CN114686582A - Application of GDF15 and ITIH3 in prediction of spontaneous abortion in early pregnancy - Google Patents

Application of GDF15 and ITIH3 in prediction of spontaneous abortion in early pregnancy Download PDF

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CN114686582A
CN114686582A CN202011591940.4A CN202011591940A CN114686582A CN 114686582 A CN114686582 A CN 114686582A CN 202011591940 A CN202011591940 A CN 202011591940A CN 114686582 A CN114686582 A CN 114686582A
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pregnancy
spontaneous abortion
gdf15
protein
itih3
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张璇
石燕
顾艳
孟楠
王健
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Shanghai Institute Of Biomedical Technology
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Shanghai Institute Of Biomedical Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Abstract

The invention relates to the technical field of medical bioengineering, and provides several secretory proteins in blood for predicting and identifying peripheral blood secretory proteins related to spontaneous abortion in early pregnancy and application thereof. Specifically, the invention screens out several blood secretory proteins which can be used for predicting and identifying the pathological pregnancy caused by spontaneous abortion in the early pregnancy by using the Elisa method. The test proves that the specific peripheral blood secretion protein can effectively predict and identify the pathological pregnancy of spontaneous abortion in the early pregnancy. The invention also relates to application of the two markers in preparing a kit for identifying spontaneous abortion in early pregnancy.

Description

Application of GDF15 and ITIH3 in prediction of spontaneous abortion in early pregnancy
Technical Field
The present invention relates to the fields of biotechnology and medicine. In particular, the invention relates to the use of GDF15 and ITIH3 for the prediction of spontaneous abortion in the early stages of pregnancy.
Background
The establishment and maintenance of normal human pregnancy need to go through a series of important links such as embryo implantation, placenta formation, embryonic development and the like, and are cooperatively regulated and controlled by different systems in multiple layers, wherein any link obstacle can cause poor pregnancy fatalities such as spontaneous abortion and the like. In 2005, the Human Society of Reproduction and Embryology (ESHRE, European Society of Human Reproduction and embryo) defined "miscarriage" as: (1) a positive urine pregnancy test or an elevated blood HCG level, but pregnancy cannot be confirmed by ultrasound or histology is "biochemical loss", which generally occurs prior to 6 weeks gestation; (2) when ultrasound or histology is able to confirm intrauterine pregnancy, it is called "clinical abortion" and can be subdivided into early abortion (before 12 weeks of gestation) and late abortion (between 12 weeks and 22 weeks of gestation). It is estimated that the biochemical loss rate in human pregnancy reaches 60%; early clinical pregnancy miscarriage rates of 15% and are significantly correlated with age, with clinical miscarriage rates of 10% at 20-24 years and miscarriage rates of 51% at 40-44 years; late-phase flow yield was typically 4%.
Recurrent spontaneous abortion (RSM), also known as rm (recurrent miscrariages) or rpl (recurrent pregnancy loss), refers to spontaneous abortion occurring 2 or more times before the 20 th week of pregnancy with the same sex partner, and the incidence rate in pregnant women is about 1-2%. RSM is complex in etiology, involves multiple aspects such as heredity, reproductive endocrine, reproductive tract anatomy, infection and female autoimmunity, and although a great deal of research focuses on the field, more than half of the reasons are still unknown. Currently, there is still a gap in the clinical use of what biomarker molecules to identify and predict the occurrence of RM.
Studies have shown that low and non-exponential increases in hCG in the peripheral blood of pregnant women early in pregnancy are a clinically recognized risk factor for abortion. In the screening of the biological marker molecules, it is most desirable to obtain the biological marker molecules by a non-invasive means.
In view of the above, it is evident from the existing studies that little is known about the methods for identifying and predicting spontaneous abortion in the early stages of pregnancy. Therefore, there is an urgent need in the art to develop a diagnostic technique for identifying spontaneous abortion in the early stage of pregnancy, which has high accuracy, high sensitivity and easy operation.
