CN110907650A - Application of nano magnetic beads enriched urine sediment PAX2 protein as biomarker for diagnosing primary vesicoureteral reflux - Google Patents
Application of nano magnetic beads enriched urine sediment PAX2 protein as biomarker for diagnosing primary vesicoureteral reflux Download PDFInfo
- Publication number
- CN110907650A CN110907650A CN201911124372.4A CN201911124372A CN110907650A CN 110907650 A CN110907650 A CN 110907650A CN 201911124372 A CN201911124372 A CN 201911124372A CN 110907650 A CN110907650 A CN 110907650A
- Authority
- CN
- China
- Prior art keywords
- urine
- protein
- pax2
- biomarker
- pax2 protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000002700 urine Anatomy 0.000 title claims abstract description 62
- 206010047370 Vesicoureteric reflux Diseases 0.000 title claims abstract description 50
- 201000008618 vesicoureteral reflux Diseases 0.000 title claims abstract description 50
- 208000031355 vesicoureteral reflux 1 Diseases 0.000 title claims abstract description 50
- 239000013049 sediment Substances 0.000 title claims abstract description 41
- 101000613577 Homo sapiens Paired box protein Pax-2 Proteins 0.000 title claims abstract description 39
- 102100040852 Paired box protein Pax-2 Human genes 0.000 title claims abstract description 39
- 239000000090 biomarker Substances 0.000 title claims abstract description 19
- 239000011324 bead Substances 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 15
- 102000011931 Nucleoproteins Human genes 0.000 claims abstract description 13
- 108010061100 Nucleoproteins Proteins 0.000 claims abstract description 13
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000001262 western blot Methods 0.000 claims description 9
- 230000001154 acute effect Effects 0.000 claims description 6
- 229940109239 creatinine Drugs 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 239000002102 nanobead Substances 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 12
- 230000002485 urinary effect Effects 0.000 abstract description 10
- 238000003745 diagnosis Methods 0.000 abstract description 9
- 230000006378 damage Effects 0.000 abstract description 5
- 208000014674 injury Diseases 0.000 abstract description 3
- 208000027418 Wounds and injury Diseases 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 12
- 238000011161 development Methods 0.000 description 6
- 238000010992 reflux Methods 0.000 description 6
- 206010061481 Renal injury Diseases 0.000 description 5
- ACTRVOBWPAIOHC-XIXRPRMCSA-N succimer Chemical compound OC(=O)[C@@H](S)[C@@H](S)C(O)=O ACTRVOBWPAIOHC-XIXRPRMCSA-N 0.000 description 5
- 230000003907 kidney function Effects 0.000 description 4
- 239000004005 microsphere Substances 0.000 description 4
- 231100000241 scar Toxicity 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 206010010356 Congenital anomaly Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 208000037806 kidney injury Diseases 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 201000005965 CAKUT Diseases 0.000 description 2
- 208000032544 Cicatrix Diseases 0.000 description 2
- 238000000729 Fisher's exact test Methods 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 206010023421 Kidney fibrosis Diseases 0.000 description 2
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 2
- 101100518992 Mus musculus Pax2 gene Proteins 0.000 description 2
- 102000007999 Nuclear Proteins Human genes 0.000 description 2
- 108010089610 Nuclear Proteins Proteins 0.000 description 2
- 108010033276 Peptide Fragments Proteins 0.000 description 2
- 102000007079 Peptide Fragments Human genes 0.000 description 2
- 206010037597 Pyelonephritis acute Diseases 0.000 description 2
- 206010065427 Reflux nephropathy Diseases 0.000 description 2
- 208000034955 Renal or urinary tract malformation Diseases 0.000 description 2
- 201000001555 acute pyelonephritis Diseases 0.000 description 2
- 208000020832 chronic kidney disease Diseases 0.000 description 2
- 208000023124 congenital anomaly of kidney and urinary tract Diseases 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 230000037387 scars Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000003932 urinary bladder Anatomy 0.000 description 2
- 208000019206 urinary tract infection Diseases 0.000 description 2
- 206010001580 Albuminuria Diseases 0.000 description 1
- 102000012192 Cystatin C Human genes 0.000 description 1
- 108010061642 Cystatin C Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 1
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 1
- 101100518991 Homo sapiens PAX2 gene Proteins 0.000 description 1
- 206010020524 Hydronephrosis Diseases 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 1
- 208000000693 Neurogenic Urinary Bladder Diseases 0.000 description 1
- 206010029279 Neurogenic bladder Diseases 0.000 description 1
- 101150085593 PAX2 gene Proteins 0.000 description 1
- 101800001646 Protein n Proteins 0.000 description 1
