Biomarker for diagnosing tethered syndrome and application thereof

Abstract

The invention relates to the technical field of gene detection, in particular to a biomarker for diagnosing tethered cord syndrome and application of the biomarker. The application finds that the GSTP1 gene is related to the disease progress of the tethered cord syndrome, and the expression of the GSTP1 gene in the tethered cord syndrome is remarkably increased, so that the GSTP1 gene can be used as a detection target to be applied to diagnosis and post-treatment prediction of the tethered cord syndrome.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and specifically to diagnostic biomarkers for tethered cord syndrome and their application in the prediction of tethered cord syndrome. Technical Background

[0002] Tethered spinal cord syndrome (TCS, or occult spinal dysraphism sequence) refers to a syndrome of neurological damage such as lower limb sensory and motor dysfunction or deformity, incontinence, etc., caused by pathological changes such as traction of the conus medullaris or / and low position of the conus medullaris due to congenital (such as maternal viruses in early pregnancy, severe folic acid deficiency due to insufficient vegetable intake, etc.) or acquired (such as lumbar spinal canal surgery) causes of lesions of the terminal filum, which leads to pathological changes such as ischemia, hypoxia, and nerve tissue degeneration at the end of the spinal cord. Clinically, symptoms such as lower limb sensory and motor dysfunction or deformity, and incontinence of urine and feces appear. Due to the involvement of multiple system organs, it can lead to serious sequelae and a high disability rate. TCS with normal cone position is more insidious onset and is more likely to be misdiagnosed or missed.

[0003] In recent years, with the development of imaging, neuroelectrophysiology, and microsurgery, great progress has been made in the understanding of this disease. In terms of morphology and ultrastructure, the applicant observed the TCS lesions with light microscopy, and the results showed that there were different degrees of fibrosis and fatty degeneration; scanning electron microscopy showed abnormal ultrastructure of the lesions, including dense collagen fibers, expansion, and twisting; at the same time, optical coherence microscopy was used to observe the lesions, and the elasticity of the lesions was reduced, fat was infiltrated, and some of them had fibrinoid degeneration. In terms of cell metabolism, Yamada et al. found that when the spinal cord is stretched, it can cause ischemic and hypoxic changes in the spinal cord, leading to abnormal metabolism of cytochrome a, a3 in the intermediate neurons, which ultimately leads to reduced ATP production, impaired energy storage and utilization in cells, and thus neuronal dysfunction and reduction in number.

[0004] Although, with the development of technology and research on the disease in recent years, it is not difficult to diagnose tethered cord syndrome based on typical medical history, clinical manifestations and auxiliary examinations. However, since the disease is often asymptomatic or the symptoms develop late in the early stages, diagnosis and surgical treatment are often late, and the filum terminale and even the cone have undergone irreversible changes, and the neurological dysfunction cannot be completely improved despite treatment. In view of the complexity of diagnosis and the untimely treatment of the disease, there is an urgent need to improve the understanding of the disease and find a method or biomarker that can diagnose the disease more effectively at an early stage, so as to greatly increase the accuracy and timeliness of early clinical diagnosis in terms of diagnosis, so as to improve the effect of subsequent patients adopting treatment options such as surgery in a timely manner. Based on this, the present application provides a biomarker for tethered cord syndrome and its application. In view of this, the present application is proposed. Summary of the invention

[0005] In view of the defects of the existing technology in disease diagnosis, the purpose of this application is to seek a new biomarker or new diagnostic method for tethered cord syndrome to solve the problems mentioned in the above background technology, such as the delay and complexity of diagnosis or prediction, which leads to the delay of treatment and poor treatment effect. To this end, the present application provides a biomarker for tethered cord syndrome, which is a gene or protein of glutathione S-transferase 1 (glutathione S-transferase pi, GSTP1).

[0006] Specifically, the detailed technical solution of this application is as follows:

[0007] The present application first provides a detection agent for obtaining the level of GSTP1 in a sample for use in the diagnosis or prediction of tethered cord syndrome;

[0008] The present application also provides an application of a detection agent for obtaining the GSTP1 level in a sample in the preparation of a product for diagnosing or predicting tethered cord syndrome.

[0009] In some aspects, the form of the product includes but is not limited to a kit form or a device form, etc.

[0010] In some aspects, the marker is highly expressed in the filum terminale tissue of patients with tethered cord syndrome; in particular, the filum terminale tissue of tethered cord syndrome has significant fibrosis changes, and the fibrosis causes the terminal filum terminale to lose elasticity and pull the spinal cord.

[0011] In some aspects, the level of GSTP1 in a filum terminale tissue sample is used to indicate the presence or severity of tethered cord syndrome.

[0012] In some aspects, the GSTP1 level includes GSTP1 gene level or protein level.

[0013] When the GSTP1 level is the GSTP1 gene level, especially the gene transcription level, the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method, of course not limited to these methods.

