CN109752558A - For diagnosing the biomarker of diabetic nephropathy early stage - Google Patents
For diagnosing the biomarker of diabetic nephropathy early stage Download PDFInfo
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- CN109752558A CN109752558A CN201810942585.7A CN201810942585A CN109752558A CN 109752558 A CN109752558 A CN 109752558A CN 201810942585 A CN201810942585 A CN 201810942585A CN 109752558 A CN109752558 A CN 109752558A
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- urine
- diabetic nephropathy
- calb1
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- 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
Abstract
The present invention provides a kind of for diagnosing the biomarker of diabetic nephropathy early stage, and the method for diagnosis diabetic nephropathy early stage.The biomarker is the CALB1 that the extracellular vesica of urine (urine extracellularvesicles urinates EVs) includes, and is significantly increased in diabetic nephropathy early stage expression quantity relative to normal person.Diagnostic method of the invention is non-intrusion type, there is huge applications prospect.
Description
Technical field
The present invention relates to Protein Detection fields, and in particular to the detection of the biomarker of diabetic nephropathy early stage.
Diabetes have become global public health problem.2015, International Diabetes Federation
(InternationalDiabetes Federation, IDF) is announced, there are 1.096 hundred million diabetics in China, and majority is 2 types
First there is impaired fasting glucose and/or impaired glucose tolerance, referred to as before its blood glucose reaches diabetes diagnostic criterion in diabetes
Prediabetes (diabetes before being otherwise known as).These patients have begun appearance although without apparent blood glucose rise
Organ injury, therefore extremely important meaning has been early diagnosed to it, the generation of diabetic complication can be more efficiently prevented from.Sugar
It urinates sick nephrosis and kidney failure is the common complication of diabetes, make a definite diagnosis diabetic nephropathy at present and punctured by biopsy, but this
It is a kind of risky invasive inspection.Microalbuminuria can diagnose the 3rd phase of diabetic nephropathy, but work as diabetic nephropathy also
When being in for 1,2 phase, do not occur microalbumin in urine also, at this moment how to diagnose early diabetic nephropathy and be still international at present
The key points and difficulties of upper this field research.That generally acknowledges not yet both at home and abroad at present can examine in early days before microalbuminuria appearance
The urinary biomarkers object of disconnected diabetic nephropathy.
The extracellular vesica of urine (extracellularvesicles, EVs) is originated from a variety of thin of entire urogenital tract
Born of the same parents are discharged into urine, and can be used as the biomarker of renal dysfunction and structural damage.
Diabetic nephropathy (DN) is the major complications of diabetes (DM), dramatically increases cardiovascular risk and associated death
Rate.The extracellular vesica of urine (EVs) is considered not only the Noninvasive source abundant of renal damage marker and whole body
The important symbol object source of organ either injury of blood vessel.Certain albumen that author of the present invention attempts to verify in urine EVs may be with
There is association in diabetic nephropathy.
There are a large amount of high-abundance proteins, such as tamm-Horsfall protein in urine under physiological status, and are able to reflect disease
Albumen in the urine of sick early stage information is often low-abundance, due to being influenced by other high-abundance proteins, is difficult special
It is different sensitively to detected.Significantly, the urine protein of these reflection disease early stage information is much present in urine
In EVs, if the method that we can find scientific and effective enrichment urine EVs, is then able to reflect disease in detection urine EVs
The protein of sick early stage information, can overcome the influence of high-abundance proteins in urine.In present invention research, it is intended to urinate
A kind of important biomarker is found and verified in liquid EVs, it may finally be assessed or is monitored, to reach DN early stage
The purpose of diagnosis.
Summary of the invention
The purpose of the present invention is to provide a kind of urine EVs inner proteins, for before microalbumin occurs in urine
Early diagnose diabetic nephropathy early stage.
