CN111257570A - Marker for early diagnosis of abortion caused by prothrombotic state and application thereof - Google Patents

Abstract

The invention discloses a group of markers for diagnosis of abortion caused by a prothrombotic state and application thereof, wherein the markers comprise the following proteins: prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecanon-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecanon-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGF R3, IL-17, BMP-4, S100A8, GROa, ADAM 8. Experiments prove that the protein has obvious differential expression in patients with abortion caused by the prothrombotic state and normal people, and the protein can be used as a diagnostic marker for abortion caused by the prothrombotic state and used for diagnosis of abortion caused by the prothrombotic state.

Description

Marker for early diagnosis of abortion caused by prothrombotic state and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a group of markers for early diagnosis of abortion caused by a prothrombotic state and application thereof.

Background

There are about 35000 genes in human, but there are as many as 100000 proteins in human, and one gene does not express only one protein. To study life phenomena and elucidate the laws of life activities, it is far from sufficient to understand the structure of genome, and it is necessary to have a deeper understanding of the importance of protein, which is a direct performer of life activities, and thus Proteomics (Proteomics), which is one of the important contents of functional genomics, has come into play. The concept of "proteome" was proposed by Wilkins and Williams in Australia in 1994 and is defined as the expression of all corresponding proteins in the genome of a cell or a tissue, which is a whole of all proteins corresponding to a genome, rather than being limited to one or several proteins, and is a new field of research that reveals the function of proteins and the laws of cell life. The protein chip method is a new technology developed with the development of gene chips in recent years. The basic principle is that various proteins are orderly fixed on a medium carrier such as a glass slide and the like to form a detection chip, then an antibody marked with a specific fluorescent substance acts on the chip, the antibody matched with the proteins on the chip is combined with the corresponding proteins, and the fluorescence on the antibody indicates the corresponding proteins and the expression quantity thereof. After washing away the antibody not complementarily binding to the protein on the chip, the fluorescence intensity of each point on the chip is measured by a fluorescence scanner or a laser copolymerization scanning technique, and the relationship between the protein and the protein is analyzed by the fluorescence intensity, thereby achieving the purpose of measuring various gene expression functions. The technology shows the capability of processing information quickly, efficiently and at high flux in the aspects of researches on antigen-antibody detection, disease diagnosis, drug development and the like. The traditional ELISA method is based on one-way reaction or single index, if a large amount of disease information is needed to be obtained, more samples need to be collected, and multiple experiments are respectively carried out. If further subtype classification is desired, more individual experiments are required. Compared with the traditional ELISA method, the test shows that the protein chip test result has good consistency with the ELISA method, and the protein chip has the characteristic of high flux, can quantitatively test a plurality of proteins at one time, is more concise, convenient and rapid compared with the ELISA method, and is worthy of clinical popularization.

At present, the protein chip technology is widely applied to the specific protein map analysis of more than ten malignant tumors such as prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, breast cancer, liver cancer and the like, and meaningful protein markers are found and used for early diagnosis of the malignant tumors; the method also has wide exploration space in the early prediction of the occurrence and prognosis of pregnancy-related diseases. stellalc et al, using protein chip technology to measure serum of 25 normal pregnant women and 20 women clinically presenting as premature birth, found that the up-regulation of protein peak with mass-to-charge ratio of 7783 and the down-regulation of protein peak with mass-to-charge ratio of 3164, and preliminarily concluded that the serum protein differentially expressed between premature birth and normal pregnancy may be the cause of premature birth initiation. BuSChA and the like use SELDI-TOF and gene chip technology to measure 24 cases of differential proteins of trisomy 21 syndrome maternal serum from 10 weeks to 14 weeks and 24 cases of normal pregnancy, preliminarily obtain 1 group of characteristic proteomes, and are considered to be capable of being used for early prediction of trisomy 21 syndrome and provide possibility for developing non-invasive prenatal diagnosis.

In the early stage of research on correlation between the thrombotic state of recurrent abortion and kidney deficiency and blood stasis, two specific proteins IGFBP-rPl and VEGF are screened by a protein chip technology, wherein IGFBP-rPl as a secretory protein can influence the growth and development of embryos by inhibiting the EMT process, so that abortion occurs; VEGF may cause vasoconstriction, promote platelet aggregation, form a prothrombotic state, and finally cause abortion by distribution of its receptor KDR protein. Based on the previous research, the inventors screened the serum of patients with abortion caused by prothrombotic state, patients with abortion caused by nonprothrombotic state and normal persons, and further studied specific markers of abortion caused by prothrombotic state and mechanisms causing abortion, so as to predict early abortion caused by prothrombotic state.

