CN110824178A - Marker and kit for early diagnosis of diabetes and application of marker and kit - Google Patents
Marker and kit for early diagnosis of diabetes and application of marker and kit Download PDFInfo
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- CN110824178A CN110824178A CN201911180411.2A CN201911180411A CN110824178A CN 110824178 A CN110824178 A CN 110824178A CN 201911180411 A CN201911180411 A CN 201911180411A CN 110824178 A CN110824178 A CN 110824178A
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- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Abstract
The invention provides a marker for early diagnosis of diabetes and application thereof, belonging to the technical field of diabetes diagnosis, wherein the marker for early diagnosis of diabetes comprises lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14: 0); the expression level of the lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) in the serum exosomes of the healthy human has a very significant difference with the expression level in the serum exosomes of the early-stage primary-diagnosis patients with diabetes; the marker for early diagnosis of diabetes provided by the invention can be used as a target for early diagnosis of diabetes; the early diagnosis of the diabetes can be effectively carried out by detecting the content of the marker in the serum exosome, and misdiagnosis and missed diagnosis are prevented.
Description
Technical Field
The invention belongs to the technical field of diabetes diagnosis, and particularly relates to a marker for early diagnosis of diabetes and application thereof.
Background
Diabetes is a metabolic disease mainly manifested by increased blood sugar, and can induce a variety of serious acute and chronic complications, of which more than 90% are Type 2 diabetes mellitus (T2 DM). T2DM has latent disease, early prevention and treatment has important significance for controlling disease progress, traditional methods such as blood sugar, glycated hemoglobin and insulin concentration detection have definite diagnosis significance for T2DM, but cannot predict or prompt the occurrence of diabetes as soon as possible, the development of help for disease prevention and treatment is not great, and it is a focus of attention of doctors and patients how to predict the occurrence of T2DM and take intervention measures as soon as possible.
Oxidative stress is one of the important mechanisms of diabetes, signal pathways such as intracellular oxidative phosphorylation and glucose autoxidation related to carbohydrate metabolism are over-activated to cause intracellular Reactive Oxygen Species (ROS) to accumulate, and excessive oxidative stress damages mitochondria, generates a large amount of inflammatory factors such as prostaglandin, impairs β cell insulin secretion function, and the like, and finally develops into T2 DM.
The traditional method has definite diagnosis significance on T2DM, but cannot predict or prompt the occurrence of diabetes as soon as possible, and is not helpful for the prevention and treatment of diseases. The method for early diagnosis of diabetes by using DNA or non-coding RNA in serum as a detection target, which is newly proposed in the prior art, is easily influenced by environmental factors, and has poor stability and poor detection effect.
The current diagnostic methods for diabetes cannot be used for effective early prediction or diagnosis of diabetes.
Disclosure of Invention
In view of the above, the present invention aims to provide a marker, a kit and applications thereof for early diagnosis of diabetes; the marker for early diagnosis of diabetes is lipid from serum exosome, can be used for early diagnosis of diabetes, and can prevent misdiagnosis and missed diagnosis.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a marker for early diagnosis of diabetes, which comprises lipid PC (P18:1(9Z)/16:0) or PE (22:2(13Z,16Z)/14: 0).
The invention provides a marker for early diagnosis of diabetes, which comprises lipid PC (P18:1(9Z)/16:0) and PE (22:2(13Z,16Z)/14: 0).
Preferably, the lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) is a lipid in serum exosomes.
Preferably, the lipid also comprises one or more of lipid PE (22:4(7Z,10Z,13Z,16Z)/16:0), LysoPC (16:0), PE (24:1(15Z)/15:0), PE (24:0/15:0), LysoPC (0:0/18:0) and PE (22:0/15: 0).
Preferably, the diabetes is type 2 diabetes.
The invention provides application of the marker in preparing a reagent for early diagnosis of diabetes.
The invention provides a kit for early diagnosis of diabetes, which comprises a reagent for detecting the marker.
Preferably, the kit comprises a serum exosome extraction reagent, an exosome pretreatment reagent and an exosome LC-MS separation detection reagent.