- 206010037596 Pyelonephritis Diseases 0.000 description 1
- 108010031374 Tissue Inhibitor of Metalloproteinase-1 Proteins 0.000 description 1
- 102000005353 Tissue Inhibitor of Metalloproteinase-1 Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 206010046580 Urinary tract malformation Diseases 0.000 description 1
- 102100038854 Uroplakin-3a Human genes 0.000 description 1
- 108050004295 Uroplakin-3a Proteins 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 239000000091 biomarker candidate Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 238000009535 clinical urine test Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 201000000523 end stage renal failure Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 208000006750 hematuria Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 210000000244 kidney pelvis Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 206010038433 renal dysplasia Diseases 0.000 description 1
- 210000005084 renal tissue Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 210000003708 urethra Anatomy 0.000 description 1
- 208000014001 urinary system disease Diseases 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- 238000007487 urography Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses application of a PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux. The PAX2 protein is derived from urine sediments. The PAX2 protein is obtained by enriching urine sediments through nano magnetic beads after cracking nucleoprotein. The urinary sediment PAX2 detection is a VUR highly-related factor, has higher value in VUR diagnosis and is a VUR more reliable biomarker. The invention detects the protein index in the urine of the infant, belongs to non-invasive examination and does not cause invasive injury to the infant; the specimen is urine, is easy to leave and take, is simple to operate and has high repeatability.
Description
Technical Field
The invention relates to the field of biomarkers, in particular to application of a nano magnetic bead enriched urine sediment PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux.
Background
Vesicoureteral reflux (VUR) is a common Congenital renal and Urinary tract malformation (CAKUT), resulting from a defect in valve development at the Congenital vesicoureteral junction, with prevalence of about 1-2% in healthy children, and as high as 19% and 25% in neonates with prenatal renal pelvis watery in the Urinary Tract and Infections (UTI) in infants with UTI).
VUR on the one hand, it has a high risk of UTI, and 5% -10% of children may suffer from persistent kidney damage and kidney scarring, which in turn leads to the formation of Reflux Nephropathy (RN), which may ultimately lead to End Stage Renal Disease (ESRD); on the other hand, the difficulty of early diagnosis, especially the newborn diagnosis, is a focus of attention and research of experts in children's kidney disease and urinary surgery at home and abroad. Voiding bladder urography (MCU) is the gold standard for VUR diagnosis and grading, but the procedure is invasive, there is radiation exposure, and the drawbacks of heavy psychological burden on parents make MCU compliance with the examination greatly affected; the 99 mTc-radionuclide renal static scanning (DMSA) is the current gold standard for diagnosing Acute Pyelonephritis (APN), detecting renal scars and evaluating renal function, and the early research of the applicant indicates that in the acute stage of febrile urinary tract infection, DMSA has better prediction capability on high-level VUR while detecting renal damage, so that the use of MCU can be reduced to a certain extent, but DMSA still has certain false negative on prediction high-level VUR, and the cost of DMSA is higher, the technical requirement on instruments is higher, and the wide development is limited.
Since VUR can cause pyelonephritis, renal scarring, and even chronic renal failure, early detection and early diagnosis of VUR are important points in the prevention and treatment of CAKUT. Because VUR lacks specific clinical manifestations in early stage, the diagnosis is still mainly based on invasive MCU. In order to reduce the invasive detection of VUR discovery and diagnosis, researchers have studied biomarkers related to their pathogenesis, and the determination of known candidate biomarkers in urine and the screening and deep research of unknown biomarkers will inevitably provide noninvasive detection means which is easy to popularize, thereby improving the prognosis and quality of life of children patients. In the research on urinary system diseases, urine is recognized as having a specific diagnostic value, is convenient to collect, can be obtained noninvasively and is stable, so that in recent years, researchers at home and abroad gradually start to research VUR molecular diagnosis strategies of early urine, and research VUR urine biomarkers are diagnosed VUR at an early stage with minimal trauma.