Disclosure of Invention
The invention aims to provide a diagnosis technology for identifying spontaneous abortion in the early pregnancy period, which has high accuracy, high sensitivity and easy operation.
It is another object of the present invention to provide a biomarker combination for the identification and prediction of spontaneous abortion in the early stages of pregnancy.
The invention also aims to provide a detection kit for identifying and predicting spontaneous abortion in early pregnancy.
In a first aspect of the invention there is provided the use of a blood marker or detection reagent therefor to prepare a diagnostic reagent or kit for:
(a) diagnosing spontaneous abortion in early pregnancy, and/or (b) predicting spontaneous abortion in early pregnancy;
wherein the blood markers are selected from: growth differentiation factor15 (GDF15) gene or protein, and/or; an inter-alpha-proteinase inhibitor heavy chain H3(inter-alpha-trypsin inhibitor heavy chain 3, ITIH3) gene or protein.
In another preferred embodiment, the early pregnancy spontaneous abortion comprises repeated spontaneous abortion.
In another preferred embodiment, the GDF15 or ITIH3 is of mammalian origin, preferably human or rodent (mouse, rat).
In another preferred embodiment, the GDF15 gene has accession number Genbank ID: NM _ 004864.4.
In another preferred embodiment, the GDF15 protein has the accession number NP _ 004855.2.
In another preferred embodiment, the ITIH3 gene has accession number Genbank ID: NM _ 002217.3.
In another preferred embodiment, the ITIH3 protein has accession number NP-002208.3.
In another preferred example, the blood markers also comprise other blood markers for diagnosing or predicting the occurrence of spontaneous abortion in early pregnancy.
In another preferred example, the other blood markers include: HCG, ITIH4, ITIH2, or a combination thereof.
In another preferred example, the other blood marker is HCG.
In another preferred example, the blood marker comprises GDF15, ITIH3 and/or HCG.
In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid, a chip (e.g., a DNA chip), or a protein chip.
In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.
In another preferred embodiment, the detection reagent is a reagent that detects a protein of GDF15, ITIH3, and/or HCG, or a gene thereof.
In another preferred embodiment, the detection reagent comprises antisense RNA, microRNA, siRNA, shRNA of GDF15, ITIH3 and/or HCG gene.
In another preferred embodiment, the detection reagent comprises an antibody specific for GDF15, ITIH3 and/or HCG, a specific binding molecule, a specific amplification primer, a probe or a chip.
In another preferred embodiment, the blood is derived from a mammal, preferably a mouse, rat or human.
In another preferred example, the blood includes peripheral blood, plasma, serum.
In another preferred embodiment, the detection comprises detection by immunohistochemistry, immunoblotting and fluorescent quantitative PCR methods.
In another preferred example, the test is a test on an ex vivo sample.
In another preferred embodiment, the detection comprises qualitative and/or quantitative detection.
In another preferred embodiment, the ex vivo sample is selected from the group consisting of: a plasma sample, a serum sample, a tissue sample, or a combination thereof.
In another preferred embodiment, the ex vivo sample is a plasma sample.
In another preferred example, the assay is an assay for the expression level of GDF15, ITIH3 and/or HCG in peripheral blood plasma.
In another preferred embodiment, said detecting is the determination of the mRNA content of GDF15, ITIH3 and/or HCG in breast tissue, ovarian tissue, uterine tissue, placental tissue.
In a second aspect of the invention, there is provided a kit for diagnosing spontaneous abortion in an early stage of pregnancy, said kit comprising a first container containing a detection reagent for detecting GDF15 gene or protein, and/or a second container containing a detection reagent for detecting ITIH3 gene or protein; and a label or instructions, wherein the label or instructions state that the kit is for use
(a) Diagnosing spontaneous abortion in early pregnancy, and/or (b) predicting spontaneous abortion in early pregnancy.