The invention has the beneficial effects that: the marker for early diagnosis of diabetes provided by the invention comprises lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14: 0); the expression level of the lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) in the serum exosomes of the healthy human has a very significant difference (P is less than 0.001) with the expression level in the serum exosomes of the early-stage primary-diagnosis patients with diabetes; the markers PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) for early diagnosis of diabetes provided by the invention can be used as targets for early diagnosis of type 2 diabetes. The early diagnosis of the diabetes can be effectively carried out by detecting the content of lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) in the serum exosome, and the misdiagnosis and missed diagnosis can be prevented.
Furthermore, the lipid in the serum exosome is used as a detection object, and the lipid is protected by the membrane structure of the exosome, has strong stability and is not easily influenced by the external environment; compared with direct detection by using whole serum, the method can prevent the detection object from covering real information due to the action of physicochemical factors and prevent misdiagnosis and missed diagnosis. The marker for early diagnosis of diabetes, namely lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0), is a final reaction product of metabolic reaction of an organism, can intuitively reflect the physiological and pathological conditions of the organism, is not covered by compensation factors, and is suitable for early diagnosis of diseases.
The LC-MS method is used for analyzing the lipid in the serum exosome, and has high detection sensitivity and good stability.
Drawings
FIG. 1 shows the exosome extraction and identification results, wherein A is the nanoparticle size distribution of the exosomes detected by Nanosight; b is Western blot detection of the classical marker expression of the surface of the exosome; c, detecting the exosome forms of the two groups of samples under a scanning electron microscope;
FIG. 2 is a statistical analysis of how far a serum exosome lipid profile is analyzed; wherein A is a healthy control group (N, red area) and an initial diagnosis T2DM group (T, green area) OPLS-DA cluster analysis chart; b is an OPLS-DA parameter test analysis chart, and R2 is 0.91; c is OPLS-DA alignment test analysis chart, Q2 ═ 0.8 (n ═ 6);
FIG. 3 is a univariate analysis of lipid profile, wherein A is the analysis of the lipid profile in healthy control group (N) and preliminary T2DM group (T) (intensity of bands is proportional to the amount of substance expressed); b is the volcano diagram analysis of the differential metabolites between the healthy group and the initial diagnosis T2DM group, wherein the baseline is above the differential substances, (n is 6);
FIG. 4 is the diagnostic marker screening results; wherein A is PC (P18:1(9Z)/16:0) B: statistical analysis of PE (22:2(13Z,16Z)/14:0) expression differences; b is a statistical analysis of the differences in PE (22:2(13Z,16Z)/14:0) expression.
Detailed Description
The invention provides a marker for early diagnosis of diabetes, which comprises lipid PC (P18:1(9Z)/16:0) or PE (22:2(13Z,16Z)/14: 0).
The invention provides a marker for early diagnosis of diabetes, which comprises lipid PC (P18:1(9Z)/16:0) and PE (22:2(13Z,16Z)/14: 0).
In the present invention, the lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) is a lipid in a serum exosome.
In the present invention, the early diagnosis of diabetes is preferably type 2 diabetes.
In the invention, the expression level of the lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) in the serum exosomes of the healthy human and the expression level in the serum exosomes of the early-stage diabetes patient are very significantly different (P <0.001), and the lipid PC (P18:1(9Z)/16:0) and can be used as a target for early diagnosis of type 2 diabetes.
The invention also provides application of the marker in preparing a reagent for early diagnosis of diabetes. In the present invention, the early diagnosis diabetes agent is preferably the marker detection agent; the specific type of the reagent is not particularly limited in the present invention, as long as the detection of lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) in serum exosomes can be achieved.