The VUR biomarker researches reported at present mainly focus on inflammatory cell-related factors IL-6 and IL-8, urothelial related proteins (UPIII mRNA and UpIb mRNA), kidney fibrosis related molecules MMP9 and an inhibitor TIMP1 thereof and the like. Related researches indicate that IL-6, IL-8 and the like are mainly related to kidney injury, and have no obvious correlation with VUR; studies related to UpIb mRNA suggest that UpIb can be used as a reference to predict its repeated UTI episodes after the first UTI episode, with no clear correlation with VUR; MMP-9 and TIMP-1 are fibrosis related indexes, and research shows that the indexes are possibly related to renal scars; in the study of the two indexes, the research object is only high-grade VUR infants, so that the correlation between the related indexes and low-grade reflux is not clear at present.
Disclosure of Invention
The invention aims to solve the technical problems and provides an application of a nano magnetic bead enriched urine sediment PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux.
The invention is realized according to the following technical scheme.
Use of a PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux.
Further, the PAX2 protein is derived from urine sediments.
Further, the PAX2 protein is obtained by applying nano magnetic beads to enrich urine sediments after cracking of nuclear proteins.
Further, the method for enriching the urine sediment PAX2 protein by using the nano magnetic beads comprises the following steps:
a. the urine of the infant patient is kept in the non-infection acute stage, and the urine creatinine is measured to determine the urine concentration;
b. centrifuging the urine at the temperature of 4 ℃ and 1500rpm for 30-45min to leave urine sediments, adding 1-1.5ml PBS to clean impurities in the sediments, centrifuging at the temperature of 4 ℃ and 1500rpm for 5-10min, and cleaning for 2 times in total;
c, extracting urine sediment nucleoprotein from the washed urine sediment, and determining the protein concentration;
d. the extracted nucleoprotein is enriched with PAA-MSP nanometer magnetic beads, and the supernatant is concentrated and then used for detecting the expression of PAX2 protein by WesternBlot.
Further, the method for enriching the urine sediment PAX2 protein by using the nano magnetic beads comprises the following steps:
a. the urine of the infant patient is kept in the non-infection acute stage, and the urine creatinine is measured to determine the urine concentration;
b. centrifuging urine at 4 deg.C and 1200rpm for 40min to collect urine sediment, adding 1ml PBS to clean impurities in the sediment, centrifuging at 4 deg.C and 1200rpm for 10min, and cleaning for 2 times;
c. extracting urine sediment nucleoprotein from the washed urine sediment, and determining the protein concentration;
d. the extracted nucleoprotein is enriched with PAA-MSP nanometer magnetic beads, and the supernatant is concentrated and then used for detecting the expression of PAX2 protein by WesternBlot.
The present invention obtains the following advantageous effects.
The invention detects the protein index in the urine of the infant, belongs to non-invasive examination and does not cause invasive injury to the infant; the specimen is urine, is easy to leave and take, is simple to operate, has high repeatability and has wide application prospect.
Drawings
FIG. 1 is a diagram showing the Western Blot detection of the PAX2 protein of the invention.
Detailed Description
The invention is further explained below with reference to the drawings and the examples.
First, grouping and basic information collection of clinical children patients
1. Patients with hydronephrosis and urinary tract infection, which were examined by MCU (microprogrammed control unit) in the department of pediatrics hospital affiliated to the university of Fudan, were treated as a case group, and VUR-confirmed children were examined by gold standard VCUG (urinary bladder urethrography), and the patients without reflux manifestation in MCU examination were treated as a control group. In order to compare VUR the relationship between the reflux level and the malformed kidney damage, the infants were further divided into no reflux group, low-grade VUR group and high-grade VUR group according to the MCU examination result; wherein the levels I and II are divided into low level VUR groups and the levels III-V are high level groups. For patients with bilateral reflux, the patients are grouped according to the side with more severe reflux.
2. Removing secondary VUR children (posterior urethra valve, neurogenic bladder, etc.), and removing hematuria and albuminuria;
3. collecting the basic data and routine detection data of the infant patients, comprising:
(1) collecting basic information of the infant patient, including name, sex, treatment age, height, weight, birth history, physical examination, auxiliary examination, regional source and the like;
(2) all children to be brought into the study need to perfect the imaging examination of urinary system B ultrasonic, MCU, DMSA and the like and the detection of renal functions (blood creatinine and cystatin-C);
(3) the specimen was left for routine results on the day of urine.