In another preferred embodiment, the kit further comprises a third container containing a detection reagent for detecting the HCG gene or protein.
In another preferred embodiment, the detection reagent comprises:
(a) an antibody specific for said protein; and/or
(b) Specific primers that specifically amplify mRNA or cDNA of the gene.
In another preferred embodiment, the assay is a serum assay.
In another preferred embodiment, the label or instructions states the following:
(a) when the ratio of the GDF15 expression C1 in the serum or the tissue of the detected object to the GDF15 expression C0 in the serum or the tissue of the control sample is less than or equal to 0.41, the probability of spontaneous abortion of the detected object in the early pregnancy period is higher than that of normal pregnant people; and/or
(b) When the ratio of the ITIH3 expression quantity B1 in the serum or the tissue of the detected object to the ITIH3 expression quantity B0 in the serum or the tissue of the control sample is more than or equal to 1.12, the probability of spontaneous abortion of the detected object in the early pregnancy period is higher than that of normal pregnant people; and/or
(c) When the ratio of the HCG expression level A1 in the serum or the tissue of the detected object to the HCG expression level A0 in the serum or the tissue of the control sample is less than or equal to 0.43, the probability of spontaneous abortion of the detected object in the early pregnancy period is higher than that of normal pregnant people.
In another preferred embodiment, the control reference value further comprises the expression level of HCG in a plasma sample from a normal pregnant group.
In another preferred example, the detection comprises protein chip method, antibody chip method, DNA chip method, RT-PCR method, real-time fluorescence quantitative PCR method, ELISA method, immunoblotting method, immunohistochemistry method, immunocytochemistry method, flow cytometry method and mass spectrometry detection.
In a third aspect of the invention, there is provided a method of (a) diagnosing spontaneous abortion in pregnancy and/or (b) predicting the occurrence of spontaneous abortion in the early stages of pregnancy, comprising the steps of:
(a) providing a test sample from a test subject ex vivo;
(b) detecting the expression level of GDF15 and/or ITIH3, HCG in the test sample; and
(c) comparing the expression level of GDF15 and/or ITIH3, HCG determined in (b) to a control reference value; wherein the control is a control of the plurality of control cells,
if the expression level of GDF15 in the plasma sample of the subject is lower than the reference value C2; and/or
The expression level of ITIH3 in the plasma sample of the test subject is higher than the reference value B2; and/or
The expression level of HCG in the subject's plasma sample is below the reference value a 2; and/or
It is suggested that the subject may be preliminarily judged that the possibility of spontaneous abortion in the early stage of pregnancy is higher than that in the normal pregnant population.
In another preferred embodiment, the sample is from a test object.
In another preferred embodiment, the test subject is a human or non-human mammal.
In another preferred embodiment, the sample is a plasma or tissue sample.
In another preferred example, the reference value is a cut-off value (cut-off value).
In another preferred embodiment, the reference value is the expression level of GDF15 and/or the expression level of ITIH3, HCG in a plasma sample from a normal pregnant human.
In another preferred embodiment, the cut-off value of the GDF15 marker is selected from 890pg/ml-1000 pg/ml.
In another preferred example, the cut-off value of the GDF15 marker is 1000 pg/ml.
In another preferred embodiment, the cut-off value of the ITIH3 marker is selected from 420000ng/ml to 423000 ng/ml.
In another preferred example, the cut-off value of the ITIH3 marker is 420000 ng/ml.
In another preferred example, the cut-off value of the HCG marker is 88000 IU/L.
In another preferred example, the detecting step (b) comprises detecting the amount of GDF15 and/or ITIH3, HCG mRNA; and/or detecting the amount of GDF15 and/or ITIH3, HCG protein.
In another preferred example, the expression level of GDF15 and/or ITIH3, HCG proteins in the sample is detected by fluorescent quantitative PCR or immunohistochemistry.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the differences in GDF15 expression in plasma of patients with spontaneous abortion at early pregnancy and normal pregnancy, wherein CV is normal pregnancy and RM is recurrent spontaneous abortion.
