WO2021167098A1 - Quantitative assessment index for fetal growth restriction - Google Patents

Abstract

The purpose of the present invention is to provide an assessment and diagnosis method for predicting neurodevelopmental disorders at an early stage, determining the optimal treatment policy, and assessing the effect of intervention treatment in newborns at an early stage, by appropriately and accurately diagnosing, with high sensitivity, perinatal brain disorders (for example, neurodevelopmental disorders) in newborns associated with fetal growth restriction. According to the present invention, provided is a method for determining, at an early stage, perinatal brain disorders of newborns caused by fetal growth restriction, the method involving (a) a process for assessing the expression levels of one or more proteins selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), neuroserpin (Serpini1), ubiquitin thioesterase OTUB1 (Otub1), and ubiquitin-like modifier activating enzyme 1 (Uba1) in a body fluid sample obtained from a subject, in comparison with the expression levels in a control body fluid.

Description

Quantitative evaluation index for fetal growth restriction

The present invention provides a biomarker for early determination, diagnosis, or assistance in diagnosis of perinatal disorder in a newborn due to fetal stunting, and early determination or early determination of perinatal disorder in a newborn using the biomarker. The present invention relates to an early diagnosis method and a method for determining the therapeutic effect of perinatal disorders at an early stage using the biomarker.

Pregnancy is associated with increased maternal cardiac output and a vast increase in uterine perfusion resulting from trophoblast-driven modification of the uterine spiral artery. Failure of this normal physiological process is also known as two of the most difficult obstetric complications, Fetal Growth Restriction (FGR) (or Intrauterine Growth Restriction; IUGR). Is involved in the causes of pre-eclampsia (pre-eclampsia) and pre-eclampsia (PET).

Fetal growth restriction affects up to 8% of all pregnancies and is associated with high perinatal mortality, long-term neurological disorders and an increased incidence of cardiovascular disease in later years, based on evidence. There is no effective treatment. Severe early-onset fetal growth restriction affects 1: 500 pregnancies and is associated with high mortality and long-term complications in survivors. In the most severe cases, a viable birth weight (at least 500 g) cannot be reached, forcing a strict choice between abortion or allowing the foetation to die in utero. Small improvements in fetal growth (eg, up to 700 g birth weight) and birth gestation (eg, 26-28 weeks) are associated with major improvements in survival and morbidity.

Current pregnancy management is designed to find women with dysgenetic foets. Many strategies are available, such as maternal serum markers and uterine artery Doppler ultrasonic testing, which can predict women who may develop these conditions.

So far, attempts have been made to perform therapeutic interventions such as stem cell therapy in order to treat perinatal brain disorders in newborns associated with fetal growth restriction, and to evaluate the therapeutic effect on neurodevelopmental disorders in infants and children thereafter. (Patent Document 1). However, at present, there is no therapeutic strategy that has succeeded in early evaluation and diagnosis of perinatal brain disorders (for example, neurodevelopmental disorders) due to fetal growth restriction.

International Publication No. 2017/199976

The present invention overcomes the above-mentioned problems and diagnoses perinatal brain disorders (for example, neurodevelopmental disorders) in newborns associated with fetal growth restriction with high sensitivity, appropriately and accurately, thereby causing neurodevelopmental disorders at an early stage. Furthermore, it is an object of the present invention to determine an optimal treatment policy and to provide an evaluation / diagnosis method for early evaluation of the effect of intervening treatment in a newborn baby. The present invention also provides information that is the basis for elucidating the pathophysiology of perinatal brain disorders associated with fetal growth restriction.

In order to solve the above problems, the present inventors comprehensively analyzed proteins in cerebrospinal fluid from a rat model of fetal growth restriction, and as a result, evaluated, diagnosed, predicted, and treated developmental disorders associated with fetal growth restriction. We have succeeded in identifying at least 6 kinds of biomarkers for the present invention, and have completed the present invention.

That is, the present invention is as follows.
[1] A method for early determination of perinatal disorders in newborns due to fetal growth restriction in mammalian subjects.
(A) α-2-Macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neurocerpin (Serpini1) ) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and one or more proteins selected from the group consisting of ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Expression levels were tested in body fluid samples obtained from subjects in comparison to expression levels in normal body fluids, post-treatment body fluids, or body fluids known to exhibit perinatal disorders; and (b). The expression level is statistically significantly different from the expression level in the normal body fluid or the body fluid after treatment, or is compared with the expression level in the body fluid known to exhibit perinatal disorders. The above method, which comprises evaluating the subject as having a perinatal disorder when it does not show a statistically significant difference.
[2] The protein is selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). 1] The method described in.
[3] The method according to [1] or [2], wherein the subject is a human patient.
[4] When all of the tested proteins show a significant difference in the body fluid sample of the subject as compared with the normal body fluid, the subject is evaluated as having a perinatal disorder, [1] to The method according to any one of [3].
[5] The method according to any one of [1] to [4], wherein the expression level is determined by an immunoassay.
[6] The method according to any one of [1] to [4], wherein the expression level is determined by liquid chromatography / mass spectrometry.
[7] The method according to any one of [1] to [4], wherein the expression level is determined using a protein array.
[8] Α-2-Macroglobulin (A2m), OX in the body fluid obtained from the subject, for early determination of neonatal perinatal disorders caused by fetal stunting in the mammalian subject. -2 Selected from the group consisting of membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), neuroserpin (Serpini1), ubiquitin thioesterase OTUB1 (Otub1), and ubiquitin-like modification activating enzyme 1 (Uba1) 1 Use of a proteomics profile of one or more protein expressions.
[9] The protein is selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). 8] Use described in.
[10] A method for early determination of the therapeutic effect of neonatal perinatal disorders caused by fetal growth restriction in a mammalian subject.
(A) α-2-Macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neurocerpin (Serpini1) ) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and one or more proteins selected from the group consisting of ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Expression levels were tested in body fluid samples obtained from the subject being treated in comparison to expression levels in normal body fluids, post-treatment body fluids, or body fluids known to exhibit perinatal disorders; (B) In the body fluid known to show no statistically significant difference in expression level compared to the expression level in the normal body fluid or body fluid after treatment, or to exhibit perinatal disorders. The above method comprising determining that treatment of perinatal disorders in the subject is effective when it shows a statistically significant difference compared to the expression level.
[11] The protein is selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). 10].

According to the present invention, by using the biomarker of the present invention, the onset of perinatal brain disorder can be predicted in the neonatal period, and the onset of neurodevelopmental disorder in infants or children can be predicted at an early stage. Therefore, it is possible to perform optimal diagnostic guidelines, treatment evaluation, and elucidation of pathological conditions in newborn babies.