The invention provides a kit for early diagnosis of diabetes, which comprises a reagent for detecting the marker. In the present invention, the reagent preferably comprises a serum exosome extraction reagent, an exosome pretreatment reagent and an exosome LC-MS separation detection reagent. The specific types of the serum exosome extraction reagent, the exosome pretreatment reagent and the exosome LC-MS separation detection reagent are not particularly limited, and the serum exosome extraction reagent, the exosome pretreatment reagent and the exosome LC-MS separation detection reagent which are conventional in the field are adopted. In the specific implementation process of the invention, the serum exosome extraction reagent is preferably VEXTMAn Exosome Isolation Reagent (from serum); the exosome pretreatment reagent preferably comprises methanol, methyl tert-butyl ether solvent, isopropanol and acetonitrile.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Interpretation of terms: t2DM (Type 2 diabetes mellitis; Type II diabetes mellitus); OPLS-DA (orthogonal partial least squares discriminant analysis); FC (FoldChange, fold test); liquid chromatography-mass spectrometry (1 acquired chromatography tandem mass spectrometry, LC-MS); VIP (Variable Projection Importance).
Example 1
Test object
The test subjects were a first-visit T2DM patient who was visited at an affiliated yixing hospital of Jiangsu university between 8 months of 2018 and 8 months of 2019, and healthy persons with similar height, weight, age and sex.
Subject general data was collected according to the following criteria: age, body mass index, blood pressure, liver and kidney function, fasting blood glucose level, 2h post-prandial blood glucose level, glycated hemoglobin level, basic medical/life history, etc. (see table 1 for details).
Initial diagnosis T2DM group inclusion criteria: the fasting venous blood sugar is more than or equal to 7.0 mmol/L; the blood sugar is more than or equal to 11.0mmol/L after 2 hours of meal; glycated hemoglobin: more than or equal to 6.5 percent; age: 18 to 75 years old; sex: without limitation.
Control group inclusion criteria: 4.16mmol/L or more and 6.44mmol/L or less of fasting venous blood glucose; the blood sugar is less than or equal to 7.8mmol/L after 2 hours of meal; glycated hemoglobin: less than or equal to 6.5 percent; the levels of sex, age, weight, etc. are similar to those of the subjects in the initial diagnosis T2DM group.
Disease exclusion criteria: patients with obvious liver and kidney function damage or gastrointestinal dysfunction, and patients with diseases of blood system or other endocrine systems; surgical or other emergency conditions; pregnant or lactating women; recent use of antibiotics; (ii) a history of psychosis or drug abuse; history of smoking or alcohol abuse.
The trial was approved by the ethical committee of the affiliated yixing hospital of Jiangsu university and informed consent of the subjects was obtained.
TABLE 1 basic conditions of the subjects
Test method
1. Collecting a specimen: collecting venous blood 10 ml/person in elbow of fasting subject, standing at room temperature for 30min, centrifuging at 4 deg.C and 1000g/min for 5min, collecting supernatant (serum), and subpackaging with 1.5ml EP tube (1.5 ml per tube), and storing at-80 deg.C.
2. Exosome extraction and identification: centrifuging the collected serum at 4 deg.C and 2000g/min for 30min to remove residual cells and debris; pipette 1ml of supernatant toIn new EP tubes, per serum volume: exosome extraction reagent volume 1ml:0.2ml (VEX)TMExosome Isolation Reagent (from serum)), and mixing the two liquids by inversion; standing the mixed solution at 2-8 deg.C for 30min, centrifuging at 4 deg.C and 10000g for 5min, and removing supernatant as much as possible with a pipette to obtain precipitate, namely serum exosome. Detecting the particle size of the exosome by a nanoparticle tracing system (NanoSight, Amesbury, UK); extracting an exosome protein sample, and detecting the expression quantity of exosome specific markers CD9, CD63, CD81 and Alix by using a polypropylene gel electrophoresis method; the exosome morphology was observed by transmission electron microscopy (FEI Tecnai 12, Philips, The Netherlands) after resuspension in 1 × PBS and mixing (see fig. 1).