FIG. 2 shows the difference in plasma expression of ITIH3 between patients with spontaneous abortion at early pregnancy and normal pregnancy, in which CV is the normal pregnancy group and RM is the recurrent spontaneous abortion group.
Detailed Description
The present inventors have extensively and intensively studied and, through extensive screening, have for the first time developed a method for identifying spontaneous abortion in early pregnancy by detecting the genes or proteins of growth differentiation factor15 (GDF15) and/or inter-alphaprotease inhibitor heavy chain H3(ITIH 3). As proved by tests, the GDF15 index alone as a peripheral blood protein marker can be used for effectively distinguishing repeated spontaneous abortion tissue samples from normal samples. In addition, experiments prove that the GDF15 index and the ITIH3 index have complementary functions in the detection of repeated spontaneous abortion, and the combination of the GDF15 index and the ITIH3 index can further improve the judgment accuracy and stability (the sensitivity can reach 88.2 percent, and the specificity reaches 86.2 percent). The present invention has been completed based on this finding.
Specifically, the inventor firstly discovers that a close correlation exists between the heavy chain H3(ITIH3) of the 15(GDF15) and/or the inter-alpha protease inhibitor and the human female early pregnancy abortion, namely, the expression quantity and/or activity of GDF15 is reduced and the expression quantity and/or activity of ITIH3 is increased in female early pregnancy patients. In addition, the inventor also finds that the combination of GDF15 and ITIH3 and peripheral blood hCG protein is more helpful to predict the possibility of early pregnancy abortion.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Recurrent spontaneous abortion
Recurrent spontaneous abortion (RSM), also known as rm (recurrent miscarriage) or rpl (recurrent pregnancy loss), is a spontaneous abortion occurring 2 or more times consecutively before the 20 th week of pregnancy with the same sex partner, and the incidence rate in pregnant women is about 1-2%. RSM is complex in etiology, involves multiple aspects such as heredity, reproductive endocrine, reproductive tract anatomy, infection and female autoimmunity, and although a great deal of research focuses on the field, more than half of the reasons are still unknown.
Spontaneous abortion in early pregnancy
Early spontaneous abortion refers to a non-human factor and terminates pregnancy before 12 weeks, early spontaneous abortion is one of the pathological pregnancies common in obstetrics, the etiology is complex, the pathogenesis is still unclear, early clinical abortion rate reaches 15%, and is significantly related to age, the clinical abortion rate reaches 10% at the age of 20-24 years, and the abortion rate reaches 51% at the age of 40-44 years.
GDF15
Human growth differentiation factor15 (GDF15) is a type of transforming growth factor beta family, whose gene is located on chromosome 19, consists of 2746 base pairs, contains 2 exons and 1 intron, GDF15 is secreted as a 40kD size propeptide, then cleaves into an active 25kD dimer, which can participate in different biological processes through multiple signaling pathways. In serum, the expression level of the polypeptide has important value in early diagnosis and prognosis prediction in combination with other tumor markers.
ITIH3
Human Alpha-proteinase Inhibitor Heavy Chain H3(Inter-Alpha-Trypsin Inhibitor Heavy Chain 3, ITIH3) is a protein-encoding gene that encodes the Heavy Chain subunit of the pre-Alpha-Trypsin Inhibitor complex. Such complexes may play an important role in the stabilization of the extracellular matrix by binding hyaluronic acid. Polymorphisms in this gene may be associated with increased risk of schizophrenia and major depression. The gene exists in the alpha-trypsin inhibitor family gene cluster of chromosome 3. Can be used as a carrier of hyaluronic acid in serum or as a binding protein between hyaluronic acid and other matrix proteins in serum, and is involved in regulating the localization, synthesis and degradation of hyaluronic acid in cells.