Disclosure of Invention

The invention aims to solve the technical problems, provides a group of early diagnosis markers for abortion caused by a prothrombotic state, application thereof and further provides related diagnosis products thereof.

The inventors screened sera of patients with abortion caused by a pre-thrombotic state, patients with abortion caused by a non-thrombotic state and normal persons, and further studied specific markers of abortion caused by a pre-thrombotic state and mechanisms causing abortion, so as to predict abortion caused by a pre-thrombotic state at an early stage.

In order to achieve the above objects, the present invention provides a set of markers for early diagnosis of abortion caused by prothrombotic state, wherein the markers comprise the following proteins: prolactin, MMP-3, Testitan 2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecanon-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecanon-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGFR3, IL-17, BMP-4, S100A8, GROa, ADAM 8.

Preferably, the Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecano-1, JAM-B, Gas 1, VEGFR1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecano-3 are down-regulated in serum of patients whose pre-thrombotic state causes abortion, and the MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGF R3, IL-17, BMP-4, S100A8, GROa, ADAM8 are up-regulated in serum of patients whose pre-thrombotic state causes abortion.

Further, the invention provides application of a reagent for detecting proteins in preparing a product for early diagnosing abortion caused by a prothrombotic state, wherein the proteins are one or more of Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecano-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecano-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGFR3, IL-17, BMP-4, S100A8, GROa and ADAM 8.

Preferably, the protein is one or more of G-CSF R, ANG-2, Syndecano-3, MIF, S100A8, GROa, EGF and ADAM 8.

Preferably, the detection reagent comprises: and a reagent for diagnosing abortion caused by the prothrombotic state by detecting the expression level of the protein through a protein chip.

Preferably, the protein chip comprises an antibody that specifically binds to the protein.

Further, the present invention provides a product for diagnosing abortion caused by a prothrombotic state, which is capable of diagnosing abortion caused by a prothrombotic state by detecting the expression level of proteins including one or more of Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecano-1, JAM-B, Gas, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecano-3, MIF, MCP-4, follistat-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGFR 45, IL-17, BMP-4, S100A8, GROa, ADAM 8.

Preferably, the product comprises a chip, kit or formulation.

Preferably, the chip is a protein chip; the protein chip comprises a solid phase carrier and an antibody specific to the protein fixed on the solid phase carrier.

Preferably, the kit is a protein immunodetection kit comprising specific antibodies for detecting the protein.

Advantageous effects

Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecanon-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecanon-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGFR3, IL-17, BMP-4, S100A8, GROa and ADAM8 differential proteins are screened by performing protein chip on blood samples of patients and normal persons who cause abortion in a prothrombotic state and application of the differential proteins in early diagnosis of prothrombotic state causing abortion is provided, and the influence of the differential proteins on the development of prothrombotic state is elucidated and the value of the differential proteins in early diagnosis of prothrombotic state causing abortion is disclosed. Therefore, the invention diagnoses whether the abortion caused by the prothrombotic state occurs or not by detecting the differential expression of the protein, and further develops a diagnosis chip, a kit or a biological agent for the abortion caused by the prothrombotic state, so that the early diagnosis can be quickly and effectively realized, and the best early intervention time can be strived for patients.

Furthermore, the inventor finds that a group of diagnostic markers Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecano-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecano-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGFR3, IL-17, BMP-4, S100A8, GROa and ADAM8 related to the prothrombotic state abortion caused by a group of prothrombotic state cause abortion, and can greatly improve the accuracy of the prothrombotic state cause abortion by simultaneously detecting a plurality of diagnostic markers related to prothrombotic state abortion.