Second, the retention and treatment of urine specimen of children patient
1. Keeping and taking urine of a group infant patient for 4 hours in a non-infection acute stage, collecting about 50-100ml urine samples according to different ages, and detecting urine creatinine to determine the urine concentration;
2. centrifuging at 4 deg.C and 1500rpm for 30min in half an hour after taking urine, collecting urine sediment, transferring the urine sediment into 1.5ml EP tube, adding 1ml PBS to clean impurities in the sediment, slightly and fully blowing, centrifuging at 4 deg.C and 1500rpm for 5min, and cleaning for 2 times;
3. the washed urine sediments are extracted by a Biyunnan cytoplasm cell nuclear protein separation and extraction kit according to the operation instruction, and protein concentration is measured by a BCA method and then protein enrichment is carried out or the obtained product is frozen in an ultra-low temperature refrigerator at minus 80 ℃;
4. the extracted nucleoprotein is enriched with PAA-MSP nano magnetic beads, and the supernatant is concentrated and then used for subsequent WesternBlot detection; applying 10mg/ml MSP @ PAA magnetic beads, adding 25ul of nucleoprotein lysate (after BCA measuring protein concentration, calculating total protein n, n-25 protein concentration), adding 10 times total nucleoprotein MSP @ PAA (volume is V, V-10 n/10mg/ml), adding ddH2And supplementing O to the total volume of 100ul, incubating for 20min at room temperature, performing magnetic separation, and concentrating the supernatant for the next protein detection experiment. MSP @ PAA magnetic beads are provided by national emphasis laboratories of the Polymer materials System of the university of Compound Dan.
Western Blot to detect the expression of PAX2 protein, and Histone 3 is used as nuclear reference protein.
Third, the result of the detection related to urinary sediment protein PAX2
(I) morning urine test conditions at present:
group 1, VUR: 60 cases.
(1) Male 39, female 21;
(2) medium & high 48, low 12;
(3) renal injury (scar or renal function difference is more than or equal to 10 percent) 26, no renal injury: 34;
con group: 40 cases.
(1) 25 male, 15 female;
(2) kidney injury (scar or renal function difference is more than or equal to 10%) 7, no kidney injury: 33;
(II) the detection condition of the Pax2 Western Blot at present:
(1)
diagnosis VUR:
fisher's exact test: p <0.001, sensitivity: 58.33%, specificity: 95.00%, PPV: 94.59%, NPV: 60.32 percent
(2)
Diagnostic high level VUR:
fisher's exact test: p ═ 0.74, sensitivity: 58.33%, specificity: 41.67%, PPV: 80.00%, NPV: 20.00 percent
(III) the detection condition of the Pax2 Western Blot at present: see FIG. 1
Fourthly, the current results and conclusions are as follows:
the separation and enrichment of low-abundance protein/peptide fragments in proteomics is an important link in the development of proteomics and peptide proteomics. In recent years, nano materials are increasingly applied to proteomics analysis due to rapid development and huge application potential, and magnetic polymer microspheres have the advantages of surface modification, good solution dispersibility, sensitive magnetic field inductivity and the like, so that the possibility of applying the nano materials to the separation and enrichment of trace peptide fragments in proteomics analysis is provided. The functional magnetic micro-nano material (magnetic microspheres) is used as the adsorbent, so that the problems that the analysis of the mass spectrometry interference machine trace sample by salt and urea in the urine sample is difficult and the like can be solved, and a new prospect is opened for VUR urine proteomics and clinical application thereof.
The target protein PAX2 detected by the invention plays a very important role in the development of the kidney in the embryonic period, the time and the level of the expression of PAX2 are very critical to the maintenance of the development of normal kidney tissues, the congenital renal dysplasia is caused by the link imbalance, trace expression or closure is realized after the birth under the normal condition, the report that the PAX2 gene is still obviously expressed in the urinary system after the birth is not found under the physiological condition, and the applicant finds that the expression of the PAX2 protein of the ureteral epithelial cells of VUR children patients is positive through earlier research, and the expression is not realized in a control group.
