CN111257569A - Marker for diagnosing recurrent abortion and application thereof - Google Patents

Abstract

The invention discloses a group of recurrent abortion diagnostic markers Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testican2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3, MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100A8 and GROa, and provides application of the markers in preparation of recurrent abortion diagnostic products. The inventor can greatly improve the accuracy rate of early diagnosis of recurrent abortion by simultaneously detecting a plurality of diagnostic markers related to recurrent abortion.

Description

Marker for diagnosing recurrent abortion and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a group of markers for diagnosing recurrent abortion 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, and initially obtain 1 group of characteristic proteomes, which are considered to be 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 inventor further studies specific markers of recurrent abortion and mechanisms causing abortion by screening serum of patients with recurrent abortion caused by prothrombotic state, patients with recurrent abortion caused by non-prothrombotic state and normal people, so as to early predict early treatment of recurrent abortion.

Disclosure of Invention

The invention aims to solve the technical problems, provides a group of markers for diagnosing recurrent abortion, application thereof and further provides a related detection product thereof.

In order to achieve the above objects, the present invention provides, in a first aspect, the use of a reagent for detecting a protein, which is one or a combination of several of Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testitan 2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, MIG, MDC, ANG-2, Syndecano-3, MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100A8, GROa, in the preparation of a diagnostic product for recurrent pregnancy.

Preferably, the Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testican2, SLAM, G-CSFR, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3 are down-regulated in serum of patients with recurrent abortion, and the MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100A8, GROa are up-regulated in serum of patients with recurrent abortion.

Preferably, the application of the reagent for detecting protein in the preparation of the diagnostic product for recurrent abortion, wherein the protein is one or more of G-CSF R, CNTF, MIG, MDC, ANG-2, Syndecano-3, HVEM, MIF, S100A8 and GROa.

Preferably, the recurrent abortion comprises recurrent abortion due to a pre-thrombotic state and recurrent abortion due to a non-thrombotic state.

Further, the present invention provides the use of an agent for detecting proteins, Furin, Prolactin, B2M, hCGb, MMP-3, TACE, IGF-1R, G-CSF, ADAM12, CEA, IL-1RII, G-CSF R, IL-17, JAM-B, BLC, VEGF R1, ADAM8, SLAM, CNTF, MIG, MDC, Syndecano-3, MIF, MCP-4, NRG1-B1, IL-15R, for the preparation of a diagnostic product for recurrent spontaneous abortion caused by a non-thrombotic state.

Preferably, the Furin, Prolactin, B2M, hCGb, MMP-3, TACE, IGF-1R, G-CSF, ADAM12, CEA, IL-1RII, G-CSF R, IL-17, JAM-B, BLC, VEGF R1, ADAM8, SLAM, CNTF, MIG, MDC and Syndecano-3 are expressed in the serum of patients with recurrent abortion caused by non-prothrombotic state, and the MIF, MCP-4, NRG1-B1 and IL-15R are expressed in the serum of patients with recurrent abortion caused by non-prothrombotic state.

Preferably, the diagnostic product is at the protein level; the diagnostic product for the protein level is to detect the protein level by immunoassay, western blotting or protein chip.

Preferably, the product comprises a protein chip or kit.

Further, the invention provides a protein chip for detecting recurrent abortion, which can detect recurrent abortion by detecting the expression level of proteins, wherein the proteins are one or a combination of G-CSF R, CNTF, MIG, MDC, ANG-2, Syndecano-3, HVEM, MIF, S100A8 and GROa.

Preferably, the protein chip comprises a solid support and an antibody specific for the protein immobilized on the solid support.

The invention has the following beneficial effects:

the invention screens Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testitan 2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3, MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100A8 and GROa differential protein by performing protein chip on blood samples of patients with recurrent abortion and normal persons, and provides the application of the differential protein in recurrent diagnostic products, elucidates the influence of the recurrent abortion on the development of the differential protein, and reveals the value of the differential protein in the diagnosis of recurrent abortion in early stage. Therefore, the invention diagnoses whether recurrent abortion occurs by detecting the differential expression of Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testitan 2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3, MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGFR3, BMP-4, IL-15R, CA15-3, HVEM, S100A8 and GROa proteins, and further develops a diagnosis chip, kit or biological agent for recurrent abortion, which can diagnose rapidly and make the best early intervention time for patients.

Furthermore, the inventor screens Furin, Prolactin, B2M, hCGb, MMP-3, TACE, IGF-1R, G-CSF, ADAM12, CEA, IL-1RII, G-CSF R, IL-17, JAM-B, BLC, VEGF R1, ADAM8, SLAM, CNTF, MIG, MDC, Syndecano-3, MIF, MCP-4, NRG1-B1 and IL-15R differential proteins by performing protein chip on blood samples of patients and normal persons with recurrent abortion caused by a non-prothrombotic state and provides application of the differential proteins in diagnostic products for recurrent abortion caused by a non-prothrombotic state.

In addition, the inventor finds that a group of diagnostic markers of recurrent abortion, such as Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testican2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3, MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100A8 and GROa can greatly improve the accuracy of early diagnosis of recurrent abortion by simultaneously detecting a plurality of diagnostic markers related to recurrent abortion.