We assume that it can be found that different protein, special in the urine EVs of the patient with diabetic nephropathy (DN)
It is not before microalbuminuria appearance.In present study, a large amount of urine specimens for DN Urinary EVs research are at this
What seminar obtained in community's molecule epidemic disease-ology research that southern community-based population carries out ISN " KHDC " project support.At this
In research, using bidirectional electrophoresis technique separation from the urine EVs with DN and the patient of health volunteer, and pass through
MALDI TOF/MS analysis carries out analysis and quantitative comparison to its protein component.
1980 protein spots in Urine in Patients EVs are isolated by electrophoretic techniques, wherein 40 protein spots and NC group
Compared to the significant difference for having at least 1.5 times.Mass Spectrometric Identification is carried out to above-mentioned 40 protein spots, finds wherein 22 kinds of protein
It may be closely related with diabetic nephropathy.
To above-mentioned differential protein carry out literature search, sufficiently retrieve urine CALB1 (Calb1 calbindin 1,
NCBI accession number P05937, molecular weight 30291.1, isoelectric point 4.7) document, account for kidney trouble renal glomerular disease (packet several greatly absolutely
Include glomerulonephritis, diabetic nephropathy) do not find report about urine CALB1.Document relevant to urine CALB1, it is main
Concentrate on features of idiopathic hypercalciuria.The above search result explanation, including the diabetic nephropathy that we study, accounts for kidney
In the most of renal glomerular disease of popular name for, the whole world does not have the report in relation to urine CALB1.
CALB1 is a kind of vitamin D-dependent calcium-binding protein (CaBP), is positioned at distal convoluted tubule and renal tubule.Lee
CT,Lien YH,Lai LW,Chen JB,et al..Increased renal calcium and magnesium
transporter abundance in streptozotocin-induced diabetes mellitus.KIDNEY INT
2006；69:1786-1791. has reported the research of the diabetes rat of streptozotocin (STZ) induction, shows cortex renis and marrow
The mRNA of CALB1 increases in matter.The mechanism how CALB1 mediates calcium metabolism is unclear.Insulin administration can be corrected effectively
The relevant hypercalciuria of hyperglycemia, and the increase for the diabetes rat calcium transport protein abundance for reversing STZ to induce.Therefore, CALB1
Increase may be offset DN kidney in high calcium load compensation mechanism.Our research reports diabetes and diabetogenous nephrosis for the first time
The increase that EVS includes CALB1 expression is urinated in patient.
The utility model has the advantages that the present invention provides a kind of extracellular vesicas of urine to include marker CALB1, it can be used for non-invasive
Diagnosis early diabetic nephropathy.
Figure of description
Five groups of subjects urine EVS of Fig. 1 include the expression quantity variation of MASP2
Specific embodiment
The present invention is explained further combined with specific embodiments below, but does not limit scope of the invention as claimed, the present invention
Agents useful for same is all commercially available unless otherwise instructed.
The transmission electron microscope (TEM) and nanoparticle trajectory analysis (NTA) of the urine of embodiment 1. EVS
Participant is divided into 5 groups: healthy control group (NC, n=15), preceding diabetes group (PreDM, n=15), albuminuria water
Put down normal diabetes group (DM, n=15), microalbuminuria diabetes group (DM-micro, n=15) and a large amount of albumin
It urinates diabetes group (DM-macro, n=15).The collects urine from all participants one morning.It had previously been established using us
Method (Musante L, Saraswat M, Ravida A, Byrne B, et al..Recovery of urinary
nanovesicles from ultracentrifugation supernatants.Nephrol Dial Transplant
2013；28:1425-1433.), it is enriched with using hydrostatic dialysis process for urine EVS in urine.
Specifically, the urina sanguinis 100ml of collection research object, no protease inhibitors after low-speed centrifugal, collect supernatant,
It is stored in -80 DEG C of refrigerators.
HETTICH CENTRIFUGE universal 320 (UK) centrifuge, 2000g RCF, room temperature are centrifuged 30min,
Remove T-H albumen, cell fragment and other sediments.Collect supernatant.