The invention applies nano-magnetic microspheres based on earlier research results and a novel magnetic nano-microsphere protein enrichment technology, and applies novel PAX2 protein in urinary sediment as a biomarker for diagnosing VUR. As a result: a total of 100 persons were enrolled, of which VUR group 60 (male 39, female 21) and Control group 40 (male 22, female 18). VUR the test results showed that 35 of 60 children had detected positive PAX2 in their urinary sediment, and 2 of 40 children had detected positive PAX2 in their control group. Using Fisher's test, the urinary sediment PAX2 protein expression was statistically different between VUR and control (P <0.001, sensitivity: 58.33%, specificity: 95.00%, PPV: 94.59%, NPV: 60.32%). The urinary sediment PAX2 protein has no obvious statistical difference in the low-grade VUR group and the medium-grade VUR group (P is 0.74, sensitivity: 58.33%, specificity: 41.67%, PPV: 80.00%, NPV: 20.00%), the detection of the urinary sediment PAX2 is a VUR highly-related factor, has higher value in VUR diagnosis, and is a VUR more reliable biomarker. The specificity of the kit in diagnosing whether UTI patients exist or not VUR is high, so that if a PAX2 positive UTI child is detected, VUR is highly suspected, and the kit can assist VUR diagnosis in clinic.
Claims (5)
1. Use of a PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux.
2. The use of a PAX2 protein as a biomarker for diagnosing essential vesicoureteral reflux according to claim 1, wherein the PAX2 protein is derived from urine sediments.
3. The use of PAX2 protein as a biomarker for diagnosing essential vesicoureteral reflux according to claim 2, wherein the PAX2 protein is obtained by using nanobead-enriched urine sediment protein after nucleoprotein cleavage.
4. The use of PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux according to claim 3, wherein the method for enriching urine sediment PAX2 protein by using nano magnetic beads comprises the following steps:
a. the urine of the infant patient is kept in the non-infection acute stage, and the urine creatinine is measured to determine the urine concentration;
b. centrifuging the urine at the temperature of 4 ℃ and 1500rpm for 30-45min to leave urine sediments, adding 1-1.5ml PBS to clean impurities in the sediments, centrifuging at the temperature of 4 ℃ and 1500rpm for 5-10min, and cleaning for 2 times in total;
c, extracting urine sediment nucleoprotein from the washed urine sediment, and determining the protein concentration;
d. the extracted nucleoprotein is enriched with PAA-MSP nano magnetic beads, and the supernatant is concentrated and then used for detecting the expression of PAX2 protein by Western Blot.
5. The use of PAX2 protein as a biomarker for diagnosing primary vesicoureteral reflux according to claim 3, wherein the method for enriching urine sediment PAX2 protein by using nano magnetic beads comprises the following steps:
a. the urine of the infant patient is kept in the non-infection acute stage, and the urine creatinine is measured to determine the urine concentration;
b. centrifuging urine at 4 deg.C and 1200rpm for 40min to collect urine sediment, adding 1ml PBS to clean impurities in the sediment, centrifuging at 4 deg.C and 1200rpm for 10min, and cleaning for 2 times;
c, extracting urine sediment nucleoprotein from the washed urine sediment, and determining the protein concentration;
d. the extracted nucleoprotein is enriched with PAA-MSP nano magnetic beads, and the supernatant is concentrated and then used for detecting the expression of PAX2 protein by Western Blot.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911124372.4A CN110907650A (en) | 2019-11-18 | 2019-11-18 | Application of nano magnetic beads enriched urine sediment PAX2 protein as biomarker for diagnosing primary vesicoureteral reflux |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911124372.4A CN110907650A (en) | 2019-11-18 | 2019-11-18 | Application of nano magnetic beads enriched urine sediment PAX2 protein as biomarker for diagnosing primary vesicoureteral reflux |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110907650A true CN110907650A (en) | 2020-03-24 |
Family
ID=69816855
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911124372.4A Pending CN110907650A (en) | 2019-11-18 | 2019-11-18 | Application of nano magnetic beads enriched urine sediment PAX2 protein as biomarker for diagnosing primary vesicoureteral reflux |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110907650A (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020142320A1 (en) * | 1999-02-20 | 2002-10-03 | Ogden Christopher William | Diagnosis and treatment of prostate cancer |
