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
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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.
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Citations (4)
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|---|---|---|---|---|
| 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 |
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| 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 * |
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