α-2-Macroglobulin (A2m) was identified by proteomics analysis as a protein with significant variation. Neuroserpin (Serpini1) has been identified by proteomics analysis as a protein with significant variation. Polyubiquitin-B (Ubb) was identified by proteomics analysis as a protein with significant variation. The OX-2 membrane glycoprotein (Cd200) was identified by proteomics analysis as a protein showing significant variation. Ubiquitin thioesterase OTUB1 (Otub1) has been identified by proteomics analysis as a protein with significant variation. Ubiquitin-like modification activating enzyme 1 (Uba1 or Ube1) was identified by proteomics analysis as a protein showing significant variation. The expression of A2m over time in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) is shown. A2m expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”)) The result of the measurement is shown. A2m expression in serum in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”) over time The measurement result is shown. The expression of Serpini1 over time in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) is shown. Serpini1 expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”)) The result of the measurement is shown. Serum expression in serum in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”) over time The measurement result is shown. The expression of Ubb over time in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) is shown. Ubb expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”)) The result of the measurement is shown. Serum Ubb expression in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”) over time The measurement result is shown. The expression of Cd200 over time in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) is shown. Over time Cd200 expression in cerebrospinal fluid in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”)) The result of the measurement is shown. Serum Cd200 expression in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”) over time The measurement result is shown. The expression of Otub1 over time in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) is shown. Time-lapse of Otub1 expression in cerebrospinal fluid in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”)) The result of the measurement is shown. The expression of Uba1 over time in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) is shown. Uba1 expression in cerebrospinal fluid over time in various fetal growth restriction model groups (sham surgery group (“Sham”), vehicle group (Vehicle)), MSC group (“MSC”), and Muse group (“Muse”)) The result of the measurement is shown. Indicates the location of the hippocampus in the brain. The results of observing the expression of various biomarkers by tissue staining in each cell constituting the hippocampus in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) are shown. The results of observing the expression of various biomarkers by tissue staining in each cell constituting the hippocampus in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) are shown. The expression intensities of various biomarkers corresponding to FIG. 13B are shown. The expression intensities of various biomarkers corresponding to FIG. 13C are shown. Indicates the location of the striatum of the brain. The results of observing the expression of various biomarkers by tissue staining in each cell constituting the striatum in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) are shown. The results of observing the expression of various biomarkers by tissue staining in each cell constituting the striatum in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) are shown. The expression intensities of various biomarkers corresponding to FIG. 14B are shown. The expression intensities of various biomarkers corresponding to FIG. 14C are shown. Indicates the location of the cerebral cortex of the brain. The results of observing the expression of various biomarkers by tissue staining in each cell constituting the cerebral cortex in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) are shown. The results of observing the expression of various biomarkers by tissue staining in each cell constituting the cerebral cortex in the fetal growth restriction model (“FGR”) and the sham surgery group (“Sham”) are shown. The expression intensities of various biomarkers corresponding to FIG. 15B are shown. The expression intensities of various biomarkers corresponding to FIG. 15C are shown.

Hereinafter, preferred embodiments will be described in detail for the purpose of explaining the present invention. It will be understood by those skilled in the art that the present invention is not limited to the following preferred embodiments, and various modifications may be made within the scope of the gist thereof.

The meanings of the technical and scientific terms described in this specification and the appended claims shall be in accordance with the meanings understood by those skilled in the art. The terms constituting the present invention will be briefly described below.

The present invention provides a biomarker for early determination, diagnosis or assistance in diagnosis of perinatal brain disorder caused by fetal stunting, and early determination or early diagnosis of perinatal disorder in a newborn using the biomarker. The present invention relates to a method and a method for determining the therapeutic effect of perinatal disorders at an early stage using the biomarker.

1. 1. Target diseases (1) Fetal growth restriction Fetal growth restriction (FGR) is greatly involved in the life and neurological prognosis of the offspring. Fetal growth restriction is defined as a condition in which fetal growth is impaired in utero for some reason and the number of weeks of growth is impaired. At present, with the development of ultrasonic inspection equipment, it is common to make a diagnosis using ultrasonic inspection.

In the past, in the 1970s, the evolution of ultrasonic diagnostic equipment and statistical processing using multiple measured fetal parameters became possible, and the formula for calculating the estimated fetal weight (EFW) was reported. In 2003, in order to standardize the formulas for calculating estimated weight that had been reported multiple times, the Japanese Society of Ultrasound created "Standardization of Ultrasonic Fetal Measurement and Japanese Standards", and in 2005, the Japan Society of Obstetrics and Gynecology. But it was officially adopted. The summarized content is described in detail in the "Estimated Fetal Weight and Fetal Growth Curve" Health Guidance Manual published in 2012. Until now, only the fetal growth curve based on the birth weight could be created, but it was extremely epoch-making that the fetal growth curve was created from the measured values in the ultrasonic examination. In particular, it is common in Japan to make a clinical diagnosis of fetal growth restriction when the fetal growth curve created based on "standardization of ultrasonic fetal measurement and Japanese standard values" falls below -1.5SD. be.

Fetal growth restriction is a heterogeneous disease, but fetal growth restriction that develops from the second trimester of pregnancy is often caused by congenital infections, genetic abnormalities, and congenital malformations, and intervention improves the prognosis of fetal growth restriction. It has been considered unlikely. On the other hand, the fetal growth restriction that develops from the second trimester of pregnancy includes a group caused by placental dysfunction including the involvement of preeclampsia (HDP). Fetal growth restriction due to placental dysfunction has a worse prognosis when it develops in the second trimester of pregnancy than in the third trimester of pregnancy. It came to be divided. These are often separated by 32 to 34 weeks, although the definition varies from report to report. Regarding the prognosis of early-onset fetal growth restriction, a large-scale prospective observational study (TRUFFLE study) was reported in 2013. Between 2005 and 2010, 503 cases of early-onset fetal growth restriction who delivered between 26 and 32 weeks were registered at a facility centered on Europe. Fetal growth restriction was defined as a case in which the estimated fetal abdominal circumference was less than the 10th percentile and the umbilical artery blood flow pulsatile index was greater than or equal to the 95th percentile. Of 503 cases, 27 (5.5%) died perinatal, 118 (24%) had severe neonatal complications (bronchopulmonary dysplasia, intraventricular hemorrhage of degree III or higher, periventricular leukomalacia, Neonatal septicemia and necrotizing enterocolitis) were observed, and the prognosis of early-onset fetal growth restriction was poor. In addition, the period from registration to delivery, perinatal mortality, and severe neonatal complications were correlated with the onset and severity of maternal HDP. In addition, the TRUFFLE study is following up on survival and neurological prognosis at 2 years of age. Among the surviving infants, 41 (10%) of the 402 patients who were evaluated neurologically had neurological abnormalities. In 2017, the prognosis of early-onset fetal growth restriction diagnosed in less than 28 weeks and delivered between 22 and 31 weeks was reported by the French Population-based cohort study (EPIPAGE 2 Study). Of the 3,698 newborns, 436 (11.8%) were diagnosed with fetal growth restriction 28 weeks ago. The survival rate by week was 66% at 25 weeks and 90% or more at 26-27 weeks. He states that the timing of fetal growth restriction is important in predicting neonatal prognosis. Late-onset fetal growth restriction has a better prognosis than early-onset fetal growth restriction, but stillbirth rates are higher and problems remain as compared to normally-developed fetal growth. In 2015, it was reported as "Shining light in dark quotes" focusing on the management method for late-onset fetal growth restriction (http://www.chugaiigaku.jp/upfile/browse/browse2521.pdf). Quote).

Fetal cell development is the period when the cell number itself increases rapidly in the first trimester of pregnancy (from the first trimester to the 16th week of pregnancy), and the cell itself as the number of cells increases in the second trimester of pregnancy (17th to 32nd week of pregnancy). The period of hypertrophy, the third trimester of pregnancy (after 33 weeks of gestation), is the period in which the number of cells hardly increases and the cells become hypertrophied. Fetal growth restriction is broadly classified into three types according to fetal cell development. The pourquoi story is given in the next section, and the relationship between clinical classification and causes is shown in Table 1 below. However, there are many exceptions, and at present, it is rarely classified into type 1 and type 2.

