of putting out a fire.Under room temperature, 5% skimmed milk power was closed after 2 hours, added primary antibodie 4 and spent night.Antigen antibody complex DAB peroxidase substrate kit (Dako) detects.Primary antibodie used is anti-ARHGAP26 (ab37618, Abcam).
2.0 statistical study
Result represents with mean ± standard deviation, and Group Design quantitative data t checks, and P<0.05 thinks statistically variant.
2.1 make Receiver Operating Characteristics (ROC) curve
ROC curve is done, to evaluate the diagnosis effect of the ARHGAP26 albumen with differential expression to the open group of arterial duct.
Two, experimental result
The western-blot of ARHGAP26 albumen the results are shown in Figure 2; The RT-PCR of ARHGAP26 albumen the results are shown in Figure 3; The ImmunohistochemistryResults Results of ARHGAP26 albumen is shown in Fig. 4.Above result shows, ARHGAP26 albumen is expressed in human artery tracheal tissue, and in open group and closed group, ARHGAP26 expressing quantity difference reaches the level of signifiance (* P<0.05), the mRNA relative content difference of ARHGAP26 albumen also reaches the level of signifiance (* P<0.05), and prompting ARHGAP26 may be that regulation and control arterial duct closes or open key factor.
Carry out ROC tracing analysis for 10 arterial ducts open patient ARHGAP26 protein level, the diagnostic value of this albumen is as shown in table 3.Result shows that ARHGAP26 albumen has higher diagnostic value to patent ductus arteriosus, and ACU can reach 0.826.
Table 3ARHGAP26 albumen relative content is to the diagnostic value of patent ductus arteriosus
| Diagnosis index | AUC | Sensitivity | Specificity |
| ARHGAP26 relative expression quantity=0.32 (ARHGAP26/ β-actin) | 0.826 | 93.6％ | 86.1％ |
| ARHGAP26 relative expression quantity=0.26 (ARHGAP26/ β-actin) | 0.728 | 85.1％ | 76.5％ |
Note: AUC is ROC area under curve; AUC, more close to 1, illustrates that diagnosis effect is better; AUC has lower accuracy 0.5 ~ 0.7 time, and AUC has certain accuracy 0.7 ~ 0.9 time, has high accuracy when AUC is more than 0.9.
Embodiment 2
To 20 doubtful patent ductus arteriosus patients with ARHGAP26 albumen relative content for mark is diagnosed.Adopt Western blotting to analyze, concrete grammar is with embodiment 1.With ARHGAP26 relative expression quantity=0.32 (ARHGAP26/ β-actin) for diagnosis critical value, be namely judged to be patent ductus arteriosus lower than above critical value, the diagnostic result of ARHGAP26 protein marker shows 14 patients for patent ductus arteriosus.Later stage, 14 patients all implemented arterial duct closure procedure, confirmed that these 14 patients are patent ductus arteriosus in operation.Other 6 patients have 5 to implement openheart surgery further, confirm 5 all non-patent ductus arteriosuss in operation.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.
Claims (6)
- The application of 1.ARHGAP26 albumen in the mark open or closed as diagnosis arterial duct.
- The purposes of 2.ARHGAP26 albumen or its specific antibody, is characterized in that, the reagent closed for the preparation of Diagnosis of Patent Ductus Arteriosus or kit.
- 3. for the kit that Diagnosis of Patent Ductus Arteriosus closes, it is characterized in that, described kit comprises the reagent detecting ARHGAP26 protein content and the reagent detecting β-actin protein content.
- 4. kit according to claim 3, is characterized in that, the reagent of described detection ARHGAP26 protein content is the specific antibody of ARHGAP26 albumen, and the reagent of described detection β-actin protein content is the specific antibody of β-actin albumen.
- 5. chip or an array, is characterized in that, described chip or array have the reagent detecting ARHGAP26 protein content and the reagent detecting β-actin protein content.
- 6. chip according to claim 5 or array, it is characterized in that, the reagent of described detection ARHGAP26 protein content is the specific antibody of ARHGAP26 albumen, and the reagent of described detection β-actin protein content is the specific antibody of β-actin albumen.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994028933A1 (en) * | 1993-06-04 | 1994-12-22 | New York University | Bispecific human monoclonal antibodies specific for human immunodeficiency virus |
| CN101528210A (en) * | 2006-12-07 | 2009-09-09 | 首尔大学校产学协力财团 | Method for screening anti-cancer compounds inhibiting function of TM4SF5 and anti-cancer composition containing chalcone compounds |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994028933A1 (en) * | 1993-06-04 | 1994-12-22 | New York University | Bispecific human monoclonal antibodies specific for human immunodeficiency virus |
| CN101528210A (en) * | 2006-12-07 | 2009-09-09 | 首尔大学校产学协力财团 | Method for screening anti-cancer compounds inhibiting function of TM4SF5 and anti-cancer composition containing chalcone compounds |
Non-Patent Citations (1)
| Title |
|---|
| QIONG WANG等: "ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding", 《RNA》 * |
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