[0014] When the GSTP1 level is the GSTP1 protein level, the detection agent is used to perform any one of the following methods: immunoassay, biomass spectrometry, amino acid sequencing and electrophoresis, but is certainly not limited to these methods.

[0015] In some aspects, the sample can be selected from at least one of a subject's blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, urine, and saliva sample.

[0016] In some aspects, the diagnosis may include: early screening, diagnosis and prognosis assessment of the disease, etc.

[0017] In some aspects, the kit also includes reagents for detecting other markers for combined detection to improve predictive or diagnostic performance.

[0018] The present application also provides a biomarker for diagnosing tethered cord syndrome and its application, wherein the marker is glutathione S-transferase Pi 1 gene or protein.

[0019] The present application also provides a product for diagnosing or predicting tethered cord syndrome, wherein the product comprises a reagent for detecting GSTP1 gene level; preferably, the GSTP1 level comprises GSTP1 gene level or protein level.

[0020] In some aspects, the product includes, but is not limited to, a kit form or a device form, etc.

[0021] Furthermore, the kit may also include a sample processing reagent, wherein the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

[0022] The present application also provides a method for diagnosing or predicting tethered cord syndrome, the method comprising:

[0023] (i) obtaining GSTP1 levels in samples from subjects;

[0024] (ii) comparing the GSTP1 level in the subject sample with that in the control sample; wherein a significant difference in the GSTP1 level in the subject sample and the control sample is an indication that the subject is at risk of developing tethered cord syndrome in the future; or,

[0025] (ii) comparing with a set threshold absolute amount; wherein the level of the subject's sample being higher than the threshold absolute amount is an indication that the subject suffers from or will suffer from tethered cord syndrome in the future.

[0026] In some aspects, the GSTP1 level includes the gene level or protein level of GSTP1.

[0027] Preferably, when the GSTP1 level is the GSTP1 gene level, especially the gene transcription level, the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

[0028] Preferably, when the GSTP1 level is the GSTP1 protein level, the detection agent is used to perform any one of the following methods: immunoassay, biomass spectrometry, amino acid sequencing and electrophoresis.

[0029] In some aspects, the sample can be selected from at least one of the subject's tissue or tissue lysate, blood, serum, plasma, cerebrospinal fluid, cell culture supernatant, urine and saliva samples; preferably, the sample is tissue, and more preferably, the filum terminale tissue.

[0030] In some specific aspects, the method may include the following specific steps:

[0031] a. Obtain Filum terminale (FT) tissue samples from patients;

[0032] b. detecting the expression level of the GSTP1 gene in the FT tissue sample using an immunohistochemistry (IHC) method;

[0033] c. comparing the detected GSTP1 gene expression level with the GSTP1 gene expression level of a normal control sample to identify the presence of tethered cord syndrome;

[0034] Furthermore, the normal control sample includes a sample of the filum terminale tissue of an aborted fetus that has been excluded from neurological malformations, and the filum terminale tissue of the tethered cord syndrome presents a fibrotic state;

[0035] In particular, the application can be used to perform early diagnosis of patients with tethered cord syndrome based on the expression level of the GSTP1 gene, and can be used as a predictive indicator after clinical treatment, and has extremely high clinical translation value.

[0036] Compared with the prior art, the advantages and positive effects of this application are:

[0037] This application first discovered that the GSTP1 gene can be used as a biomarker for the diagnosis of tethered cord syndrome; compared with normal controls, the expression level of the GSTP1 gene in the filum terminale tissue of tethered cord syndrome was significantly upregulated; immunohistochemistry showed that the GSTP1 gene derived from the filum terminale tissue has a good ability to diagnose tethered cord syndrome. The method proposed in this application has a good diagnostic efficacy for the early diagnosis of tethered cord syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 , pathological changes of the filum terminale in the tethered cord syndrome group; A is H&E staining, original magnification 10×10; B is H&E staining, original magnification 10×10; C is H&E staining, original magnification 10×40).

[0040] Figure 2 , Pathological changes of the filum terminale tissue in the tethered cord (TC) group, among which, AC is H&E staining, original magnification 10×40 times.

[0041] Figure 3 , pathological changes of the filum terminale in the aborted fetus group; A is H&E staining, original magnification 10×10; B is H&E staining, original magnification 10×40.

[0042] Figure 4 , KEGG enriched pathway statistical results.

[0043] Figure 5 , differential protein interaction network analysis diagram.

[0044] Figure 6 , GSTP1 immunohistochemical results; (A) Fetal group FT: GSTP1 weak expression, a large range of tissue staining can be seen under the microscope as light yellow; (B) TCS group FT: a large range of cell cytoplasm staining can be seen under the microscope as brown (↑), indicating strong positive expression of GSTP1, and the fibrous tissue is severely collagenized and not stained (←); (CD) Fetal group FT: GSTP1 is positively expressed in FT tissue, brown-yellow tissue staining can be seen, and a large number of differentiated and mature glial cells can be seen in FT under high-power microscope (↓). (AC: Masson staining, original magnification 10×10; D: Masson staining, original magnification 10×40).