(I) Type 1 (symmetrical type)
When the foetation is damaged in the early stages of pregnancy due to abnormalities of the foetation itself such as chromosomal abnormalities or TORCH syndrome, cell division and cell proliferation of the fetal organs are inhibited, so the size of the cells constituting the organs is normal. It exhibits hypoplasia with a small number of cells and is classified as type 1. The hypoplastic type is characterized by well-proportioned growth in which the head and trunk are similarly suppressed because of the small number of foets, and the well-proportioned dysgenesis is called the symmetric type. Type 1 accounts for about 20% of all fetal growth restriction.

(Ii) Composite type (intermediate type)
Fetal growth restriction, which is impaired during the period when the cells themselves enlarge as the number of cells increases, is considered to be a complex type and accounts for about 10% of the total fetal growth restriction. Causes include preeclampsia, chronic nephritis, hypertension, and placental umbilical cord factors that develop early in pregnancy.

(Iii) Type 2 (Asymmetric type)
When a disorder occurs in the third trimester of pregnancy, cell division is already completed and cell hypertrophy is suppressed. When the number of cells is normal but the cells themselves are small, a malnutrition state called type 2 occurs. It is often caused by pathological abnormalities of the placenta caused by maternal diseases such as preeclampsia and diabetes that develop in late pregnancy. When fetal placenta circulation deteriorates, a vasodilatory effect (blood flow redistribution) occurs to protect important organs such as the brain, heart, and adrenal gland, and maintenance of cerebral blood flow is prioritized, so head growth is maintained. However, blood flow to internal organs such as the trunk, liver, and intestinal tract is reduced, and the abdomen has a small subcutaneous fat and a thin body with little subcutaneous fat. Type 2 (asymmetric type) accounts for about 70% of all fetal growth restriction. However, it should be noted that when the brain sparring effect collapses and the growth of the head is impaired, it shifts to the symmetrical type (http://www.chugaiigaku.jp/upfile/browse/browse2521.pdf). Quoted from).

(2) Perinatal encephalopathy "Perinatal encephalopathy" refers to cerebral disorders that occur during the perinatal period (from 22 weeks gestation to less than 7 days after birth in humans), for example, low during labor. It means a brain disorder associated with oxygen-ischemic encephalopathy or systemic inflammatory reaction syndrome secondary to viral or bacterial infection. The present invention also covers the time when neurodevelopmental disorders caused by fetal growth restriction are embodied as symptoms (for example, 2 to 3 years old in humans). More specifically, according to the present invention, perinatal brain disorders occur in human neonates (within 28 days of age), infants (less than 1 year of age), and infants (1 to 6 years of age). Target for improvement and treatment of brain disorders.

"Cerebral palsy" refers to irreversible cerebral palsy that occurs during the developmental period of the brain (in humans, it refers to the age of 13 days to 48 days after birth), and has non-progressive lesions, the symptoms of which are It is based on motor dysfunction, and most develop by the age of three. Specifically, it generally refers to brain damage that occurs by the neonatal period. The causes are divided according to the time of occurrence of the disorder. (a) Prenatal causes include intrauterine infection, placental insufficiency, fetal cerebrovascular accident, hereditary, etc. (b) Birth causes include , Mechanical damage during labor, cerebral hemorrhage, anoxia, hypoxia, cerebral circulatory disorder, etc. (c) Postnatal causes include severe kernicterus (kernicterus), intracranial infection, cerebral hemorrhage, etc. Classification is based on the content of the paralysis. The contents of muscle tone include strong (convulsive) straightness, toughness, ataxia, athetosis (involuntary movement that appears when maintaining a certain posture or trying to exercise), non-tension, etc. , Extremity paralysis, hemiplegia, diplegia, paraplegia, double hemiplegia, monoplegia, etc. Complications include intellectual disability (including learning disabilities), seizures, neurological disorders, and speech disorders. Brain lesions caused by hypoxic-ischemic encephalopathy in mature infants are often cerebral cortical layer necrosis, basal ganglia necrosis, cerebral infarction, leukomalacia, bridge-shaped circumflex necrosis, and brain stem necrosis. Basal ganglia necrosis causes atetose-type cerebral palsy, and cerebral infarction causes spastic limb palsy and hemiplegia. Brainstem necrosis often causes poor prognosis and death in infancy, and even if it survives, it causes dysphagia and respiratory dysregulation.

For example, head ultrasound imaging, MRI, CT, electroencephalogram, and laser Doppler blood flow meters can be used to obtain physiological findings of the brain. Perinatal brain damage can be suspected if abnormal findings are obtained using these devices. In addition, it may be possible to make a diagnosis by directly observing learning disabilities and motor disorders in the subject.

3. 3. Method for early determination, diagnosis or diagnosis of perinatal disorder in newborns by the biomarker of the present invention According to the present invention, perinatal brain disorders in newborns caused by fetal stunting in a mammalian subject can be detected. A method for early determination, diagnosis or assistance (hereinafter, may be simply referred to as "early diagnosis method") is characterized in that one or more of six types of biomarkers described later are used. The biomarkers (proteins) used are the expression level of proteins present in the body fluids of newborns who are thought to have the above-mentioned diseases, the expression levels of proteins present in normal body fluids or body fluids after treatment, or the diseases. It is identified by comparing the expression level of the protein present in the body fluid of the subject known to show the presence or absence of the difference, or by identifying the protein that is upregulated or downregulated. Generally, the total amount of proteins present in a biological sample (eg, body fluid, tissue, organism, or cell culture) at a given time point is referred to as a "proteome." The expression level of the protein can be determined by using an immunoassay, mass spectrometry, or a protein array.

The expression comparison as described above may be a study of overall changes in protein expression in a sample (also referred to as "proteomics" or "expression proteomics"). Proteomics generally refers to (1) separation of individual proteins in a sample by two-dimensional gel electrophoresis; (2) identification of individual proteins recovered from the gel, such as liquid chromatography, mass spectrometry or N-terminal sequencing. Includes steps of singing and (3) analyzing data using bioinformatics. Proteomics methods can be a useful complement to other gene expression profiling methods.

The present invention mainly performs proteomic analysis of body fluids, but the "body fluids" used are not limited to cerebrospinal fluid (CSF), cord blood, cervical-vaginal fluid (CVF), amniotic fluid, and the like. Examples include blood, serum, plasma, urine, breast milk, mucus, saliva and sweat. In the present invention, cerebrospinal fluid is preferable as the body fluid for diagnosing perinatal brain disorder associated with fetal growth restriction, which is a risk of neurodevelopmental disorder. This is because the information on biomolecules circulating in the brain due to the inflow and outflow of cerebrospinal fluid sensitively reflects the state of brain tissue and its changes. In addition, serum, which is the outflow destination of cerebrospinal fluid, is useful because it is non-invasive and can be easily collected.