3. Sample treatment and LC-MS analysis condition settings: 1) melting the exosome sample in ice bath at 4 ℃, adding 300 mu l of methanol solution, and carrying out vortex oscillation for 2 min; 1ml of methyl tert-butyl ether solvent (MTBE) was added, extracted with shaking for 1 hour, then 200. mu.l of water was added and mixed well. 12000rpm, 4 ℃,10 min of centrifugation, 400 u L MTBE layer solution evaporation and concentration, adding 100 u l isopropanol: and (3) shaking acetonitrile at a ratio of 1:1 to redissolve, centrifuging at 12000rpm for 10min, taking the upper layer solution (lipid substance), placing the upper layer solution in a 250 mu l inner lining tube, and detecting. 2) The chromatographic separation is performed by using Dionex of ThermoFisher ScientificTMUltiMateTMSamples were analyzed by 3000 reverse phase chromatography on a Waters UPLC BEH C8 column (1.7um 2.1mm 100 mm). The column temperature was 40 ℃ and the flow rate was 0.300[ ml/min ]]. Mobile phase: equal to "acetonitrile: 6:4 of water; 0.1% formic acid; 5mM ammonium acetate "; b. equal ═ isopropanol: 9:1 of acetonitrile; 0.1% formic acid; 5mM ammonium acetate "(see Table 2 for chromatographic conditions). The mass spectrometry adopts a quadrupole rod orbit ion trap mass spectrometer containing a thermal electrospray ion source, the voltages of a positive ion source and a negative ion source are respectively 3.7kv and 3.5kv, the heating temperature of a capillary tube is 320 ℃, the pressure of rising gas is 30psi, the pressure of auxiliary gas is 10psi, the volume heating evaporation temperature is 300 ℃, the rising gas and the auxiliary gas are both nitrogen, the collision gas is nitrogen, and the pressure is 1.5 mTorr. The first-order full scan parameters are: resolution 70000, automatic gain control target of 1 × 106Maximum isolation time 50ms, mass to charge ratio scan range 50-1500.
TABLE 2 chromatographic conditions
4. Data preprocessing and metabolite identification: the collected data are processed by Progenetics QI software, and the method comprises the steps of importing original data, aligning peaks, extracting peaks, normalizing, and finally forming a table of retention time, mass-to-charge ratio and peak intensity. Metabolite identification primary molecular weight matching was performed using the human metabolome database and the lipid database.
5. Statistical analysis: data were imported into MetabioAnalyst 3.0, and an orthogonal partial least squares discriminant analysis (OPLS-DA) was used to distinguish the overall differences in metabolic profiles between groups and output a cluster map. Measuring the influence strength and the interpretability of the expression pattern of each metabolite on the classification and discrimination of each group of samples by calculating Variable Projection Importance (VIP) (the greater the VIP value, the greater the contribution of the Variable to distinguish differences among groups), a binding multiple test (Fc) and T test analysis, and screening of auxiliary marker metabolites (the VIP value is greater than 1.0, the FC is greater than 1.5, and the P is less than 0.05 as a screening standard).
Results of the experiment
1. Serum exosomes extraction and identification exosomes (exosomes) were isolated from serum by kit and centrifugation. The Nanosight visible nanoparticle analyzer measures the sizes of the serum-derived exosomes of the initial T2DM patient and healthy control, the average diameter of the exosomes of the T2DM patient is about 110nm, and the average diameter of the exosomes of the healthy control is about 108nm (A in FIG. 1). Western Blot analysis showed that the exosome marker proteins CD9, CD81, CD63 and Alix were expressed (B in FIG. 1). Observed under a transmission electron microscope, the diameter of the exosome is about 110nm, the exosome has a complete cell membrane, and a round or oval membrane vesicle structure (C in figure 1) with low electron density components is formed in the cavity. The result shows that the quality of the extracted serum exosome meets the requirement and can be used for subsequent experiments.
2. The detection of serum exosome lipid metabolites and multivariate statistical analysis of the tested group are based on LC-MS results, the analysis is carried out by Progenetics QI software, and the derivation of an original data matrix comprises the following steps: sample information, retention time, mass-to-nuclear ratio, and mass spectral response intensity (peak area), among others. And comparing the extracted peak information with a database, performing full-database retrieval on three databases of MassBank, Respect and GNPS, and importing the obtained information data into MetabioAnalyst 3.0 for multivariate statistical analysis. The results show that the OPLS-DA model can completely distinguish the initial T2DM group (T) from the healthy control group (N) (a in fig. 2). To prevent overfitting of the model, the quality of the PLS-DA model was examined using seven cycles of cross-validation (parametric test) and 100 response ranking tests (ranking test), which showed R2 to be close to 0.91 and Q2 to 0.80. The values of R2 and Q2 respectively reflect the interpretation rate and the prediction rate of the OPLS-DA model, and the closer to 1, the more stable the model is, the more reliable the result is, so that the model can be selected as a model for separating two groups of samples to better screen the differential lipid of the sample (B and C in figure 2).