[0045] Figure 7 , GSTP1 immunohistochemistry score (IHC Score) comparison bar chart; TCS refers to the TCS children group; TC refers to the TC children group; fetus refers to the aborted fetus group; * indicates P < 0.05, with significant difference; ns indicates no statistically significant difference. DETAILED DESCRIPTION

[0046] The embodiments of the present application will be described in detail below in conjunction with the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be considered to limit the scope of the present application. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0047] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0048] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0049] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" is used, this includes a plural of that noun.

[0050] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0051] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0052] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. It will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment. Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0053] According to one aspect of the present application, the present application provides a use of a GSTP1 level detection agent in the preparation of a product for diagnosing or predicting tethered cord syndrome. Preferably, changes in GSTP1 levels are a sensitive indication for the diagnosis or prediction of tethered cord syndrome.

[0054] The term "product" refers to the process for detecting GSTP1 levels configured into a corresponding product form. It is understood that such product forms are diverse, including but not limited to kit forms, device forms, etc., but any product form containing corresponding components belongs to the protection scope of this application.

[0055] The "GSTP1" or "glutathione-S-transferase 1" of the present application is a member of the glutathione S-transferase (GSTs) family and belongs to the pi class. Its main function is to enable various electrophilic compounds, such as drugs, environmental toxins, oxidation chain products, etc., to bind to glutathione and enter the next metabolic step. GSTP1 plays an important role in cell resistance to damage and cancer, and is one of the most important phase II metabolic enzymes for biotransformation in the body. The GSTP1 gene species can be mammals; preferably, primates; more preferably, humans.

[0056] The "tethered spinal cord syndrome", "Tethered spinal cord syndrome, TCS" or "occultspinal dysraphism sequence" in this application have the same meaning, which refers to the pathological changes such as spinal cord ischemia, hypoxia, and nerve tissue degeneration caused by congenital (such as viruses in the mother's early pregnancy, insufficient vegetables and severe folic acid deficiency, etc.) or acquired (such as lumbar spinal canal surgery) reasons, resulting in spinal cord traction or / and low cone, resulting in ischemia, hypoxia, and nerve tissue degeneration in the spinal cord, and clinical symptoms of lower limb sensory and motor dysfunction or deformity, incontinence and other neurological damage. In some aspects, the filum terminale tissue of patients with tethered cord syndrome in this article is highly expressed; in particular, the filum terminale tissue of tethered cord syndrome has significant fibrosis changes, and the fibrosis causes the terminal filum terminale to lose elasticity and traction on the spinal cord. This application found through research that there is a significantly increased GSTP1 gene expression in the filum terminale tissue of tethered cord syndrome, and through molecular systems and clinical tests, it is confirmed that GSTP1 can be used as a diagnostic indicator of tethered cord syndrome. Therefore, any detection method that can reflect the GSTP1 level in a patient and thus achieve the diagnosis of tethered cord syndrome falls within the scope of this application.

[0057] The detection of GSTP1 level can be any means in the art, because GSTP1 level can include two dimensions, gene level and protein level, and therefore any means in these two dimensions are within the scope of this application.

[0058] In some embodiments, the detection agent detects at the gene level.

[0059] Gene level includes DNA or RNA level. The detection agent of DNA or RNA level can be selected from reagents known to those skilled in the art, such as nucleic acids (usually probes or primers) that can hybridize with the DNA or RNA and are labeled with fluorescent markers. It is also easy for those skilled in the art to detect cDNA after reverse transcription of mRNA into cDNA, and the conventional replacement of these technical means does not exceed the protection scope of this application.

[0060] For example, in some embodiments, the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

[0061] In some embodiments of the present application, the nucleic acid sequencing method can be transcriptome sequencing or genome sequencing. In some other embodiments of the present application, the nucleic acid sequencing method is high-throughput sequencing, also known as second-generation sequencing ("NGS"). NGS is different from "Sanger sequencing" (first-generation sequencing), which is based on the electrophoretic separation of chain termination products in a single sequencing reaction. NGS is a revolutionary change to traditional Sanger sequencing technology, and can sequence hundreds of thousands to millions of nucleic acid molecules at a time. The sequencing platform of the available NGS of the present application is commercially available, including but not limited to Roche / 454FLX, Illumina / Solexa GenomeAnalyzer and Applied Biosystems SOLID system, etc. Transcriptome sequencing can also quickly and comprehensively obtain almost all transcripts and gene sequences of a certain species specific cell or tissue in a certain state through a second-generation sequencing platform, which can be used to study gene expression, gene function, structure, alternative splicing and new transcript prediction, etc. In some other embodiments of the present application, the nucleic acid sequencing method can be single-molecule real-time sequencing. Single-molecule DNA sequencing technology is a new generation of sequencing technology developed in the past 10 years, also known as the third-generation sequencing technology, including single-molecule real-time sequencing, true single-molecule sequencing, single-molecule nanopore sequencing and other technologies.