In comparative analysis, it is of course important to treat, for example, normal and biological samples accurately and in the same way, in order to accurately represent the relative expression level or amount of protein and obtain accurate results. be. The total protein requirement depends on the analytical technique used and is easily measured by one of ordinary skill in the art. Proteins present in biological samples are generally separated by two-dimensional gel electrophoresis according to pI and molecular weight. Proteins are first separated by their charge using isoelectric focusing (one-dimensional gel electrophoresis). This step may be performed using, for example, a commercially available fixed pH gradient (IPG) stripe. The second dimension can be a conventional SDS-PAGE analysis, where concentrated IPG stripes are used as the sample. After separation, proteins can be visualized by conventional dyes such as Coomassie blue or silver stain and can be imaged using known techniques and equipment such as Bio-Rad GS800 densitometer and PDQUEST software. can. The individual spots are then excised from the gel, decolorized and trypsined. The peptide mixture may be analyzed by mass spectrometry (MS). Alternatively, the peptides may be separated, for example by capillary high performance liquid chromatography (HPLC), or analyzed separately or in combination by MS.

The mass spectrometer consists of an ion source, a mass analyzer, an ion detector and a data acquisition unit. The fragmented peptide is ionized at the ion source. The ionized peptides are then separated by mass / charge ratio in a mass spectrometer to detect different ions. In particular, mass spectrometry has been widely used in protein analysis since the development of matrix-assisted laser desorption / ionization / time-of-flight (MALDI-TOF) and electrospray ionization (ESI) methods. For example, MALDI-TOF and quadrupole-TOF, or an ion trap mass spectrometer connected to ESI are exemplified.

Protein arrays fix proteins on solid surfaces such as glass, silicon, microwells, nitrocellulose, PVDF membranes and microbeads using a variety of covalent and non-covalent attachment chemistries well known in the art. It is formed by doing. The solid support is chemically stable before and after the coupling procedure, allows for good spot morphology, represents very small non-specific bonds, does not affect the background of the detection system, and does not affect the background of the detection system. Must be compatible with different detection systems.

The diagnostic method of the present invention may be carried out in the form of various immunoassay formats, which are well known in the art. There are two main types of immunoassays, homologous and heterogeneous. In an allogeneic immunoassay, the immune response and detection between an antigen and an antibody is carried out in an allogeneic reaction. The heterologous immunoassay comprises at least one separation step of separating the reaction product from the unreacted reagent.

The six proteins used in the present invention are proteins (or peptides) found in all 601 proteins based on the comprehensive analysis by the above proteomics. Specifically, α-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neurocerpin. (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Information on the amino acid sequences of these proteins is available using publicly available databases (eg, UniProt; https://www.uniprot.org/).

In the early diagnosis of perinatal brain damage in newborns of the present invention, at least one, preferably at least two, three, four or five, or all of the above proteins is used. As shown in Examples described later, four of the above six proteins (that is, α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (that is, polyubiquitin-B) Ubb) and neuroserpin (Serpini1)) are proteins that are present in cerebrospinal fluid and have been significantly and chronically fluctuated due to fetal stunting. In addition, these proteins have been shown to improve their expression fluctuations from an early stage by administration of pluripotent stem cells (Muse cells). Of these four proteins, at least three (ie, α-2-macroglobulin (A2m), polyubiquitin-B (Ubb), and neuroserpin (Serpini1)) are also found in neonatal serum. It was confirmed that there was a significant change in expression associated with fetal growth restriction and improvement of the change was observed by administration of pluripotent stem cells (Muse cells).

Fluctuations in the expression levels of the above 6 or 4 proteins in body fluid samples are known to indicate normal body fluids, post-treatment body fluids (due to drugs, pluripotent stem cells, etc.), or perinatal disorders. It is done by comparing with the expression level in the body fluid. When the body fluid used as the comparison standard is a normal body fluid or a body fluid after treatment, the expression level of the fluctuation is 1.1 times, 1.2 times, 1.3 times, 1.4 times based on these. , 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 The increase or decrease may be fold, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, or more. When the body fluid used as the comparison standard is a body fluid known to exhibit perinatal disorders, the difference from the body fluid is 100%, 90%, 80%, 70%, 60 of the expression level of the reference body fluid. %, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0 .8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less, or preferably 0%. ..

According to another aspect of the present invention, there is provided a method for early determination of the therapeutic effect of a neonatal perinatal disorder caused by fetal growth restriction in a mammalian subject. The method for early determination of the therapeutic effect of perinatal disorders is as described above for α-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-. B (Ubb) (SEQ ID NO: 3), neurocerpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Of the proteins of, at least one, preferably at least two, three, four or five, or all are used and the expression level of the protein is normal in body fluid samples obtained from the subject being treated. Tested in comparison to expression levels in normal body fluids, post-treatment body fluids, or body fluids known to exhibit perinatal disorders; and said expression levels as well as expression levels in said normal body fluids or post-treatment body fluids. The subject when it does not show a statistically significant difference in comparison or shows a statistically significant difference in comparison with the expression level in the body fluid known to show perinatal disorders. It can be determined that the treatment of perinatal disorders in Ubiquitin is effective.

Fluctuations in the expression level of the above proteins in body fluid samples include expression levels in normal body fluids, post-treatment body fluids (due to drugs, pluripotent stem cells, etc.), or body fluids known to exhibit perinatal disorders. It is done by comparison. When the reference body fluid is a normal body fluid or a body fluid after treatment, the difference from the standard body fluid is 100%, 90%, 80%, 70%, 60%, 50%, 40% of the expression level of the reference body fluid. , 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7 %, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less, or preferably 0%. In addition, when the body fluid used as a comparison standard is a body fluid known to exhibit perinatal disorders, the expression level of the fluctuation is 1.1 times, 1.2 times, and 1. 3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, The increase or decrease may be 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, or more.

The present invention provides a method for early diagnosis of brain disorders (eg, neurodevelopmental disorders) in post-growth infants and children by capturing mild perinatal brain disorders that are difficult to detect with current diagnostic methods. Can be provided. According to the present invention, it is possible to diagnose a brain disorder that occurs after growth, which has been overlooked so far, in the neonatal period, and to capture a perinatal brain disorder that has not been diagnosed so far.

As described in Examples described later, it is obtained by comprehensively analyzing proteins in cerebrospinal fluid from a rat model of fetal growth restriction, and is used as a marker for perinatal brain damage due to fetal growth restriction or a therapeutic marker treatment. The characteristics of the six associated proteins are outlined below.

(A) A2m (α-2-macroglobulin)
Features: A type of acute response protein. Increased in response to cytotoxicity. In the case of the brain, it is elevated by the breakdown of the blood-brain barrier (BBB) and damage to nerve cells.
Results: Significant increases were confirmed in serum and cerebrospinal fluid both 5 days and 14 days after birth. In addition, suppression of expression by Muse cells was confirmed on both 5 days and 14 days after birth. In the results of cerebrospinal fluid, the inhibitory effect of Muse cells could not be confirmed at the stage of 5 days after birth, but what was confirmed in serum was A2 m due to fetal growth restriction even in organs other than the brain (lungs, etc.). It is considered that the upregulation of serum was induced and the Muse cells acted on the organ earlier than the brain.

(B) Serpini1 (neuroserpin)
Features: A type of serine protease inhibitor, neuroserpin is expressed in large amounts in the brain. In addition to suppressing proteolytic degradation by inhibiting proteolytic enzymes, it also controls inflammation. It is said that nerve cells are important for normal functioning and control memory and emotions. It has also been reported to have a neuroprotective effect. Dysfunction of this protein induces dementia.
Results: Significant increases were confirmed in serum and cerebrospinal fluid both 5 days and 14 days after birth. As with cerebrospinal fluid, the degree of increase in serum samples is greater at 14 after birth. Furthermore, compared with cerebrospinal fluid, it was hardly confirmed in the control, so the rate of increase was large. In addition, suppression of expression by Muse cells and mesenchymal stem cells (MSC) was confirmed on both 5 and 14 days after birth. The inhibitory effect is more remarkable in the Muse cell group, and it is considered that the inhibitory effect is sustained for a long period of time.