HCG
Human Chorionic Gonadotropin (HCG) is a glycoprotein hormone synthesized by syncytiotrophs, has a molecular weight of about 39kD and contains two subunits, alpha and beta. At present, the traditional Chinese medicine composition is widely applied to the aspects of clinical pregnancy diagnosis, other disease diagnosis, curative effect evaluation, prognosis evaluation and the like, and has very important functions of reducing birth defects and improving population quality.
Natural abortion related protein set
As used herein, the terms "peripheral blood secreted protein in the present invention", "protein associated with spontaneous abortion in early pregnancy in a female", "blood marker of the present invention", "protein associated with recurrent spontaneous abortion" are used interchangeably and refer to a secreted protein in the peripheral blood of a female mammal (particularly a human), in particular, a peripheral blood secreted protein expressed in breast tissue, ovarian tissue, uterine tissue, placental tissue. Related molecular biological materials include (but are not limited to): muteins, arbitrary fragments of proteins, genes and arbitrary DNA fragments thereof, mutated genes, mRNA, interfering RNA, antibodies, and the like. Representative peripheral blood secreted proteins include GDF15, ITIH3, HCG, ITIH4, ITIH 2.
The inventor detects the protein expression level in the peripheral blood plasma of the early spontaneous abortion sample and the normal sample plasma, and unexpectedly finds that the expression level of GDF15 in the peripheral blood plasma of the early spontaneous abortion sample is remarkably reduced and the expression level of ITIH3 is remarkably increased compared with that of a normal pregnancy group for the first time. The content of peripheral blood hCG has been reported in the literature to be useful in predicting the likelihood of an early pregnancy abortion occurring. By detecting the expression of GDF15, ITIH3 and hCG in the sample, the tissue sample of spontaneous abortion in the early pregnancy can be effectively distinguished from the normal sample.
In particular, the peripheral blood secreted proteins described above can be used alone or in combination with biomarker molecules for blood identification of recurrent abortions. In a particular embodiment, the detection means is the Elisa method, which is well known to those skilled in the art.
Protein chip and application thereof in detection of human blood and other samples
The invention also provides a protein chip for detecting spontaneous abortion in the early pregnancy. The chip can qualitatively or quantitatively detect the content of the peripheral blood secreted protein in a sample. These detection results can be used for auxiliary judgment of the cause of spontaneous abortion in the early pregnancy or repeated spontaneous abortion, for example.
The terms "protein array" or "protein chip" are used interchangeably to refer to an array of capture reagents capable of binding protein markers, including liquid chips. Typically, the capture reagent is a polyclonal or monoclonal antibody that binds to a specific protein. After the marker is captured, the marker is detected by using a labeled detection molecule, so that the purpose of qualitative or quantitative (using a standard substance) detection is achieved.
The protein chip of the present invention is characterized in that the chip is provided with a detection spot for peripheral blood secreted protein, and as used herein, the term "detection spot" refers to a detection spot located on the protein chip for detecting a protein, such as a detection spot for detecting GDF15 protein, and is usually formed by spotting a monoclonal antibody against GDF15 protein onto a chip substrate or coupling the monoclonal antibody to magnetic beads or plastic beads.
The protein chip suitable for use in the present invention is not particularly limited, and blank protein chips of various structures known in the art may be used. Typically, these protein chip carriers include: magnetic beads, plastic beads, immunomicrospheres, glass sheets, plastic sheets, nitrocellulose membranes, PVDF membranes, and the like, and among them, magnetic beads, plastic beads, immunomicrospheres, and various substrates are particularly preferable. The method comprises the steps of sequentially fixing and coupling polypeptide, protein or antibody on various carriers by adopting an in-situ synthesis method, a mechanical spotting method or a covalent bonding method to form a chip for detection, enabling the fluorescence-labeled antibody or other components to interact with the chip combined with a sample to be detected, washing away components which cannot be complementarily combined with the protein on the chip by rinsing, and measuring the fluorescence intensity of each point on the chip or other carriers by utilizing a fluorescence scanner or a laser confocal scanning technology to analyze the content of each protein, thereby achieving the purpose of measuring various proteins.