Drawings

FIG. 1 is a summary of differences between protein chips in 48 serum samples;

FIG. 2 is a clustering analysis of abundance of differentially expressed cytokines between the pre-thrombotic state group + the non-thrombotic state group and the normal group, wherein black represents up-regulated expression and gray represents down-regulated expression;

FIG. 3 is a biological process of FUNRICH analysis;

FIG. 4 is a signal pathway involved in a differential protein;

FIG. 5 is the differential protein interaction analysis between the disease group and the normal group;

FIG. 6 is clustering analysis of abundance of differentially expressed cytokines between the pre-thrombotic status group and the normal group, wherein black represents up-regulated expression and gray represents down-regulated expression;

FIG. 7 is a cluster analysis for verifying the abundance of differentially expressed cytokines between the disease and normal groups, with black indicating up-regulated expression and gray indicating down-regulated expression;

FIG. 8 is a graph showing the pre-thrombotic state group and normal group differentially expressed cytokine abundance clustering analysis, wherein black represents up-regulated expression and gray represents down-regulated expression;

FIG. 9 is a ROC plot of 6 serum markers of the invention used to distinguish patients with abortion due to a pre-thrombotic state from normal controls;

FIG. 10 is a ROC curve analysis of 33 serum markers.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The invention extracts 12 serum samples (normal group) of women with normal pregnancy history, 24 serum samples (prothrombotic state group) of recurrent abortion patients caused by prothrombotic state, and 12 serum samples (prothrombotic state group) of recurrent abortion patients caused by non-prothrombotic state. The protein expression profiles of the normal group, the prothrombotic state group and the nonthrombogenic state group were studied using a raybiotech quantitative protein chip.

Screening differential proteins of a normal group and a prothrombotic state group, and carrying out significant functional analysis on the differential proteins, namely GO/GO-net analysis and KEGGpathway/pathway-net analysis to obtain the protein related to habitual abortion caused by the prothrombotic state.

By screening the serum of patients with abortion caused by the prothrombotic state, patients with abortion caused by the non-prothrombotic state and normal people, the specific markers of abortion caused by the prothrombotic state and the mechanism of abortion caused by the prothrombotic state are further discussed, so that the abortion caused by the prothrombotic state can be predicted early.

The invention adopts a novel bioinformatics analysis technology and an unsupervised clustering method for analysis. Since the diagnosis of clinical diseases is often performed by referring to various indexes, such as imaging, pathology, physiological and biochemical indexes, etc., the diagnosis is confirmed. The meaning of the single index is not strong, and the clinical requirement can not be met. The inventor adopts a multi-index multi-parameter establishment analysis model, and distinguishes different groups in a clustering mode, so that the clinical coincidence rate can be greatly improved, and the modeling mode has important significance for clinical accurate diagnosis and treatment.

Example 1 differential protein screening

1. Clinical data

1.1 case origin:

1.2 case grouping: 12 serum samples of women with normal pregnancy history (normal group), 24 serum samples of patients with recurrent abortion caused by prothrombotic state (prothrombotic state group), and 12 serum samples of patients with recurrent abortion caused by non-prothrombotic state (prothrombotic state group).

1.3 case number:

protein chip detection: 12 cases in the normal group, 24 cases in the pre-thrombotic state group, and 12 cases in the non-thrombotic state group.

2. Materials and instruments used in the invention

2.1 kit: a QAH-CAA-440 kit comprising the contents of:

the kit is stored at the temperature of-20 ℃, after the kit is used, the slide chip, the cytokine standard mixed powder, the detection antibody mixture and the Cy 3-streptavidin are stored at the temperature of-20 ℃, and other reagents are stored at the temperature of 4 ℃ to avoid repeated freeze thawing.

The box composition is shown in table 1.

TABLE 1 kit Components

2.2 materials and instruments required except for the kit:

plastic centrifuge tubes (2-5ml, 50 ml); shaking table; plastic preservative films; aluminum foil paper; double distilled water; innoscan300Microarray Scanner fluorescence Scanner; thermo Scientific Wellwash Versa chip washer.

2.3 sample: 48 sera.

3. Experimental procedure

3.1 complete drying of slide chips

Taking out the slide chip from the box, balancing at room temperature for 20-30min, opening the packaging bag, uncovering the sealing strip, and then placing the chip in a vacuum drier or drying at room temperature for 1 hour.

3.2 configuration of standards

(1) Cytokine standards were diluted in gradient.

(2) Add 500. mu.l of sample diluent to the vial of cytokine standard mixture and redissolve the standard. Before opening the tube, the powder was rapidly centrifuged and gently pipetted up and down to dissolve the powder, and the tube was labeled Std 1.

(3) The 6 clean centrifuge tubes were labeled Std2, Std3 to Std7, respectively, and 200 μ l of sample diluent was added to each vial.