[0062] For example, in other embodiments, the polymerase chain reaction method of the present application includes primers for GSTP1 gene amplification, wherein the primer refers to a primer that can specifically amplify the GSTP1 coding gene or mRNA, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least a portion of the template sequence and serve as a starting point for synthesizing primer extension products. In some specific embodiments, the probe hybridization method includes a GSTP1 probe, wherein the probe refers to a probe that can specifically identify the GSTP1 coding gene, which is a molecule that can bind to a specific sequence or subsequence or other part of another molecule, generally referring to a nucleic acid probe that binds to another nucleic acid (i.e., a specific sequence of GSTP1 as a target nucleotide) through complementary base pairing.

[0063] In some embodiments, the detection agent detects at the protein level.

[0064] In some embodiments, the detection agent is used to perform any of the following methods: biological mass spectrometry, amino acid sequencing, electrophoresis, and antibody immunoassay. For example, the antibody immunoassay can further be immunoprecipitation, immunocoprecipitation, immunohistochemistry, ELISA, and Western Blot. In some specific embodiments, the immunoassay uses a specific antibody, wherein the specific antibody refers to an antibody that can specifically recognize the GSTP1 protein, specifically including but not limited to at least one of a monoclonal antibody, a polyclonal antibody, a multivalent antibody, and a multispecific antibody.

[0065] In some embodiments, the kit further includes a sample processing reagent; further, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent, and a sample nucleic acid extraction reagent.

[0066] In some embodiments, the sample is selected from at least one of blood, serum, plasma, pleural effusion, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, semen and saliva samples of the lung adenocarcinoma patient, preferably tissue, more preferably filum terminale tissue.

[0067] According to another aspect of the present application, the present application also provides a method for diagnosing or predicting tethered cord syndrome, the method comprising: using the detection agent as described above to detect the presence or content of GSTP1.

[0068] The diagnostic or predictive methods based on markers, as known in the art, can generally include the following basic steps or ideas:

[0069] (i) obtaining GSTP1 levels in samples from subjects;

[0070] (ii) comparing the GSTP1 level in the subject sample with that in the control sample; wherein a significant difference in the GSTP1 level in the subject sample and the control sample is an indication that the subject is at risk of developing tethered cord syndrome in the future; or,

[0071] (ii) comparing with a set threshold absolute amount; wherein the level of the subject's sample being higher than the threshold absolute amount is an indication that the subject suffers from or will suffer from tethered cord syndrome in the future.

[0072] Likewise, the GSTP1 level includes the GSTP1 gene level or protein level.

[0073] In some embodiments, when the GSTP1 level is the GSTP1 gene level, such as when the GSTP1 level is the GSTP1 gene transcription level, the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method;

[0074] In other embodiments, when the GSTP1 level is the GSTP1 protein level, the detection agent is used to perform any one of the following methods: immunoassay, biological mass spectrometry, amino acid sequencing and electrophoresis.

[0075] In some embodiments, the sample can be selected from at least one of the subject's blood, serum, plasma, cerebrospinal fluid, tissue or tissue lysate, cell culture supernatant, urine and saliva samples; preferably, the sample is tissue, and more preferably, the filum terminale tissue.

[0076] In a specific embodiment of the present application, the diagnosis or prediction method comprises the following specific steps:

[0077] 1) Obtaining filum terminale tissue samples from patients;

[0078] 2) using immunohistochemistry (IHC) method to detect the expression level of GSTP1 gene in the filum terminale tissue sample;

[0079] The detected GSTP1 gene expression level is compared with the GSTP1 gene expression level of a normal control sample to identify the presence of tethered cord syndrome;

[0080] Preferably, the normal control sample is selected from the filum terminale tissue of aborted fetuses excluding nervous system malformations, and the filum terminale tissue of tethered cord syndrome presents a fibrotic state.

[0081] In some specific embodiments of the present application, a set value of GSTP1 level can be given, and the set value can be determined based on the expression level of GSTP1 of normal samples of normal people and / or non-TCS patients. For example, the average value of the expression level of GSTP1 of normal samples of an appropriate number of samples is selected, or a reasonable multiple is set based on the average value, such as 0.9 times, 0.8 times, 0.7 times, 0.6 times, 0.5 times, etc. When the expression level of GSTP1 of the subject is higher than the set value, it is judged as TCS. It can be understood that the set value determined based on the average value or the multiple of the average value needs to have good classification significance, and the known samples can be tested by using the commonly used statistical test method through the classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment standard.