Drawings

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

FIG. 2 is clustering analysis of abundance of differentially expressed cytokines between disease and normal groups, with black indicating up-regulated expression and gray indicating 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 a clustering analysis of abundance of differentially expressed cytokines between the non-thrombotic pre-status group and the normal group, black for up-regulated expression and gray for 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 clustering analysis of the abundance of differentially expressed cytokines between the non-thrombotic status group and the normal group, wherein black indicates up-regulated expression and gray indicates down-regulated expression;

FIG. 9 is a ROC plot of the serum markers of the invention used to differentiate patients with recurrent abortion from normal controls;

FIG. 10 is a ROC curve analysis of 38 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, and the reagents used are commercially available.

The invention screens 38 abortion-specific proteins by using a protein chip from 12 women with normal pregnancy history, 24 recurrent abortion patients caused by prothrombotic state and 12 recurrent abortion patients caused by non-prothrombotic state. An unsupervised clustering model is established by analyzing and establishing the expression of the 38 differential proteins, and a disease diagnosis model is established, namely, a multi-parameter mathematical model is utilized to predict the risk of abortion with normal and unknown reasons, so that a basis is provided for clinically developing accurate treatment. Subsequently, the inventors further analyzed the functions of the differential proteins by using bioinformatics technology, and found that the differential proteins are mostly involved in the development process of cells or embryos, and that 26 proteins are down-regulated and 12 proteins are up-regulated in the disease group.

Downregulated proteins Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testican2, SLAM, G-CSFR, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3. Wherein, like MMP-3 matrix glycoprotein, can promote the remodeling of tissues; prolactin can fully develop mammary gland in the gestation period, and a duct at the end of a lobular end of the mammary gland develops into a small acinus; G-CSF R is a polypeptide factor promoting proliferation of hematopoietic cells, and the combination of G-CSF and G-CSF R can regulate proliferation and differentiation of granulocytes and enhance the function of mature granulocytes; desmoglein2 cell-cell desmosome junction components, involved in the interaction of plaque proteins and intermediate filaments mediating cell-cell adhesion; participating in EMT regulation and control; the vast majority are associated with cell adhesion, proliferation, cell differentiation, epithelial-mesenchymal transition, microvascular formation, etc., suggesting that down-regulation of these proteins increases the risk of miscarriage.

To further confirm the preliminary screening results, the inventors selected 20 proteins for corresponding validation. The test samples were used in 58 cases, 34 cases in the nonthrombotic state group and 25 cases in the normal group. Compared with the normal group, 10 proteins in the disease group (prothrombotic state + nonthrombogenic state) are differentially expressed proteins, 4 of which are up-regulated and 6 of which are down-regulated.

Further, the inventors plotted a ROC curve to compare the diagnostic ability of the 38 serum markers described above to distinguish recurrent abortion patients from healthy controls. 38 serum markers were found to have good specificity and sensitivity.

The inventor of the test adopts a novel bioinformatics analysis technology and carries out analysis by an unsupervised clustering method. 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. A multi-index multi-parameter establishment analysis model is adopted, different groups are distinguished in a clustering mode, the clinical coincidence rate can be greatly improved, particularly the coincidence rate between a non-thrombus antenatal abortion group and a normal group is completely consistent, and therefore 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 plate 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 tubule, it is centrifuged rapidly and gently pipetted up and down to dissolve the powder, marking the tubule as 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 10 seconds of 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; 31390Carbonne 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 analytical method is modertedt-statistics, the data packet is limma, from R/Bioconductor; screening differential proteins by adopting adjustp value (p value after correction by BH method) and logFC (expression difference multiple, 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 detection of protein chips in the non-Thrombus Pre-State group and Normal group

The samples tested were 24 in the pre-thrombotic state group and 12 in the normal group. The detection results of the non-thrombus pre-state group and the normal histone chip are analyzed, and the results show that: the 46 cytokines were significantly differentially expressed between the non-prothrombotic group and the normal group. Compared with the normal group, the expression of 32 cytokines in the serum of the non-thrombus state group is obviously reduced, the expression of 14 cytokines is obviously increased, and the expression of the cytokines is shown in tables 5 and 6.

The 46 differential proteins were subjected to cluster analysis, and the results (fig. 6) of unsupervised cluster-stratification analysis showed that the normal group and the non-prothrombotic state group were clearly divided into two major groups, disease group 24/24 and normal group 10/12, with agreement rates of 100% and 83.3%, respectively. The overall consistency rate is 84.4%, which indicates that the 46 protein indexes can effectively distinguish a normal group from a non-prothrombotic state group, and provides reference basis for accurate clinical treatment.