Nanometer film ultrafiltration and concentrating sample: using homemade nanometer film condensing device, connects 1000kDa nanometer film, SDS
Cleaning, the cleaning of miliQ water are checked without leakage gas leakage, after device work is intact, start to process sample, eight samples per treatment,
Divide all diabetes of several batch processeds and normal control's urine.During nanometer film concentrating sample, close observation device is
No operation is intact, and if any the phenomenon of leakage, the nanometer film more renewed in time handles sample again.Our nanometer film condensing device
Run it is intact, there is no this phenomenon occur.When ultrafiltration is to 6-8ml, 10-20mlmiliQ water is added, collects filtered solution, it is repeatedly dense
Three times, 200mlmiliQ pure water is then added in contracting filtering, continues ultrafiltration concentration.When urine is concentrated to 3-5ml, sample is collected
It saves stand-by.
Urine by transmission electron microscope (TEM) identification from 5 groups urinates EVS.Nanoparticle trajectory analysis is carried out, is made
With the quantity and size distribution of the NanoSight NS300 camera estimation urine EVS equipped with sCMOS.In Bradford protein
5 micrograms, which are separated, using SDS-PAGE in measurement urinates EVS protein.Gel is transferred on polyvinylidene fluoride (PVDF) film, with
Specific rabbit anti-human's antibody TSG101 is incubated for, and then the secondary antibody with horseradish peroxidase conjugation is incubated for.Finally, with Kodak IS
4000R image station observes sample.
It is uniformly distributed the result shows that urine EVS has, film encapsulating structure, cupuliform or circle, diameter 40-100nm is presented.
More fully urine EVS size is carried out to each group using NTA to analyze.The curve shows main peak between 55 and 110nm.
Embodiment 2. carries out protein electrophoresis and Mass Spectrometric Identification to the urine EVS that embodiment 1 obtains
The internal standard protein (TSG101) in each sample is marked with equivalent using Cy2.Cy3 and cy5 dyestuff mark is used respectively
Remember the protein from control group and patient.Equal electricity are carried out using IPG immobilineTM drystrip (24cm, pH4-7)
It focuses (IEF), and carries out isoelectric focusing in IPGphor (GE Healthcare), carried out under 1000V (2 hours) gradient；
8000V (2 hours)；Dielectrophoresis program (2D-DIGE) is carried out 5-6 hours with 17W/ gel in the dark.Use Typhoon
9410 scanners (GE Healthcare) scan gel.It is cut by Ettan Spot Picker (GE Healthcare)
Consistently change between two groups (preDM, DM, DM-micro and DM-macro are compared with NC) in all these protein point diagrams
Differential protein particle (| ratio |>1.5, p<0.05), then thoroughly cleaned in ultrapure water and carry out tryptose in gel
Enzymic digestion.Protein identification passes through 4800 proteome analysis instrument MALDI-TOF-MS (Applied of ABI
Biosystems it) carries out.Existed using Global Proteome Server Explorer software (Applied Biosystems)
Mass Spectrometric Identification is searched in Swiss Prot database is used for protein identification.
Data are compared using ExoCarta database (www.exocarta.org) to analyze.Use gene ontology software
(GO) molecular function and biological process of the protein of these identifications are analyzed.It has been finally screened by retrieval bibliography and has not been sent out
Existing some potential source biomolecule markers as diabetes and diabetic nephropathy.
As a result as shown in Figure 1, Western blotting show TSG101 be present in 5 groups, but DM, DM-micro and
DM-macro group TSG101 intensity is significantly stronger than NC and PreDM group.Perhaps this may be due to the glycosuria with and without injury of kidney
The patient ratio NC and more urine EVS of PreDM patient's secretion.