[0082] As used herein, the terms "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment or therapy.

[0083] As used herein, the terms "sample", "specimen", "test sample", "subject sample", etc. include various sample types obtained from patients, individuals or subjects and can be used for diagnosis or monitoring assays. Patient samples can be obtained from healthy subjects, patients with spinal cord tethering syndrome, or patients with symptoms associated with spinal cord tethering syndrome. In addition, the sample obtained from the patient can be divided into parts, and only one part can be used for diagnosis. In addition, the sample or a part thereof can be stored under conditions that maintain the sample for subsequent analysis. This definition specifically includes tissue samples and other liquid samples of biological origin (including but not limited to cells (including cell culture supernatant), body fluids, serum, plasma, whole blood, urine, etc.). In a specific embodiment, the sample includes a tissue sample. In another embodiment, the sample includes a blood sample. This definition also includes samples that are manipulated in any way after the sample is obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of certain cell populations. These terms also include clinical samples, and also include cells in culture, cell supernatants, tissue samples, organs, etc. Samples may also include fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or studies by immunohistochemistry.

[0084] In addition, it can also be understood that in order to further enhance the diagnostic or predictive effect of the disease, in the diagnostic or predictive process, in addition to the GSTP1 of the present application, the markers may also include other nucleic acid fragments, proteins, metabolites, etc. with corresponding diagnostic capabilities that are already well known to those skilled in the art, and used as joint markers. In this way, the combined use of multiple biomarkers is achieved, and they cooperate with each other to achieve better diagnostic or predictive effects on TCS, thereby achieving a more effective evaluation purpose. Therefore, in addition to being an independent indication of tethered cord syndrome, the GSTP1 can also be combined with other existing known diagnostic markers for detection, thereby improving diagnostic efficiency.

[0085] The following are specific examples to further illustrate the content of this application.

[0086] 1. Clinical samples

[0087] Thirty-six samples of filum terminale tissue were screened from pregnant women and children who visited the Henan Children's Congenital Spinal Cord Malformation Center and Obstetrics Department of the Third Affiliated Hospital of Zhengzhou University from August 2019 to August 2022. Among them, the group of children with tethered cord syndrome (TCS): 14 children with typical symptoms of tethered cord syndrome underwent filum terminale segmentation; the group of children with tethered cord (TC): 14 children without typical symptoms of tethered cord syndrome but clinically diagnosed with low spinal cord underwent filum terminale segmentation during surgical treatment of major diseases; the group of aborted fetuses: 8 aborted fetuses with no neurological malformations had their spinal canals and dural sacs opened to remove the filum terminale tissue. (This project was approved by the Ethics Committee of this hospital and the pregnant women, children or their families signed informed consent, ethics number 2023-(Pre)062). The inclusion and exclusion criteria are as follows in Table 1:

[0088] Table 1. Inclusion and exclusion criteria

[0089]

[0090]

[0091] Note: MRI: Magnetic resonance imaging; TCS: Tethered cord syndrome; TC: Tethered cord; CM: Conus medullaris.

[0092] Example 1: Study on pathological changes of the filum terminale

[0093] 1. Sample preparation

[0094] The filum terminale tissues collected in this study were all freshly excised filum terminale. The samples in the experimental group were freshly cut filum terminale near the end of the conus medullaris during the spinal cord tethering release operation. Immediately after excise, 1 cm of the filum terminale was taken from the head end and temporarily stored in liquid nitrogen; the samples in the control group were freshly induced aborted fetuses enrolled in the group, which were immediately sent to the pathology department for sampling, and 1 cm of the filum terminale was taken from the end of the conus medullaris. All samples were stored in a -80℃ refrigerator until use in the experiment.

[0095] 2. Sample fixation: Fix the FT samples in an appropriate fixative, such as 10% neutral buffered formaldehyde solution, to preserve the tissue structure.

[0096] 3. Dehydration: Dehydrate the fixed samples with alcohol in stages to remove moisture from the tissue.

[0097] 4. Transparency: Treat the sample with a transparent agent (such as xylene or Xylene) to prepare for paraffin embedding.

[0098] 5. Paraffin embedding: Immerse the transparent sample in melted paraffin for embedding.

[0099] 6. Sectioning: Use a microtome to cut the embedded sample into thin slices (usually 4-6 microns thick).

[0100] 7. Dewaxing to water: Treat the paraffin sections with xylene and graded alcohol to remove the paraffin until the sections reach a hydrated state.

[0101] 8. Hematoxylin staining: Immerse the sections in hematoxylin staining solution for staining. The staining time is determined according to the laboratory standard operating procedures.

[0102] 9. Washing: After staining, gently rinse the sections with tap water to remove excess dye.

[0103] 10. Eosin staining: Immerse the sections in eosin staining solution for contrast staining.