(C) Cd200 (OX-2 Membrane Glycoprotein)
Features: A protein that mainly suppresses cells of the immune system and regulates inflammation. It is highly expressed in nerve cells in the brain. Anti-inflammatory cytokines are secreted by Cd200 / Cd200R signal transduction, and inflammation is suppressed.
Results: A significant increase was confirmed 5 days after birth, but the inhibitory effect of stem cells remained on the trend.

(D) Ubb (polyubiquitin-B)
Characteristic: A protein that functions as a label given when removing unnecessary proteins or abnormal proteins. Increased expression of this protein means an increase in unwanted proteins. In addition, abnormal expression fluctuations cause neurodegenerative diseases.
Results: A significant increase was confirmed on both 5 and 14 days after birth. In addition, suppression of expression by Muse cells was confirmed on both 5 days and 14 days after birth. The effect is faster in Muse cells than in MSCs.

(E) Uba1 (ubiquitin-like modification activating enzyme 1)
Features: A type of ubiquitin activating enzyme, which is a protein for activating ubiquitin for use in the next proteolysis of ubiquitin used for proteolysis. Like Ubb, it is an important molecule for protein quality control. Uba1 may be referred to as "Ube1".

(F) Otub1 (ubiquitin thioesterase OTUB1)
Features: Deubiquitinating enzyme. It is a protein for desorbing ubiquitin from ubiquitinated proteins and chromatin. Like Ubb, it is an important molecule for protein quality control.

Other embodiments of the present invention include α-2-macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3). , Neurocerpin (Serpini1) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Provided for use in the production of a proteomics (or protein) profile of body fluid for early diagnosis of neonatal perinatal disorders due to fetal stunting in a mammalian subject of one or more of the proteins of.

In certain embodiments, the proteomics profile contains information on at least two, at least three, at least four, at least five, or all expression levels of the above proteins in any combination.

In one embodiment, the proteomics profile contains information on the level of expression of the protein, and one or more of the proteins tested statistically compared to the level of expression in normal or post-treatment body fluids. The subject is diagnosed with perinatal disorder if it shows a significant difference or does not show a statistically significant difference compared to the expression level in the body fluid known to show perinatal disorder. do.

In the present invention, the "treatment" when the body fluid as a comparative reference is the body fluid after treatment is not limited, but is a treatment method for perinatal brain disorder (for example, hypothermia) as recognized by those skilled in the art. Therapies) or stem cell therapies (eg, umbilical cord blood stem cells, Muse cells).

"Muse (Multigeneage-Differentating Stress Enduring) cells" are bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)) It can be obtained from skin tissues such as dermal connective tissue and is scattered in the connective tissues of each organ. In addition, these cells are cells having the properties of both pluripotent stem cells and mesenchymal stem cells. For example, the respective cell surface markers "SSEA-3 (Stage-specific embryonic antigen-3)" and " Identified as double positive for "CD105". Therefore, a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using these antigen markers as an index, for example. In addition, Muse cells are stress resistant and can be concentrated from mesenchymal tissues or cultured mesenchymal cells by various stress stimuli. For the cell preparation of the present invention, a cell fraction in which Muse cells are concentrated by stress stimulation can also be used. Details such as a method for separating Muse cells, a method for identifying them, and their characteristics are disclosed in International Publication No. WO2011 / 007900. In addition, as reported by Wakao et al. (2011, supra), when mesenchymal cells are cultured from bone marrow, skin, etc. and used as a population of Muse cells, all SSEA-3 positive cells are CD105. It is known to be a positive cell. Therefore, when separating Muse cells from living mesenchymal tissues or cultured mesenchymal stem cells, Muse cells can be simply purified and used using SSEA-3 as an antigen marker. In the present specification, using SSEA-3 as an antigen marker, a cell population containing pluripotent stem cells (Muse cells) or Muse cells isolated from living mesenchymal tissues or cultured mesenchymal tissues is simply referred to as "Muse cells". It may be described as "SSEA-3 positive cells". Further, in the present specification, the “non-Muse cell” refers to a stem cell contained in a living mesenchymal tissue or a cultured mesenchymal tissue, and is a stem cell other than the “SSEA-3 positive cell”. A cell population obtained by removing SSEA-3 and CD105-positive cells from MSC can be used as non-Muse cells according to the method described in WO2011 / 007900 on Isolation and Identification of Human Muse Cells.

Briefly, a Muse cell or a stem cell population containing Muse cells uses a living tissue (eg, an antibody against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively). , Mesenchymal tissue). Here, the "living body" refers to a living body of a mammal. In the present invention, the living body does not include a fertilized egg or an embryo at a developmental stage before the blastogenic stage, but includes an embryo at a developmental stage after the blastogenic stage including a foetation or a blastoblast. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats and guinea pigs, rabbits, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets and the like. Be done. Muse cells used in the cell preparations and pharmaceutical compositions of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are separated directly from living tissues using markers. The "mesenchymal tissue" refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligaments, tendons, dental pulp, umbilical cord, and cord blood, and tissues existing in various organs. For example, Muse cells can be obtained from bone marrow, skin and adipose tissue. For example, it is preferable to collect mesenchymal tissue of a living body, separate Muse cells from this tissue, and use it. In addition, Muse cells may be separated from cultured mesenchymal cells such as fibroblasts and bone marrow mesenchymal stem cells by using the above-mentioned separation means. In the cell preparation and pharmaceutical composition of the present invention, the Muse cells used may be autologous or allogeneic to the recipient.

As described above, Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, SSEA-3 positive and double positive of SSEA-3 and CD105 as indicators, but human adult skin. Is known to include various types of stem cells and progenitor cells. However, Muse cells are not the same as these cells. Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural ridge stem cells (NCSC), melanoblasts (MB), perivascular cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC). ). Muse cells can be isolated using the "non-expression" of a marker unique to these cells as an index. More specifically, Muse cells include CD34 (markers for EP and ADSC), CD117 (c-kit) (markers for MB), CD146 (markers for PC and ADSC), CD271 (NGFR) (markers for NCSC), NG2 (PC marker), vWF factor (Fonville brand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and At least one of 11 markers selected from the group consisting of Dct (MB marker), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 The non-expression of 11 or 11 markers can be separated as an index. For example, without limitation, the non-expression of CD117 and CD146 can be used as an index, and the non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be used as an index. The non-expression of 11 markers can be separated as an index.