US20100029560A1 (en) * | 2007-01-16 | 2010-02-04 | Phigenix, Inc | Compositions and methods for diagnosing, treating, and preventing prostate conditions |
US20120157508A1 (en) * | 2005-10-14 | 2012-06-21 | Phigenix, Inc. | Targeting en2, pax2, and/or defb1 for treatment of prostate conditions |
CN109270276A (en) * | 2018-11-09 | 2019-01-25 | 北京师范大学 | The urine protein marker and its diagnostic uses of oophoroma |
-
2019
- 2019-11-18 CN CN201911124372.4A patent/CN110907650A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020142320A1 (en) * | 1999-02-20 | 2002-10-03 | Ogden Christopher William | Diagnosis and treatment of prostate cancer |
US20120157508A1 (en) * | 2005-10-14 | 2012-06-21 | Phigenix, Inc. | Targeting en2, pax2, and/or defb1 for treatment of prostate conditions |
US20100029560A1 (en) * | 2007-01-16 | 2010-02-04 | Phigenix, Inc | Compositions and methods for diagnosing, treating, and preventing prostate conditions |
CN109270276A (en) * | 2018-11-09 | 2019-01-25 | 北京师范大学 | The urine protein marker and its diagnostic uses of oophoroma |
Non-Patent Citations (2)
Title |
---|
YIBING ZHENG等: "The significance of Pax2 expression in the ureter epithelium of children with vesicoureteric reflux", 《HUMAN PATHOLOGY》 * |
甘肃省兰州兽医研究所: "《兽医手册》", 甘肃人民出版社 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Murakami et al. | Urinary screening of elementary and junior high-school children over a 13-year period in Tokyo | |
EP1340078B1 (en) | Method for differentiating irritable bowel syndrome from inflammatory bowel disease (ibd) and for monitoring persons with ibd using total endogenous lactoferrin as a marker | |
CN106405104B (en) | A kind of new cirrhosis or hepatic fibrosis markers | |
Sheu et al. | The role of procalcitonin for acute pyelonephritis and subsequent renal scarring in infants and young children | |
AU2002220029A1 (en) | Method for differentiating between irritable bowel syndrome and inflammatory bowel disease using lactoferrin as marker | |
CN112540180B (en) | ELISA detection kit for detecting human amyloid-beta double-antibody sandwich | |
Ghasemi et al. | Predictive accuracy of urinary neutrophil gelatinase associated lipocalin (NGAL) for renal parenchymal involvement in children with acute pyelonephritis | |
CN113049814A (en) | Application of rheumatoid arthritis marker detection reagent in serum and diagnostic kit | |
Cruz-Monserrate et al. | Biomarkers of Chronic Pancreatitis: A systematic literature review | |
CN103487493A (en) | Applications of urine molecule | |
JP2018080943A (en) | Method of detecting nash | |
Rahimzadeh et al. | Serum procalcitonin level for prediction of high-grade vesicoureteral reflux in urinary tract infection | |
CN110907650A (en) | Application of nano magnetic beads enriched urine sediment PAX2 protein as biomarker for diagnosing primary vesicoureteral reflux | |
CN107271655A (en) | A kind of kit and method for detecting urine excretion body load miRNAs | |
Jianguo et al. | Serum and urinary procollagen III aminoterminal propeptide as a biomarker of obstructive nephropathy in children | |
CN111856035B (en) | Application of TNC protein detection reagent and kit | |
CN111413502A (en) | New application of GP73 and liver tissue inflammation activity degree detection kit based on GP73 | |
Bazić-Đorović et al. | Technetium-99m-dimercaptosuccinic acid renal scintigraphy can guide clinical management in congenital hydronephrosis | |
CN110687285A (en) | Diagnostic kit and application of MAK16 in preparation of early diagnosis reagent for systemic lupus erythematosus | |
CN117031042B (en) | Biomarker for screening and diagnosing congenital heart disease fetus and application thereof | |
ZareMehrjardi et al. | Comparison of fecal calprotectin level in inflammatory bowel disease and irritable bowel syndrome | |
CN116047082B (en) | Application of FGL1 protein in preparing kit for diagnosing chronic kidney disease | |
CN114381529B (en) | Application of ACTR10 and CA125 combination in ovarian cancer detection and kit | |
CN113466469B (en) | Biomarker, detection kit and detection system for autism detection | |
CN114509573B (en) | Diabetes kidney disease early warning model established based on synchronous detection of urine markers |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200324 |