3. The lipid metabolism spectrum difference analysis of the peripheral serum exosomes of the tested group draws a heat map (A in figure 3) according to the expression quantity of lipid components in the exosomes, and visually displays the difference of the types and the contents of the lipids between two groups of samples. And simultaneously performing univariate analysis on various lipids by adopting a T test and a fold test (dividing the average value of the lipid expression quantity of each group of the healthy control group by the corresponding substance of the initial diagnosis T2DM group to obtain fold difference (FC)), and drawing a volcano chart on the obtained result, wherein a black point above a base line in the chart represents a differential metabolite, and the greater the dispersion, the greater the difference (B in FIG. 3).
4. Screening of differential lipids in peripheral serum exosomes of test groups differential lipids between groups were screened according to the method combining multivariate analysis and univariate analysis, the screening criteria were that VIP value of the first main component of OPLS-DA model was >1, p value of T-test (test's T test) was <0.05, and FC value of fold test was greater than 1.5, to obtain differential lipids (see table 3) mainly including Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), lysophosphatidylcholine (LysoPC), Cholesterol Ester (CE), Triacylglycerol (TG), Sphingomyelin (SM), etc. The larger the FC value, the lower the expression of this metabolite in the initial T2DM group, and conversely the higher the expression, the 3 lipids showing a reduction in expression in the initial T2DM group according to the test data, and the others showing an increase in expression, with 8 different phospholipid fatty acids, the most significant lipid reduction being PC (P18:1(9Z)/16:0), and the most significant increase being PE (22:2(13Z,16Z)/14: 0). The PC (P18:1(9Z)/16:0) and the PE (22:2(13Z,16Z)/14:0) are selected as the markers for early diagnosis of T2DM, and the statistical result shows that the expression levels of the two substances are obviously different between the serum exosomes of the healthy group and the serum exosomes of the initial diagnosis T2DM group (P <0.001) (A and B in figure 4). PC (P18:1(9Z)/16:0) and PE (22:2(13Z,16Z)/14:0) are suggested as new targets for early diagnosis of type 2 diabetes.
Table 3 differential lipid list (VS initial diagnosis T2DM group, n ═ 6 in healthy control group)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A marker for early diagnosis of diabetes, which comprises lipid PC (P18:1(9Z)/16:0) or PE (22:2(13Z,16Z)/14: 0).
2. A marker for early diagnosis of diabetes mellitus, comprising the lipids PC (P18:1(9Z)/16:0) and PE (22:2(13Z,16Z)/14: 0).
3. The marker according to claim 1 or 2, characterized in that the lipid PC (P18:1(9Z)/16:0) and/or PE (22:2(13Z,16Z)/14:0) is a lipid in serum exosomes.
4. The marker of claim 1 or 2, further comprising one or more of lipid PE (22:4(7Z,10Z,13Z,16Z)/16:0), LysoPC (16:0), PE (24:1(15Z)/15:0), PE (24:0/15:0), LysoPC (0:0/18:0) and PE (22:0/15: 0).
5. The marker of claim 1 or 2, wherein the diabetes is type 2 diabetes.
6. Use of the marker of any one of claims 1 to 5 for the preparation of a reagent for the early diagnosis of diabetes.
7. A kit for early diagnosis of diabetes comprising a reagent for detecting the marker of any one of claims 1 to 5.
8. The kit of claim 7, comprising a serum exosome extraction reagent, an exosome pretreatment reagent and an exosome LC-MS separation detection reagent.
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| CN113009162B (en) * | 2021-02-26 | 2022-07-12 | 南芯芯仪(广州)制造有限公司 | Serum metabolic marker for diagnosing gestational diabetes and application thereof |
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