In addition, Muse cells having the above characteristics are as follows:
(I) Low or no telomerase activity;
(Ii) Has the ability to differentiate into cells of any of the three germ layers;
It may have at least one property selected from the group consisting of (iii) no neoplastic growth; and (iv) capable of self-renewal. In one aspect of the present invention, the Muse cells used in the cell preparations and pharmaceutical compositions of the present invention have all of the above properties. Here, with respect to the above (i), "the telomerase activity is low or absent" means that, for example, when the telomerase activity is detected using TRAPEZE XL telomerase detection kit (Millipore), it is low or cannot be detected. say. "Low" telomerase activity means, for example, telomerase having the same level of telomerase activity as somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less of that of Hela cells. It means having activity. Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endoderm lineage, mesodermal lineage, and ectodermal lineage) in vitro and in vivo, and are, for example, induced and cultured in vitro. Can differentiate into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. It may also show the ability to differentiate into three germ layers when transplanted into the testis in vivo. Furthermore, it has the ability to migrate and engraft in organs (heart, skin, spinal cord, liver, muscle, etc.) damaged by transplantation into a living body by intravenous injection, and to differentiate into cells according to tissues. Regarding (iii) above, Muse cells proliferate at a growth rate of about 1.3 days in suspension culture, but grow from one cell in suspension culture to form embryo-like cell clusters, and the growth slows down in about 14 days. However, when these embryo-like cell clusters are brought into the adhesive culture, cell proliferation is started again, and the cells proliferated from the cell cluster spread. Furthermore, when transplanted into the testis, it has the property of not becoming cancerous for at least half a year. In addition, with respect to the above (iv), Muse cells have a self-renewal (self-renewal) ability. Here, "self-renewal" means that the differentiation of cells contained in embryoid body-like cell clusters obtained by culturing one Muse cell in suspension culture into three germ layer cells can be confirmed, and at the same time, at the same time, it can be confirmed. By bringing the cells of the embryo-like cell mass to the suspension culture again with one cell, the next generation embryo-like cell mass is formed, and from there, the embryos in the three germ layer differentiation and suspension culture are again formed. It means that a skeletal cell mass can be confirmed. Self-renewal may be repeated one or more cycles.

In addition, the cell fraction containing Muse cells gives an external stress stimulus to the mesenchymal tissue or cultured mesenchymal cells of the living body, kills cells other than the cells resistant to the external stress, and causes the surviving cells. It may be a cell fraction enriched with SSEA-3 positive and CD105 positive pluripotent stem cells having at least one, preferably all of the following properties, obtained by a method involving recovery.
(I) SSEA-3 positive;
(Ii) CD105 positive;
(Iii) Low or no telomerase activity;
(Iv) Has the ability to differentiate into three germ layers;
(V) does not show neoplastic growth; and (vi) has self-renewal ability.

The above external stresses are protease treatment, culture at low oxygen concentration, culture under low phosphoric acid condition, culture at low serum concentration, culture under low nutritional condition, culture under heat shock exposure, low temperature. Culturing in, freezing treatment, culturing in the presence of harmful substances, culturing in the presence of active oxygen, culturing under mechanical stimulation, culturing under shaking treatment, culturing under pressure treatment or physical impact It may be any or a combination of two or more. For example, the treatment time with the protease is preferably 0.5 to 36 hours in total in order to give external stress to the cells. The protease concentration may be any concentration used when peeling the cells adhered to the culture vessel, breaking up the cell mass into a single cell, or recovering a single cell from the tissue. The protease is preferably serine protease, aspartic protease, cysteine protease, metal protease, glutamate protease or N-terminal threonine protease. Further, it is preferable that the protease is trypsin, collagenase or dispase.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1. Preparation of fetal growth restriction model animals and animal management 60 SD rats (11 weeks old) on the 4th day of gestation were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). Fetal growth restriction model group (vehicle group, n = 15) to which each rat is administered with a sham operation group (sham group, n = 15), equilibrium salt solution administration, and fetal growth restriction model group (MSC) to which mesenchymal stem cells are administered. The group was randomly divided into a group, n = 15), and a fetal growth restriction model group (Muse group, n = 15) to which Muse cells were administered. One pregnant rat in each group was bred at room temperature 22-24 ° C., humidity 50-60%, 12-hour light-dark cycle, and free access to food and water. On the 17th day of gestation of pregnant rats in the vehicle, MSC, and Muse groups, intrauterine hypoperfusion treatment was performed by attaching Ameloid Constrictors (Research Instruments SW, CA, US) to the uterine and ovarian arteries. The uterus exposed when the Ameroid Constrictor was worn was wrapped in gauze soaked with saline warmed to 37 ° C to prevent dryness and decrease in body temperature. As for the pregnant rats in the sham group, only the laparotomy and uterine exposure treatment were performed on the 17th day of pregnancy as in the vehicle group, and the abdomen was closed without wearing the Ameloid Constrictor.

Birth of pups was confirmed from each gestation rat on the 21st day of gestation, and decimation was performed randomly from among female pups so that each mother rat would have 10 pups. Evaluate body weight transition from day 3 to day 14 of all male pups, and remove cerebrospinal fluid from pups on days 4, 5, 7, 10 and 14 of birth and pups on days 5 and 14 of birth. Blood was collected from rats. On the 4th day of birth, a balanced salt solution was administered to the sham and vehicle groups from the jugular vein, mesenchymal stem cells were administered to the MSC group, and 1.0 × 10 ⁴ Muse cells were administered to the Muse group. All animal experiments were carried out in accordance with "Animal Research: Reporting In vivo Experiments guidelines for the care and use of laboratory analogs", and were approved by the Nagoya University Animal Experiment Ethics Committee (approval numbers 30082 and 1107). All surgery and sampling were performed in the anesthesiology department with isoflurane, and every effort was made to minimize pain and suffering.

Example 2. Collection of cerebrospinal fluid A capillary (DRM Microcap, 1-000-0500) was purchased for collection of cerebrospinal fluid and sharpened by crushing the tip. The posterior fossa of the rat pups asleep with isoflurane was exposed, a sharpened capillary was punctured into the cisterna magna, and cerebrospinal fluid was collected by capillary action. The collected cerebrospinal fluid was placed in a tube to which a proteolytic enzyme inhibitor had been added in advance, immediately frozen in liquid nitrogen, and stored frozen at -80 ° C until use.

Example 3. Serum collection 500 μL of blood was collected from the heart of a rat pups asleep with isoflurane and placed in a microvascular (BD Microtainer SST) containing a coagulation promoter and a serum separator. After 5 inversion mixing, blood cell components were precipitated by centrifugation at 10000 × g, and serum was collected. A proteolytic enzyme inhibitor was added to the collected serum, and the serum was stored frozen at −80 ° C. until use.