(4) 100. mu.l of Std1 was added to Std2 and mixed gently, and then 100. mu.l was added to Std3 from Std2, so that it was diluted to Std7 in a gradient.

(5) Draw 100. mu.l of sample dilution into another new centrifuge tube, labeled CNTRL, as a negative control.

Note: since the initial concentration of each cytokine was different, the serial concentrations of each cytokine were different after gradient dilution from Std1 to Std 7.

3.3 chip operation flow

(1) Add 100. mu.L of sample dilution to each well, incubate for 1h on a shaker at room temperature, and block the quantitative antibody chip.

(2) Buffer was removed from each well, 60 μ L of standard and sample were added to the wells and incubated overnight on a shaker at 4 ℃ (sample diluted 2.5 fold loaded).

(3) Cleaning:

the slide was washed with a Thermo Scientific Wellwash Versa chip washer in two steps, first with 1 Xwash I, 250. mu.L of 1 Xwash I per well for 10 times with 10s shaking each time with high shaking intensity, and 20 Xwash I was diluted with deionized water. Then, the washing is carried out by changing to 1 Xwashing liquid II channel, 250 mu L of 1 Xwashing liquid II is washed for 6 times, each time the washing is carried out for 10s, the shaking intensity is selected to be high, and 20 Xwashing liquid II is diluted by deionized water.

(4) Incubation of the detection antibody mixture:

the test antibody mixture vials were centrifuged and then 1.4ml of sample diluent was added, mixed well and then centrifuged quickly again. Add 80. mu.l of detection antibody to each well and incubate for 2 hours on a shaker at room temperature.

(5) Cleaning: the same as the step (3).

(6) Cy 3-streptavidin incubation:

the Cy 3-streptavidin vial was centrifuged, then 1.4ml of sample diluent was added, mixed well and centrifuged quickly again. Add 80. mu.l of Cy 3-streptavidin to each well, wrap the slide with aluminum foil and incubate in the dark for 1 hour on a shaker at room temperature.

(7) Cleaning: the same as the step (3).

(8) Fluorescence detection:

1) the slide frame was removed, taking care not to touch the antibody-printed side of the slide by hand.

2) The signal is scanned with a laser scanner such as InnoScan300, using either Cy3 or green channel (excitation frequency of 532 nm):

the instrument model is as follows: innoscan300Microarray Scanner;

the manufacturer: innopsys;

3) the producing area: parc d' Activies ActiveStre; 31390 Carbonne France;

4) scanning parameters are as follows: WaveLengh; 532 nm; resolution: 10 μm.

(9) Data analysis was performed using the data analysis software of QAH-CAA-440.

4. Statistical method

After Normalization of the raw data with software, Normalization data was selected for analysis. The analysis method is modulated t-statistics, the data packet is limma and comes from R/Bioconductor; screening differential proteins by adopting adjust p value (p value after correction by BH method) and logFC (multiple of expression difference, base 2), and selecting conditions as follows:

logFC > log2(1.2), the difference threshold is 1.2.

Corrected p-value: val < 0.05.

Using cluster analysis and intersection analysis-Venn Diagram (Venn Diagram) to find the differentially regulated protein; and performing GO/GO-net analysis and KEGG pathway/pathway-net analysis on the differential protein to obtain a protein related to recurrent abortion caused by a prothrombotic state.

5. Protein chip detection

5.1 disease group and Normal human proteomics Difference

The test adopts protein chip to quantitatively detect 440 cytokines in 48 serum samples in total, and the detected samples are 24 cases in the pre-thrombus state group, 12 cases in the non-thrombus state group and 12 cases in the normal group. The results are shown in FIG. 1.

5.1.1 Cluster analysis of disease groups and Normal groups

First, the inventors performed differential analysis of cytokine expression measured between the normal group and the disease group (prothrombotic state + non-prothrombotic state abortion), and 38 proteins showed significant difference between the two groups. Then, the inventor carries out cluster analysis on the 38 differential proteins, and results of unsupervised cluster hierarchical analysis show (figure 2) that the normal group and the disease group are obviously divided into two major groups, namely a disease group 36/36 and a normal group 9/12, and the consistency rates are 100% and 75% respectively. The overall consistency rate is 93.75%, which indicates that the 38 protein indexes can effectively distinguish a normal group from a disease group, and provides a reference basis for accurate clinical treatment.