[0104] 11. Dehydration and sealing: The stained sections are dehydrated again through graded alcohol and transparent agent, and then sealed with sealing medium.

[0105] 12. Microscopic examination and image acquisition and analysis: Use an optical microscope to observe the sections and evaluate the morphological structure and staining characteristics of the tissue. Use a microscope photography system to collect images for further analysis.

[0106] Experimental results:

[0107] TCS group: Microscopically, a large amount of adipose tissue in the FT was abnormally proliferated, and the fibrous tissue showed a dense and chaotic structure. Figure 1 Large areas of abnormal fibrous tissue proliferation can be seen in the FT, which is dense and disordered. The secreted collagen accumulates between the fibroblasts, and the fibrous tissue changes into collagen (↓). Only in some fibroblasts can blue spindle nuclei be seen (see Figure 1 A); Nerve fiber bundles (→) and surrounding proliferating fibrous tissue (←) can be found in the FT, and the structure is dense and chaotic (see Figure 1 BC).

[0108] TC group: Microscopically, the FT tissue structure was basically the same as that of the TCS group. Microscopically, the fibrous tissue was hyperplastic and disordered, with twisted and disordered fibers, localized hyperplasia of fat infiltration, and nerve fiber bundles. Compared with the TCS group, the TC group had localized hyperplasia of fat tissue, the degree of collagenization of fibrous tissue was lower than that of the TCS group, and more blue spindle-shaped nuclei of fibroblasts were observed (see Figure 2 A, ←); The fibrous tissue around the arterioles and venules in the FT tissue of the child showed obvious collagen changes and was arranged in a disordered manner (see Figure 2 BC). Small blood vessels can be seen under high magnification, with obvious endothelium, thick walls, small lumens and round shapes, which are arterioles (see Figure 2B, →); small veins with large, flat and irregular lumens (see Figure 2 C,↓).

[0109] Aborted fetus group: Spinal cord-like tissue was observed inside the FT of this group under the microscope. A large amount of loose connective tissue was arranged in a regular and orderly manner. The reticular area formed by the interweaving of nerve fibers, dendrites, axons, synapses of nerve cells, processes of glial cells, and capillaries was the neuropil (see Figure 3 A); Observing the neuropil under a high-power microscope, we can see that nerve fibers, glial cells (↑) and synapses are densely interwoven (see Figure 3 B).

[0110] In summary, compared with the normal control group, the tethered cord syndrome progressively develops dense fibrous connective tissue or fibrosis in the filum terminale tissue.

[0111] Example 2: Proteomic Differences of Filum Terminale Tissue

[0112] This example is based on previous studies, specifically a randomized controlled study, in which samples selected according to the standard are divided into two groups, namely the experimental group (EG) and the control group (CG). The experimental group is a random selection of multiple terminal filum samples from TCS children who underwent surgical treatment, and the control group is a random selection of multiple terminal filum samples from fetuses that were induced for other reasons without neurological malformations.

[0113] For all samples, 1 cm of the filum terminale near the end of the conus medullaris was taken for protein extraction. The differential proteins between the two groups of samples were screened using label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS / MS) technology, and bioinformatics analysis was performed to conduct related pathway analysis.

[0114] According to the experimental results, 253 differentially expressed proteins were identified in the two groups of samples, including 101 up-regulated proteins and 152 down-regulated proteins (Table 2).

[0115] Table 2. Statistics of differentially expressed proteins

[0116]

[0117] Further bioinformatics analysis such as enrichment analysis and protein interaction analysis was performed on the differentially expressed proteins. The results of the enrichment analysis are shown in Figure 4Through the enrichment results, it can be seen that all differentially expressed proteins are involved in 18 different signaling pathways, including 8 pathways related to metabolism, namely carbon metabolism, amino acid biosynthesis, fatty acid metabolism, pyruvate metabolism, unsaturated fatty acid biosynthesis, fatty acid separation, glycine, serine and threonine metabolism, and vitamin B6 biological metabolism. The number of genes involved in carbon metabolism is the highest at 13%. There are 4 pathways involved in genetic information processing, namely spliceosome translation, ribosome conversion, endoplasmic reticulum processing, and protein export, of which 31% of genes are involved in ribosome processing. There is one pathway involved in environmental signal analysis interaction, which is EMC receptor interaction, covering 8% of genes. There are 3 pathways involved in cellular processes, namely actin cell skeleton regulation, focal adhesion movement, and tight junction, of which 14% of genes are involved in focal adhesion movement. There are 2 pathways involved in biological systems, namely complement and coagulation cascades, and leukocyte transendothelial cell migration, and 13% of genes are involved in complement and coagulation cascades.