Example 4. Comprehensive analysis of proteins in cerebrospinal fluid by proteomics The protein concentration in the collected cerebrospinal fluid was measured by the BCA method and unified to a concentration of 50 μg / 100 μL. The abundance of total protein contained in cerebrospinal fluid was measured by a liquid chromatograph / mass spectrometer (LC / MS), and a protein profile was obtained. The Spearman correlation coefficient between the abundance of all 601 proteins detected in the cerebrospinal fluid and the body weight on the third day of birth was calculated, and the p-value and false discovery rate (FDR) were calculated based on the correlation coefficient. ) Was calculated. It has already been confirmed that the body weight on the third day of birth correlates with the body weight at birth. As for the proteins showing p <0.05 and FDR <0.10, 140 kinds on the 4th day of birth and 123 kinds on the 5th day, a total of 212 kinds (51 kinds of duplication) were confirmed, and these 212 kinds of proteins were confirmed. It was found as a protein that fluctuates with fetal growth restriction. A group of proteins satisfying the conditions of p <0.05, Fold Engineering> 2, and Symbols number> 3 was extracted by functional analysis of the 212 proteins using Gene Ontology. Among the 140 proteins on the 4th day of birth, 333 proteins related to biological processes, 42 proteins related to cell components, 50 proteins related to molecular function, and 123 proteins on the 5th day of birth , 191 proteins related to biological processes, 27 proteins related to cell components, and 35 proteins related to molecular function were extracted. Among these terms, the ones extracted at the top as showing particularly remarkable fluctuations are proteins related to the differentiation of neural stem cells and glial cells, neurogenesis and control of cell death on the 4th day of birth, and the 5th day. In, it was a group of proteins related to synaptogenesis, neural circuit development, and behavioral function. Furthermore, protein groups responsible for inflammatory / immune response, cell adhesion, and protein structure management were commonly extracted on the 4th and 5th days of birth. As a result of extracting proteins that increased or decreased in common at both the time points on the 4th and 5th days of birth from the protein group that fluctuated in common on the 4th and 5th days of birth, 14 kinds of proteins Was found. Furthermore, among these 14 types, those observed to be expressed in the brain were investigated using The Human Protein Atlas, and as a result, 6 types of proteins, α-2-macroglobulin (A2m) (Fig. 1), and neurocerpin (Fig. 1) were used. Serpini1) (Fig. 2), Polyubiquitin-B (Ubb) (Fig. 3), OX-2 membrane glycoprotein (Cd200) (Fig. 4), Ubiquitin thioesterase OTUB1 (Otub1) (Fig. 5), Ubiquitin-like modification activation Enzyme 1 (Uba1) (Fig. 6) was found.

Example 5. Quantification of proteins by polyacrylamide gel electrophoresis and Western blotting Cerebral spinal fluid and serum proteins sample denaturing buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 4% w / v sodium dodecyl sulfate [SDS] ], 0.001% w / v bromophenol blue and 10% mercaptoethanol), and the protein was denatured by heating at 95 ° C. for 5 minutes. For each sample, 5 μg of protein per lane was added to a 10% SDS-polyacrylamide gel and first electrophoresed at 80 V, 10 ° C. for 120 minutes. The proteins separated by electrophoresis were transferred onto a polyvinylidene difluoride membrane (Merck Millipore, MA, USA) at room temperature and 400 mA for 1 hour. After blocking the protein on the membrane with 5% skim milk dissolved in Tris buffered physiological saline (pH 7.4) (TBS-T) containing 0.1% Tween-20, the primary antibody, rabbit anti-albumin antibody (Proteintech:). 16475-1-AP, 1: 5000), rabbit anti-A2m antibody (Abcam: ab58703, 1: 500), rabbit anti-Cd200 antibody (Proteintech: 14057-1-AP, 1: 200), rabbit anti-Ube1 (Uba1) antibody. (Proteinthch: 5912-1-AP, 1: 400), rabbit anti-Otub1 antibody (Abcam: ab101471, 1: 500), rabbit anti-neuroselpin antibody (Abcam: ab33077, 1: 200), or rabbit anti-Ubb antibody (Stressmarq). Bioscience: SPC-119, 1: 500) was reacted overnight at 4 ° C. Then, the secondary antibody, Horseradish peroxidase goat anti-rabbit IgG antibody (Rockland: 611-603-122, albumin 1: 10000, others 1: 1000) was reacted at room temperature for 1 hour on the membrane on which the primary antibody was reacted. I let you. During each step, the membrane was washed with TBS-T. Secondary antibody binding was visualized and detected by chemiluminescence using the Immobilon Westem Chemiluminescence HRP substrate (Merck Millipore). To quantify the detected signal, images were scanned using ImageQuant LAS 4000 (GE healthcare, IL, US) and analyzed using ImageJ software. In polyacrylamide gel electrophoresis, the rat albumin molecule is 66 kDa, the A2m molecule is 163 kDa, the neuroselpin molecule is 46 kDa, the Ubb molecule is 34 kDa, the CD200 molecule is 41 kDa, the OTUB1 molecule is 31 kDa, and the UBE1 molecule is. The appearance of the band was confirmed at 117 kDa. The density of each detected band was quantified by background subtraction, and the quantified values of A2m, neuroserpin, Ubb, Cd200, OTUB1, and UBE1 were corrected by the quantified values of albumin of each sample (FIGS. 7A to 12A).

The results of measuring the abundance of each protein expressed in various fetal growth restriction model groups (sham surgery group (sham group), vehicle group, MSC group, and Muse group) are shown in FIGS. 7B and C, 8B and C, FIG. 9B and C, FIGS. 10B and C, FIG. 11B, and FIG. 12B). 7B, 8B, 9B, 10B, 11B, and 12B show the abundance of each protein in cerebrospinal fluid ( births 5, 7, 10, and 14 days), while FIGS. 7C, 8C, 9C, and 10C indicates the abundance of each protein in serum (5th and 14th day of birth).

(A) A2m (α-2-macroglobulin)
Significant increases were confirmed in serum and cerebrospinal fluid both 5 days and 14 days after birth. In addition, suppression of expression by Muse cells was confirmed on both 5 days and 14 days after birth (FIGS. 7B and C). In the results of cerebrospinal fluid, the inhibitory effect of Muse cells could not be confirmed at the stage of 5 days after birth (Fig. 7B), but what was confirmed in serum was the fetus in organs other than the brain (lungs, etc.). It is considered that the growth restriction induces the upregulation of A2m, and the Muse cells act on the organ earlier than the brain.

(B) Serpini1 (neuroserpin)
Significant increases were confirmed in serum and cerebrospinal fluid both 5 days and 14 days after birth. As with cerebrospinal fluid (Fig. 8B), the serum sample (Fig. 8C) increased more significantly 14 days after birth. Furthermore, compared with cerebrospinal fluid, it was hardly confirmed in the control, so the rate of increase was large. In addition, suppression of expression by Muse cells and mesenchymal stem cells (MSC) was confirmed on both 5 and 14 days after birth (Fig. 8C). The inhibitory effect is more remarkable in the Muse cell group, and it is considered that the inhibitory effect is sustained for a long period of time.

(C) Ubb (polyubiquitin-B)
A significant increase was confirmed on both 5 and 14 days after birth. In addition, suppression of expression by Muse cells was confirmed on both 5 days and 14 days after birth. The effect is faster in Muse cells than in MSCs (FIGS. 9C and D).

(D) Cd200 (OX-2 Membrane Glycoprotein)
A significant increase was confirmed 5 days after birth, but the inhibitory effect of stem cells remained on the trend (FIGS. 10B and C).

(E) Otub1 (ubiquitin thioesterase OTUB1)
In cerebrospinal fluid, the expression of Muse cells was confirmed to be suppressed by Muse cells and MSCs on both 5 days and 14 days after birth. The effect is faster in Muse cells than in MSCs (Fig. 11B).

(F) Uba1 (ubiquitin-like modification activating enzyme 1)
In cerebrospinal fluid, the expression of Muse cells was confirmed to be suppressed by Muse cells and MSCs on both 5 days and 14 days after birth. The effect is faster in Muse cells than in MSCs (Fig. 12B).