5.1.2 differential protein analysis between disease and Normal groups

Among the values of the concentration of each histone measured, pvale, logFC, FDR, logCPM were calculated, FDR <0.05, logFC >1 was set as a screening condition, and the disease group had 26 proteins whose expression was significantly down-regulated and 12 proteins whose expression was significantly up-regulated compared to the normal group (see tables 2 to 3).

TABLE 2 Difference protein (down) between Normal and disease groups

TABLE 3 differential protein (up) between the Normal and disease groups

5.1.3 differential protein signaling pathway analysis between disease and Normal groups

By string analysis of 38 differential proteins (table 4), it was found that the differential proteins are mainly exhibited in regulation of biological processes, regulation of cellular processes, cellular communication, and the like. The FUNRICH assay (FIG. 3) differential proteins are associated with cell growth, adhesion resistance to apoptosis, and the like. These analysis results indicate that the differential protein may be associated with changes in early embryonic growth or endometrial changes.

TABLE 4 biological Processes involved in differential proteins

By analyzing the signaling pathway (fig. 4), the inventors found that the differential protein is involved in the signaling pathway of cell surface interaction of blood vessel wall, epithelial-mesenchymal transition, uPA-mediated thrombolysis, hydrolysis of cell adhesion protein, angiogenesis, etc. These involved signaling pathways suggest that they may be of some relevance to clinical miscarriage symptoms.

5.1.4 differential protein interaction assay (PPI) between disease and Normal groups

To explore the interaction relationship between the differential proteins in the disease group and the normal group, the inventors performed PPI analysis. The results show (figure 5) that among the 38 different proteins, protein 22 constitutes an interaction network that plays a role in the biological process (development) of the cell, and that most of the proteins are involved in the central regulation of CD274, SDC1, IL1B, CXCL13, ADAM17, MMP 3. In addition, FLT1-FLT4(VEGFR1-VEGFR4) plays an important role in negative feedback regulation of vascular endothelial cell proliferation.

5.2 protein chip detection of Pre-Thrombus State group and Normal group

The detection results of the prothrombotic state group and the normal histone chip are respectively analyzed, and the results show that: between the prothrombotic state group and the normal group, 33 cytokines were expressed with significant differences. Compared with the normal group, the expression of 20 cytokines in the serum of the group with the prothrombotic state is obviously reduced, the expression of 13 cytokines is obviously increased, and the expression conditions of the cytokines are shown in tables 5 and 6.

The 33 different proteins were subjected to cluster analysis, and unsupervised cluster-hierarchical analysis was performed, and the results showed (fig. 6) that the normal group and the prothrombotic state group were clearly divided into two major groups, i.e., disease group 24/24 and normal group 11/12, with agreement rates of 100% and 91.7%, respectively. The overall consistency rate is 97.2%, which indicates that the normal group and the prothrombotic state group can be effectively distinguished by adopting the 33 protein indexes, and a reference basis is provided for clinical accurate treatment.

Through analysis of 33 differential proteins, the differential proteins are basically consistent with those of a disease group and a normal group and are mainly shown in regulation of biological processes, regulation of cell processes, cell communication and the like. FUNRICH assay differential proteins are associated with cell growth, adhesion resistance to apoptosis, and the like. In terms of signal pathways, the signal pathways involved by differential proteins include vascular wall cell surface interaction, epithelial-mesenchymal transition, uPA-mediated thrombolysis, hydrolysis of cell adhesion proteins, angiogenesis, and the like. These involved signaling pathways suggest that they may be of some relevance to clinical miscarriage symptoms. These analysis results indicate that the differential protein may be associated with changes in early embryonic growth or endometrial changes.

TABLE 5 differential proteins (down) between the prothrombotic group and the normal group

TABLE 6 differential protein (up) between prothrombotic and Normal groups

Example 2 differential protein validation

1. The verification of the different proteins of the non-thrombus state group, the thrombus state group and the normal group was verified by the protein chip method, referring to example 1.

In the results of the preliminary screening, the inventors found that 38 proteins in the disease group were significantly up-regulated or down-regulated compared with the normal group, and in order to further confirm the differential expression of the proteins, the inventors selected 20 proteins for verification. The samples tested were 58 cases in the pre-thrombotic state group, 34 cases in the non-thrombotic state group, and 25 cases in the normal group.

The results show that: in the results of the validation, 10 proteins in the disease group compared to the normal group were differentially expressed proteins, 4 of which were up-regulated and 6 of which were down-regulated, as shown in table 7.