[0118] STRING was further used to perform protein interaction network analysis. In order to make the results more accurate, proteins without interactions were excluded, and proteins with no empty item values ​​were selected for analysis. The protein interaction network diagram was constructed using Cytoscape software. Figure 5 This is a partial interaction network analysis diagram between the two groups. It is drawn based on four functional analyses: protein / gene fold change, protein-protein interaction, KEGG pathway enrichment analysis, and biological process enrichment. Round nodes represent proteins / genes, red represents upregulation, and green represents downregulation. Rectangular nodes represent KEGG Pathway / Biological process, and significant P-values ​​are represented by yellow-blue gradient colors, with yellow representing a smaller P value and blue representing a larger P value. By analyzing this application, 47 interaction network center nodes can be obtained.

[0119] In summary, the differentially expressed proteins are involved in lipid metabolism, fiber metabolism and dissolution, coagulation cascade, ribosome conversion and carbon metabolism. α-Enolase (ENO1), bigamycin (BGN), glutathione S transferase P (GSTP1), and actin (ACTC1) are all involved in fiber metabolism. ENO1 is a multifunctional enzyme that, in addition to playing a role in glycolysis, is also involved in a variety of processes such as growth control, hypoxia resistance and allergic reactions. It may also play a role in the fibrinolytic system within and around the cell; BGN is involved in collagen fiber assembly; GSTP1 prototype glutathione conjugation with a large number of exogenous and endogenous hydrophobic electrophiles, weakens CDK5 activity to prevent neurodegeneration, and participates in the negative regulation of fibroblast proliferation; ACTC1 is a highly conserved protein that participates in various types of cell movement, is expressed in all nucleated cells, and participates in the regulation of stress fiber assembly.

[0120] There are different types of interactions and co-expression between the above proteins. In previous ultrastructural studies of diseased filum terminale, both light microscopy and scanning electron microscopy showed different degrees of fibrosis, which is consistent with the experimental results. Therefore, it is believed that these protein interactions may cause fibrotic changes in the structure of the filum terminale, thereby leading to the occurrence of the disease. The conclusions on fiber formation are consistent with those found in the histopathology of the filum terminale. In the process of enrichment analysis and protein interaction screening research, this application found that the glutathione S transferase Pi 1 (GSTP1) gene is significantly expressed in the fiber metabolism process, and it is speculated that it may play a vital role in the pathogenesis of tethered cord syndrome.

[0121] Example 3: Immunohistochemical verification of the disease prediction performance of GSTP1 in the filum terminale tissue

[0122] In this example, the aforementioned tissue samples were used to detect the expression of GSTP1 by immunohistochemistry to further verify the significant difference in the expression of GSTP1 in FT tissues of TCS children, TC children and aborted fetuses.

[0123] As in Example 1, this example also uses freshly isolated terminal filum terminale from the clinic. The samples in the experimental group are fresh terminal filum terminale severed near the end of the conus medullaris during spinal cord tethering and release. Immediately after separation, 1 cm terminal filum terminale is taken from the head end and temporarily stored in liquid nitrogen; the samples in the control group are freshly induced aborted fetuses enrolled in the group, which are immediately sent to the pathology department for sampling, and 1 cm terminal filum terminale is taken near the end of the conus medullaris. All samples are stored in a -80°C refrigerator until use in the experiment.

[0124] IHC operation process:

[0125] Dewax paraffin sections to water → antigen repair → block endogenous peroxidase → serum blocking → add primary antibody: (PBS and 1:50 recombinant Anti-GST3 / GST Pi) → add secondary antibody: (1:200 prepared goat anti-rabbit IgG secondary antibody, HRP labeled) → DAB color development → counterstain cell nuclei → dehydrate and seal sections → microscopic examination, image acquisition and analysis.

[0126] IHC scoring method:

[0127] Immunohistochemistry score (IHC Score) = staining intensity × positive cell ratio

[0128] Staining intensity: 0 (0 points): no positive staining is negative; 1 (1 point): light yellow is weakly positive; 2 (2 points): brownish yellow is positive; 3 (3 points): brownish brown is strongly positive;

[0129] Positive cell ratio: grade 0 is 0-5%, grade 1 is 6%-25%, grade 2 is 26%-50%, grade 3 is 51%-75%, and grade 4 is >75%.

[0130] Determination of the positive grade of the section: The immunohistochemical score is obtained by multiplying the staining intensity and the positive cell ratio to determine the positive grade of the section staining: (1) 0 points for negative (-); (2) ≤3 points for weak positive (+); (3) 4-5 points for positive (++); (4) ≥6 points for strong positive (+++).

[0131] Data analysis Graphpad Prism 9.1 and SPSS 21 were used to analyze and plot the obtained data. With P < 0.05 as the test standard, one-way analysis of variance Turkey method was used to analyze the inter-group correlation of GSTP1 expression, IHC score, age, and fibrosis in the three groups of FT, and a scatter bar graph was drawn based on the statistical results.