The protein chip of the present invention may contain one or more of the peripheral blood secreted proteins of the present invention, for example, 1, 2, 3 of the detection points (GDF15, ITIH3, HCG) of the peripheral blood proteins of patients in early pregnancy as first disclosed herein. The 3 detection points can be used independently or in any combination, or combined with the disclosed early pregnancy spontaneous abortion occurrence index, and can be used for early pregnancy spontaneous abortion occurrence evaluation, diagnosis, etc.
Detection method
In the present invention, methods for qualitatively or quantitatively detecting the peripheral blood secretory protein and its related molecular biological material of the present invention are also provided, which include (but are not limited to): human peripheral blood secretory protein detection, gene mutation detection, gene transcribed mRNA detection, regulation related miRNA detection, mutant protein detection and protein activity detection. Biological samples for detection include (but are not limited to): urine, blood, tissue.
Representative detection methods include (but are not limited to): protein chip, antibody chip, DNA chip, liquid chip, ELISA method, immunoblotting method, RT-PCR, real-time fluorescence quantitative PCR method, immunohistochemistry method, immunofluorescence method, flow cytometry method, mass spectrometry method, capillary electrophoresis method, immunoprecipitation method, enzyme activity detection method, etc.
The main advantages of the invention include
(1) The inventor firstly discovers that the expression levels of GDF15 and ITIH3 are related to spontaneous abortion in the early pregnancy, and can be used as specific markers of spontaneous abortion in the early pregnancy.
(2) Through the detection of peripheral blood samples, the expression conditions of GDF15 and ITIH3 are obtained, so that the diagnostic and/or prediction of spontaneous abortion pathological pregnancy in the early pregnancy can be realized.
(3) The invention has the advantages of high sensitivity and high specificity through Elisa in vitro detection, and has important application value.
(4) The peripheral blood as the biomarker detection sample has the advantages of convenient material taking, simple operation steps, continuous in vitro detection and the like.
The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and alterations to the present invention, which fall within the scope of the invention.
Experimental procedures in the following examples, in which specific conditions are not specified, can be carried out by methods conventional in the art, for example, with reference to the molecular cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989, or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.
Example 1: screening of peripheral blood secretory proteins
The method comprises the following steps:
the subjects were recruited according to the following inclusion criteria, and 34 recurrent abortions were collected after informed consent from the subjects, selected from the patients who had recurrent abortion at the scheduled birth clinic of the hospital.
Figure BDA0002868902910000101
Experimental methods
1. Plasma separation: whole blood samples were collected in EDTA anticoagulation tubes. Gently inverting, mixing, centrifuging at 4 deg.C for 10min at 1600 Xg for 10min, and subpackaging the supernatant (blood plasma) into multiple centrifuge tubes to absorb blood plasma while avoiding leukocyte absorption to the middle layer;
GDF15 and ITIH3 assays
Human growth differentiation factor15 (GDF15) enzyme-linked immunosorbent assay kit (Wuhan Huamei bioengineering Co., Ltd., product number: CSB-E12009H) and human alpha protease inhibitor heavy chain H3(ITIH3) enzyme-linked immunosorbent assay kit (CSB-EL011896HU) were used, and the procedures were carried out according to the instructions.
Taking out the subpackaged plasma sample from a refrigerator at the temperature of-80 ℃, thawing at room temperature, centrifuging at the temperature of 4 ℃ for 15min at 1000g, and taking the supernatant.
2) And (3) balancing various reagents in the kit for 30min at room temperature, and respectively preparing a standard substance and a diluted sample.
3) Taking out a standard product, performing instantaneous centrifugation at 10000rpm for 30s, adding 1ml of sample diluent, sucking and beating for 5 times, standing for 5min, and sequentially diluting by times according to instructions.