Through analysis of 46 differential proteins, the differential proteins are basically consistent with those of a disease group and a normal group and are mainly shown in regulation and control 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 Difference protein (down) between non-Thrombus Pre-State group and Normal group

TABLE 6 differential protein (up) between the non-prothrombotic State group and the Normal group

The inventors found that Tie-1, Periostin, BMPR-IA, LRIG3, IL-15R, IL-1F5 are upregulated in the differential proteins of the non-prothrombotic state group, where Tie-1 is associated with angiogenesis and Periostin plays a role in cell adhesion, enhancing the integration of BMP1 in the fibronectin matrix of connective tissue. BMPR-IA is a bone morphogenetic receptor and plays an important role in embryonic development. The down-regulated differential proteins FOLR1 (folate receptor), FetuinA (fetoprotein A), G-CSF (granulocyte colony stimulating factor), CEA (carcinoembryonic antigen), which may be associated with abortion, are disclosed.

Example 2 differential protein validation

The differential protein assay was the same as in example 1.

1. Verification of non-prothrombotic state group, prothrombotic state group and normal group differential protein

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.

Table 7 verification of differential proteins between disease and Normal groups

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.

2. Verification of differential protein between non-prothrombotic state group and normal group

In order to understand the difference between the non-thrombus state group and the normal group, the inventors analyzed the results of the protein chips of 34 cases and 25 cases of the non-thrombus state group and the normal group in the test samples.

The results show that: in the results of the validation, 14 proteins in the non-prothrombotic state group compared to the normal group were differentially expressed proteins, 6 of which were up-regulated and 8 of which were down-regulated, as shown in table 8.

Furthermore, unsupervised cluster analysis showed (FIG. 8) that 59 samples tested were clustered into two distinct groups, the non-prothrombotic state group and the normal group, and the clustering results were identical to the clinical classification. The 14 differential proteins were suggested to distinguish the non-prothrombotic state group from the normal group well.

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

Example 3 Receiver Operating Characteristic (ROC) Curve analysis

ROC curves were constructed to compare the diagnostic ability of 38 serum markers to distinguish repeat miscarriage patients from 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 38 serum markers is between 0.741 and 0.920, the specificity is between 0.603 and 0.845, and the AUC value is between 0.750 and 0.901.

The AUC for the 38 serum markers in combination can reach 0.966, with sensitivity and specificity of 1.00 and 0.862 respectively (as shown in fig. 10). These results indicate that the 38 markers in combination have higher sensitivity and specificity for the detection of early stage of recurrent abortion compared to the 38 markers of recurrent abortion 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. The application of a reagent for detecting protein in preparing a diagnostic product for recurrent abortion, wherein the protein is one or a combination of several of Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testitan 2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecano-3, MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100A8 and GROa.

2. The use of claim 1, wherein Prolactin, MMP-3, Furin, IGF-1R, B2M, hCGb, Testican2, SLAM, G-CSF R, JAM-B, Desmoglein2, CEA, ADAM12, IL-1RII, ADAM8, BLC, TACE, VEGF R1, IL-17C, Syndecan-1, ANGPTL4, CNTF, MIG, MDC, ANG-2, Syndecan-3 are down-regulated in serum of patients with recurrent abortion, and wherein MIF, MCP-4, NRG1-B1, MCP-3, B7-H1, VEGF R3, BMP-4, IL-15R, CA15-3, HVEM, S100a8, GROa are up-regulated in serum of patients with recurrent abortion.

3. The use according to claim 2, wherein the protein is one or more of G-CSF R, CNTF, MIG, MDC, ANG-2, Syndecano-3, HVEM, MIF, S100A8, GROa.

4. The use of claim 1, wherein the recurrent abortion comprises recurrent abortion in a pre-thrombotic state and recurrent abortion in a non-thrombotic state.

5. Use of a reagent for detecting proteins Furin, Prolactin, B2M, hCGb, MMP-3, TACE, IGF-1R, G-CSF, ADAM12, CEA, IL-1RII, G-CSFR, IL-17, JAM-B, BLC, VEGF R1, ADAM8, SLAM, CNTF, MIG, MDC, Syndecano-3, MIF, MCP-4, NRG1-B1, IL-15R, for the preparation of a diagnostic product for recurrent spontaneous abortion caused by a non-thrombotic state.

6. The use according to claim 5, wherein Furin, Prolactin, B2M, hCGb, MMP-3, TACE, IGF-1R, G-CSF, ADAM12, CEA, IL-1RII, G-CSF R, IL-17, JAM-B, BLC, VEGF R1, ADAM8, SLAM, CNTF, MIG, MDC, Syndecano-3 are down-regulated in the serum of patients with recurrent abortion in the non-prothrombotic state, and MIF, MCP-4, NRG1-B1, IL-15R are up-regulated in the serum of patients with recurrent abortion in the non-prothrombotic state.

7. The use of claim 1 or 5, wherein the diagnostic product is a diagnostic product at protein level; the diagnostic product of the protein level detects the protein level through immunodetection, Western blotting or a protein chip.

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

9. The protein chip for detecting recurrent abortion is characterized in that recurrent abortion can be detected by detecting the expression level of proteins, wherein the proteins are one or a combination of G-CSF R, CNTF, MIG, MDC, ANG-2, Syndecano-3, HVEM, MIF, S100A8 and GROa.

10. The protein chip of claim 9, wherein said protein chip comprises a solid support and an antibody specific for said protein immobilized on the solid support.

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