Example 6. Evaluation of expression localization and expression fluctuation of various protein molecules in the brain by immunofluorescence (Method) For evaluation of the localization of various proteins, the brains of 5 day-old pups (n = 5 / group) were used. bottom. Anesthetized rats were transcentricly perfused with phosphate buffered saline (PBS) and then perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer for fixation. Brain samples were post-fixed with 4% PFA dissolved in 0.1 M phosphate buffer for 24 hours and then phosphate buffered sucrose containing 0.1% sodium azide (10% sucrose, 4-6 hours, 20% sucrose, 4-6 hours, 30% sucrose, 12-36 hours) was cryoprotected and rapidly frozen at −80 ° C. After freezing, brain sections were sliced in the coronal direction to a thickness of 40 μm. The prepared section was blocked with 10% normal goat serum for 1 hour at room temperature, and then the primary antibody rabbit anti-A2m antibody (Abcam: ab58703, 1:50) and rabbit anti-CD200 antibody (Proteintech: 14057-1-AP, 1:50). 50), rabbit anti-Ube1 (Uba1) antibody (Proteintech: 15912-1-AP, 1:50), or rabbit anti-neuroseperpine antibody Abcam: ab33077, 1: 100) was reacted at 4 ° C. for 20 hours. After washing 3 times with PBS for 5 minutes, ATTO 488 conjugated secondary antibody goat anti-rabbit IgG (Rockland, 611-152-122, 1: 500) was reacted at 4 ° C. for 12 hours. Then, it was washed with PBS three times for 5 minutes, and the primary antibody mouse anti-NueN antibody (Merck: MAB377, 1: 100) and mouse anti-Olig2 antibody (Proteintech: 66513-1-Ig: 14057-1-AP, 1: 100). , Mouse anti-S100 antibody (Abcam: ab4066, 1: 100) or mouse anti-Iba1 antibody (Abcam: ab15690, 1: 100) was reacted at 4 ° C. for 20 hours. Each of these antibodies was used as a marker for nerve cells, oligodendrocytes, astrocytes, and microglia. After washing 3 times with PBS for 5 minutes, ATTO 550 conjugated secondary antibody goat anti-mouse IgG (Rockland, 611-152-122, 1: 500) was reacted at 4 ° C. for 12 hours. After washing with PBS 3 times for 5 minutes, the cells were passed through purified water once and encapsulated with nuclear staining using Prolong Gold with DAPI. After drying, the observation was performed using a confocal laser scanning microscope (TiE-A1R, Nikon, Japan). Select 10 sections nasally from the intersection of the sagittal and coronal sutures (Bregma) of the brain, and observe the hippocampus (Fig. 13A), striatum (Fig. 14A), and cerebral cortex (Fig. 15A) of each section. bottom. 100 cells were randomly selected from each brain region, and the expression levels of the four biomarker candidate proteins in NeuN-positive cells, Olig2-positive cells, S100-positive cells, and Iba1-positive cells were relative to the fluorescence intensity. Quantified. Of the 100 cells, the one in which the four candidate proteins showed the highest intensity was set to 100, and the non-expressing cell was set to 0.

(Results) As shown in FIGS. 13B to 13B, 14B to E, and 15B to E, neuroserpin was strongly expressed in NeuN-positive cells (nerve cells) in both the sham surgery group and the FGR group, and other neuroserpins were observed. Almost no expression was observed in cells. In addition, the expression intensity was significantly increased in the FGR group. While A2m did not show strong strength in the sham surgery group as a whole, an increase in highly expressed cells was observed in NeuN-positive cells, S100-positive cells (astrocytes) and Oligo2-positive cells (oligodendrocytes) in the FGR group. .. CD200 showed strong expression in S100-positive cells in both the sham-surgery group and the FGR group, followed by NeuN-positive cells. In these cells, CD200 was more strongly expressed in the FGR group than in the sham surgery group. Ubb was detected in NeuN-positive cells, S100-positive cells and Olig2-positive cells, and its expression intensity was significantly increased in the FGR group. In addition, these results were very similar in all brain regions of the cerebral cortex, hippocampus, and striatum. From these results, it was clarified that the four biomarker candidate proteins were significantly upregulated in the brain tissue. It was also shown that the expression variation of the candidate protein depends not on the brain region but on the type of brain cells.

The biomarker of the present invention can be used for early diagnosis of perinatal disorders in newborns due to fetal growth restriction in mammalian subjects, and is applied to the treatment of neurodevelopmental disorders in infants and children. be able to.

All publications and patent documents cited herein are incorporated herein by reference in their entirety. Although specific embodiments of the present invention have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. It will be easily understood.

Claims (11)

1. A method for early determination of neonatal perinatal disorders due to fetal growth restriction in mammalian subjects.
   (A) α-2-Macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neurocerpin (Serpini1) ) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and one or more proteins selected from the group consisting of ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Expression levels were tested in body fluid samples obtained from subjects in comparison to expression levels in normal body fluids, post-treatment body fluids, or body fluids known to exhibit perinatal disorders; and (b). The expression level is statistically significantly different from the expression level in the normal body fluid or the body fluid after treatment, or is compared with the expression level in the body fluid known to exhibit perinatal disorders. The above method, which comprises evaluating the subject as having a perinatal disorder when it does not show a statistically significant difference.
2. The protein according to claim 1, wherein the protein is selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). The method described.
3. The method according to claim 1 or 2, wherein the subject is a human patient.
4. Any of claims 1 to 3, wherein the subject is evaluated as having a perinatal disorder when all of the tested proteins show a significant difference in the body fluid sample of the subject as compared with the normal body fluid. The method according to item 1.
5. The method according to any one of claims 1 to 4, wherein the expression level is determined by an immunoassay.
6. The method according to any one of claims 1 to 4, wherein the expression level is determined by liquid chromatography / mass spectrometry.
7. The method according to any one of claims 1 to 4, wherein the expression level is determined using a protein array.
8. Α-2-Macroglobulin (A2m), OX-2 membrane in body fluid obtained from the subject to determine early neonatal perinatal disorders due to fetal stunting in a mammalian subject One or more selected from the group consisting of glycoprotein (Cd200), polyubiquitin-B (Ubb), neuroserpin (Serpini1), ubiquitin thioesterase OTUB1 (Otub1), and ubiquitin-like modification activating enzyme 1 (Uba1). Use of a proteomic profile of protein expression in Ubiquitin.
9. 8. The protein is selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). Use of description.
10. A method for early determination of the therapeutic effect of neonatal perinatal disorders due to fetal growth restriction in mammalian subjects.
    (A) α-2-Macroglobulin (A2m) (SEQ ID NO: 1), OX-2 membrane glycoprotein (Cd200) (SEQ ID NO: 2), polyubiquitin-B (Ubb) (SEQ ID NO: 3), neurocerpin (Serpini1) ) (SEQ ID NO: 4), ubiquitin thioesterase OTUB1 (Otub1) (SEQ ID NO: 5), and one or more proteins selected from the group consisting of ubiquitin-like modification activating enzyme 1 (Uba1) (SEQ ID NO: 6). Expression levels were tested in body fluid samples obtained from the subject being treated in comparison to expression levels in normal body fluids, post-treatment body fluids, or body fluids known to exhibit perinatal disorders; (B) In the body fluid known to show no statistically significant difference in expression level compared to the expression level in the normal body fluid or body fluid after treatment, or to exhibit perinatal disorders. The above method comprising determining that treatment of perinatal disorders in the subject is effective when it shows a statistically significant difference compared to the expression level.
11. 10. The protein is selected from the group consisting of α-2-macroglobulin (A2m), OX-2 membrane glycoprotein (Cd200), polyubiquitin-B (Ubb), and neuroserpin (Serpini1). The method described.

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