4) Add 20. mu.l of plasma sample to a tube containing 100. mu.l of sample diluent and mix well.
5) Sample adding: adding 100 mul of standard substance or sample to be tested into each hole, shaking gently and mixing, covering the plate with a film, and incubating in a constant temperature box at 37 ℃ for 2 hours.
6) Discarding the liquid, and drying the liquid without washing.
7) Add 100. mu.l of biotin-labeled antibody working solution to each well, cover the new plate with a film, incubate in 37 ℃ incubator for 1 h.
8) Discarding liquid in the hole, spin-drying, adding 250 μ l of washing solution into each hole, gently shaking by a horizontal shaking table for 2min, spin-drying, patting on absorbent paper, and washing for 3 times.
9) 100 mul of horse radish peroxidase labeled avidin working solution is added to each well, covered with a new plate film, and incubated for 1h at 37 ℃.
10) Washing is carried out for 5 times.
11) Add 90. mu.l of substrate solution to each well and incubate at 37 ℃ in the dark for 20min until the color gradient of the standard is apparent.
12) The reaction was terminated by adding 50. mu.l of a reaction-stopping solution to each well in sequence.
13) Measuring OD450 value and OD630 value with enzyme labeling instrument within 5 min.
14) A standard Curve was generated using Curve Expert and the sample concentration was calculated.
Example 2: preparation of protein chips
(1) Directly and densely spotting each protein sample (antibody against the protein of the invention) in a multi-well plate on a chemically treated glass slide with active aldehyde groups on the surface by using a high-speed spotting instrument, wherein each array corresponds to GDF15, ITIH3 and HCG protein, and the diameter of each spot is 0.4 mm;
(2) incubating for 1 hour at room temperature overnight or at 37 ℃ to allow the amino groups of the protein to undergo condensation reactions with the aldehyde groups on the slide glass to immobilize the sample;
(3) reducing the active aldehyde groups on the glass slide which are not occupied by the protein, fully washing and drying at room temperature;
(4) and finally, sealing and packaging the mixture by using a light-resistant material and storing the mixture in an environment at 4 ℃.
Example 3: preparation of the kit
The chip prepared in example 2 was packaged and placed in a box together with instructions for use to construct a kit.
Example 4: detection and verification of chip
A plurality of spontaneous abortion samples (including 34 spontaneous abortion serum samples and 30 normal samples) obtained from hospitals were prepared and labeled with proteins according to the methods of examples 1-2, and the chips prepared according to the method of example 3 were tested by a double-blind method. The sample is judged according to the existence of the blood marker and the up-regulation and down-regulation conditions. Detecting the expression difference of GDF15 and ITIH3 in the plasma of a patient group with spontaneous abortion in the early pregnancy and a normal pregnancy group.
Data processing:
(1) differential expression of GDF15 in plasma of repeated spontaneous abortion (RM) and normal pregnancy (CV)
Figure BDA0002868902910000121
Figure BDA0002868902910000131
TABLE 1 expression level of GDF15 (in pg/ml) in plasma of recurrent spontaneous abortion (RM) and normal pregnancy (CV)
The results of the experiment are shown in fig. 1 and table 1.
When a GDF15 marker is singly adopted to judge a plurality of spontaneous recurrent abortion samples (including 34 spontaneous recurrent abortion serum samples and 30 normal samples) obtained from a hospital, the accuracy and the repeatability stability of the GDF15 marker are good (the sensitivity can reach 85.3 percent and the specificity can reach 80.0 percent) when the cut-off value is 1000pg/ml, and the spontaneous recurrent abortion samples and the normal samples can be effectively distinguished.
(2) Differential expression of ITIH3 in plasma of repeated spontaneous abortion (RM) and normal pregnancy (CV)
Figure BDA0002868902910000132
Figure BDA0002868902910000141
TABLE 2 expression level (in ng/ml) of ITIH3 in plasma of recurrent spontaneous abortion (RM) and normal pregnancy (CV)
The results of the experiment are shown in fig. 2 and table 2.