[0132] According to the IHC test results (see Tables 3-5 and Figure 6 ): (A) Fetal group FT: GSTP1 was weakly expressed, and a large area of ​​tissue was stained light yellow under the microscope; (B) TCS group FT showed a large area of ​​cytoplasm stained brown under the microscope (↑), indicating strong positive expression of GSTP1, and the severely collagenized area of ​​fibrous tissue was not stained (←); (CD) GSTP1 was positively expressed in FT tissue of TC group, and brown-yellow tissue staining was observed. A large number of differentiated and mature glial cells were distributed in FT under high-power microscope (↓).

[0133] Table 3. Main symptoms, FT lesions and GSTP1 expression in TCS children

[0134]

[0135]

[0136] Note: CM location: the vertebra corresponding to the end of the conus medullaris; L1: the first lumbar vertebra; L2: the second lumbar vertebra; L3: the third lumbar vertebra; L4: the fourth lumbar vertebra; L5: the fifth lumbar vertebra; +: weakly positive; ++: positive; +++: strongly positive.

[0137] Table 4. Main diagnoses, FT lesions and GSTP1 expression in the TC group

[0138]

[0139]

[0140] Note: CM location: the vertebra corresponding to the end of the conus medullaris; L2: the second lumbar vertebra; L3: the third lumbar vertebra; L5: the fifth lumbar vertebra; L2-L3: between the lower edge of L2 and the upper edge of L3; GSTP1: Glutathione-S-transferase Pi 1; +: weakly positive; ++: positive; +++: strongly positive.

[0141] Table 5. Main disease information, FT lesions and GSTP1 expression in the fetal group

[0142]

[0143] Note: MRI: Magnetic resonance imaging; FT: Filum terminale; GSTP1: Glutathione-S-transferase Pi 1; +: weakly positive; ++: positive; +++: strongly positive.

[0144] After testing, 10 FT (71.4%) GSTP1 positively expressed in the TCS group, 8 FT (57.1%) GSTP1 positively expressed in the TC group, and only 1 FT (14.3%) GSTP1 positively expressed in the aborted fetus group. Figure 7 ), the positive expression ratio of GSTP1 in FT of TCS children was higher than that of the other two groups, and the expression of GSTP1 in FT of TCS children was significantly higher than that of the control aborted fetus group, which was statistically significant (P=0.0155), so it can be used to distinguish between TCS and aborted fetus groups.

[0145] In summary, high-level expression of GSTP1 gene is involved in the fibrosis process of filum terminale tissue in children and plays an important role in it. The expression level of GSTP1 gene is closely related to the degree of tethered cord syndrome and can be used to predict tethered cord syndrome.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a detection agent for obtaining GSTP1 levels in a sample in the diagnosis or prediction of tethered cord syndrome, or in the preparation of a product for the diagnosis or prediction of tethered cord syndrome.

2. The use according to claim 1, characterized in that: The GSTP1 level includes the GSTP1 gene level or protein level.

3. The use according to claim 1, characterized in that: When the GSTP1 level is the GSTP1 gene level, the detection agent is used to perform any of the following methods: polymerase chain reaction, denaturing gradient gel electrophoresis, nucleic acid sequencing, nucleic acid typing chip detection, denaturing high performance liquid chromatography, in situ hybridization, biological mass spectrometry and HRM method.

4. The use according to claim 1, characterized in that: When the GSTP1 level is the GSTP1 protein level, the detection agent is used to perform any one of the following methods: immunoassay, biological mass spectrometry, amino acid sequencing and electrophoresis.

5. The use according to any one of claims 1 to 4, characterized in that: In the tethered cord syndrome, the filum terminale tissue is in a fibrotic state.

6. Application of GSTP1 gene or protein as a biomarker for the diagnosis of tethered cord syndrome.

7. A biomarker for diagnosing tethered cord syndrome, characterized in that: The marker is GSTP1 gene or protein.

8. A product for diagnosing or predicting tethered cord syndrome, characterized in that: The product comprises a reagent for detecting the GSTP1 gene level; preferably, the product is in the form of a kit or a device; more preferably, the GSTP1 level comprises the GSTP1 gene level or protein level.

9. A method for diagnosing or predicting tethered cord syndrome, characterized in that: The following steps are involved: (i) obtaining GSTP1 levels in samples from subjects; (ii) comparing the GSTP1 level in the subject sample with that in the control sample; wherein a significant difference in the GSTP1 level in the subject sample and the control sample is an indication that the subject is at risk of developing tethered cord syndrome in the future; or, (ii) comparing with a set threshold absolute amount; wherein the level of the subject's sample being higher than the threshold absolute amount is an indication that the subject suffers from or will suffer from tethered cord syndrome in the future.

10. The method for diagnosis or prediction according to claim 9, characterized in that: The control sample is selected from the filum terminale tissue of aborted fetuses with excluded nervous system malformations, and the filum terminale tissue of tethered cord syndrome presents a fibrotic state.

Images