Furthermore, unsupervised cluster analysis results showed (fig. 7) that 117 samples tested were clustered into two distinct groups, normal versus disease, and that the clustering results were identical to the clinical classification. The 10 differential proteins are suggested to be capable of well distinguishing a normal group from a disease group.

Table 7 demonstrates the differential proteins between the normal and disease groups

2. Verification of different proteins between prothrombotic state group and normal group

In order to understand the difference between the pre-thrombus state group and the normal group, the inventors analyzed the results of the protein chips of 58 cases and 25 cases of the normal group in the test sample pre-thrombus state group.

The results show that: in the results of the validation, 8 proteins in the prothrombotic state group compared to the normal group were differentially expressed proteins, 5 of which were up-regulated and 3 of which were down-regulated, as shown in table 8.

In addition, unsupervised cluster analysis showed (fig. 8) that 83 samples tested were clustered into two distinct groups, the normal group was identical to the pre-thrombotic state group, and the clustering results were identical to the clinical classification. The 8 differential proteins are suggested to be capable of well distinguishing the normal group from the prothrombotic state group.

Table 8 demonstrates the differential proteins between the prothrombotic group and the normal group

Example 3 Receiver Operating Characteristic (ROC) Curve analysis

ROC curves were constructed to compare the diagnostic ability of 33 serum markers to discriminate between prothrombotic states resulting in recurrent abortion patients and healthy controls. Due to the large number, curves specific to the 6 serum markers ROC were given as reference examples, as shown in fig. 9. At the optimal cutoff value, the sensitivity of 33 serum markers is between 0.621 and 0.920, the specificity is between 0.675 and 0.948, and the AUC value is between 0.688 and 0.841.

The 33 serum markers combined AUC reached 0.948, with sensitivity and specificity of 1.00 and 0.828 respectively (as shown in fig. 10). These results indicate that the combination of 33 markers has higher sensitivity and specificity for early detection of abortion caused by the pre-thrombotic status compared with the 33 markers of abortion caused by the pre-thrombotic status alone.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (10)

1. A group of early diagnosis markers of abortion caused by prothrombotic state, which is characterized in that the markers are composed of the following proteins: prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecanon-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecanon-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGF R3, IL-17, BMP-4, S100A8, GROa, ADAM 8.

2. The marker of claim 1, wherein Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, syndecano-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, syndecano-3 are down-regulated in serum of patients whose pre-thrombotic state causes abortion, and MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGF R3, IL-17, BMP-4, S100a8, GROa, ADAM8 are up-regulated in serum of patients whose pre-thrombotic state causes abortion.

3. The application of a reagent for detecting protein in preparing a product for early diagnosing abortion caused by a prothrombotic state, wherein the protein is one or more of Prolactin, MMP-3, Testican2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecanon-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecanon-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGF R3, IL-17, BMP-4, S100A8, GROa and ADAM 8.

4. The use according to claim 3, wherein the protein is one or more of G-CSF R, ANG-2, Syndecano-3, MIF, S100A8, GROa, EGF, ADAM 8.

5. The use of claim 3, wherein the detection reagent comprises: and a reagent for diagnosing abortion caused by the prothrombotic state by detecting the expression level of the protein through a protein chip.

6. The use of claim 5, wherein the protein chip comprises an antibody that specifically binds to the protein.

7. A product for diagnosing abortion caused by a pre-thrombotic state, which is capable of diagnosing abortion caused by a pre-thrombotic state by detecting the expression amount of proteins including one or more of Prolactin, MMP-3, Testitacan 2, hCGb, IGF-1R, SLAM, Furin, IL-17C, APRIL, B2M, Syndecano-1, JAM-B, Gas 1, VEGF R1, Desmoglein2, CEA, BLC, G-CSF R, ANG-2, Syndecano-3, MIF, MCP-4, Follistatin-like 1, MCP-3, NRG1-B1, EGF, CA15-3, VEGF R3, IL-17, BMP-4, S100A8, GROa, ADAM 8.

8. The product of claim 7, wherein the product comprises a chip, kit, or formulation.

9. The product of claim 8, wherein the chip is a protein chip; the protein chip comprises a solid phase carrier and an antibody specific to the protein fixed on the solid phase carrier.

10. The product of claim 8, wherein the kit is a protein immunoassay kit comprising specific antibodies for detecting the protein.

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