Multiple spontaneous abortion samples (including 34 spontaneous abortion serum samples and 30 normal samples) were obtained from hospital when
(1) When the combined judgment of the detection results of the GDF15 marker in (1) and the ITIH3 marker in (2) is carried out, the cut-off value of the GDF15 marker is 1000pg/ml, and the cut-off value of the ITIH3 marker is 420000ng/ml, so that the accuracy and the repeatability stability are good (the sensitivity can reach 88.2 percent, and the specificity can reach 86.2 percent), and the spontaneous recurrent abortion sample and the normal sample can be effectively distinguished.
In conclusion, the inventor establishes a method for identifying and detecting the repeated abortion by detecting the expression of GDF15 and ITIH3 in the peripheral blood, plasma or serum of a sample subjected to repeated abortion.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. Use of a blood marker or a detection reagent thereof for the preparation of a diagnostic reagent or kit for:
(a) diagnosing spontaneous abortion at an early stage of pregnancy, and/or (b) predicting the occurrence of spontaneous abortion at an early stage of pregnancy;
wherein the blood marker is selected from: growth differentiation factor15 (growth differentiation factor15, GDF15) gene or protein, and/or; an inter-alpha-proteinase inhibitor heavy chain H3(inter-alpha-trypsin inhibitor heavy chain 3, ITIH3) gene or protein.
2. The use of claim 1, wherein the early-pregnancy spontaneous abortion comprises repeated spontaneous abortion.
3. The use of claim 1, wherein the blood markers further comprise other blood markers useful for diagnosing or predicting the onset of spontaneous abortion in the early stages of pregnancy.
4. The use of claim 1, wherein the diagnostic reagent comprises an antibody, primer, probe, sequencing library, nucleic acid, chip (e.g., DNA chip) or protein chip.
5. The use of claim 1, wherein the detection reagent is a reagent that detects a protein of GDF15, ITIH3, and/or HCG, or a gene thereof.
6. A kit for diagnosing spontaneous abortion in an early stage of pregnancy, which comprises a first container containing a detection reagent for detecting GDF15 gene or protein, and/or a second container containing a detection reagent for detecting ITIH3 gene or protein; and a label or instructions, wherein the label or instructions state that the kit is for use
(a) Diagnosing spontaneous abortion at an early stage of pregnancy, and/or (b) predicting the occurrence of spontaneous abortion at an early stage of pregnancy.
7. The kit of claim 6, further comprising a third container containing a detection reagent for detecting HCG gene or protein.
8. The kit of claim 6, wherein the detection reagent comprises:
(a) an antibody specific for said protein; and/or
(b) Specific primers that specifically amplify mRNA or cDNA of the gene.
9. A method for (a) diagnosing spontaneous abortion, and/or (b) predicting the occurrence of spontaneous abortion in the early stages of pregnancy, comprising the steps of:
(a) providing a test sample ex vivo from a test subject;
(b) detecting the expression level of GDF15 and/or ITIH3, HCG in the test sample; and
(c) comparing the expression level of GDF15 and/or ITIH3, HCG determined in (b) to a control reference value; wherein the control is a control of the animal,
if the expression level of GDF15 in the plasma sample of the subject is lower than the reference value C2; and/or
The expression level of ITIH3 in the plasma sample of the test subject is higher than the reference value B2; and/or
The expression level of HCG in the subject's plasma sample is below the reference value a 2; and/or
It is suggested that the subject may be preliminarily judged that the possibility of spontaneous abortion in the early stage of pregnancy is higher than that in the normal pregnant population.
10. The method of claim 9, wherein the detecting step (b) comprises detecting the amount of GDF15 and/or ITIH3, HCG mRNA; and/or detecting the amount of GDF15 and/or ITIH3, HCG protein.
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