And two dimension DIGE (2D-DIGE) analysis shows, in 5 groups, compared with NC group, have nearly 40 in 1980 protein spots
A albumen at least 1.5 times of significant difference between big group of PreDM, DM, DM-micro and DM-, | ratio |>1.5, p<
0.05.Above-mentioned differential protein particle is selected to identify by mass spectrography.According to our analysis, 22 kinds of specific proteins have been selected
Next step data analysis is carried out, as shown in table 1 below.
Table 1: differential protein
Data are analyzed by ExoCarta, Gene Ontology database, to disclose the albumen consistent with EVS is urinated
Matter group, especially urine source and relevant to diabetic nephropathy.According to ExoCarta database, previously in normal urine
The differential protein of all identifications is reported in EVS.As shown in Figure 1, discovery CALB1 shows increased expression, Calb1
Calbindin 1, NCBI accession number P05937, molecular weight 30291.1, isoelectric point 4.7 carry out literature search, do not find to urinate
EVS includes Calb1 and diabetes/diabetic nephropathy correlation research.Newfound urine EVS, which includes Calb1, can be used for identifying
The albuminuria " preceding microalbuminuria " of diabetic.
Claims (5)
1. the biomarker for diagnosing diabetic nephropathy early stage, the biomarker urine EVs is included
CALB1, NCBI accession number are P05937.
2. application of the CALB1 that urine EVS is included in diagnosis diabetic nephropathy early stage, the CALB1 is relative to normal person
Expression quantity significantly increases.
3. application according to claim 2, the CALB1 increases 30% or more relative to normal person's expression quantity.
4. application according to claim 3, the CALB1 increases 50% or more relative to normal person's expression quantity.
5. application according to claim 4, the CALB1 increases 100% or more relative to normal person's expression quantity.
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| CN201810942585.7A CN109752558A (en) | 2018-08-17 | 2018-08-17 | For diagnosing the biomarker of diabetic nephropathy early stage |
| PCT/CN2019/100819 WO2020035033A1 (en) | 2018-08-17 | 2019-08-15 | Biomarker calb1 for diagnosing early stages of diabetic nephropathy |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020035033A1 (en) * | 2018-08-17 | 2020-02-20 | 南方医科大学 | Biomarker calb1 for diagnosing early stages of diabetic nephropathy |
| CN111398595A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Application and detection method of protein TNFAIP8 in plasma small cell outer vesicle |
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| CN101393165A (en) * | 2007-09-20 | 2009-03-25 | 复旦大学附属华山医院 | Method for detecting discrepancy expressed protein spectrum in blood serum sample of diabetes and kidney disease patient |
| CN101561440A (en) * | 2008-04-16 | 2009-10-21 | 中国科学院上海生命科学研究院 | Application of fiber gelatinized protein 3 |
| CN105181973A (en) * | 2015-09-10 | 2015-12-23 | 付冬霞 | Diabetes and nephropathy early detection marker composition, kit and using method thereof |
| CN107247102A (en) * | 2017-06-21 | 2017-10-13 | 中国药科大学 | Citric acid method for quantitatively determining and its application in diabetogenous nephrosis disease early diagnosis in serum |
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| CN103237901B (en) * | 2010-03-01 | 2016-08-03 | 卡里斯生命科学瑞士控股有限责任公司 | For treating the biomarker of diagnosis |
| CA2827894A1 (en) * | 2011-02-22 | 2012-08-30 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Circulating biomarkers |
| CN109752558A (en) * | 2018-08-17 | 2019-05-14 | 南方医科大学 | For diagnosing the biomarker of diabetic nephropathy early stage |
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| CN105181973A (en) * | 2015-09-10 | 2015-12-23 | 付冬霞 | Diabetes and nephropathy early detection marker composition, kit and using method thereof |
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Cited By (2)
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|---|---|---|---|---|
| WO2020035033A1 (en) * | 2018-08-17 | 2020-02-20 | 南方医科大学 | Biomarker calb1 for diagnosing early stages of diabetic nephropathy |
| CN111398595A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Application and detection method of protein TNFAIP8 in plasma small cell outer vesicle |
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