Marker composition for evaluating inflammatory enteritis treatment effect of mesenchymal stem cells and application of marker composition

Abstract

The invention provides a marker composition for evaluating the inflammatory enteritis treatment effect of mesenchymal stem cells and application of the marker composition, and relates to the technical field of biology. The marker composition is prepared from a Syt8 gene, a Gabrg2 gene, an Fpr1 gene and an Hs3st4 gene. The marker composition is applied to preparation of products for evaluating the treatment effect of inflammatory enteritis of subjects treated by mesenchymal stem cells, and the problem that in the prior art, a method for evaluating the treatment effect of IBD treated by the mesenchymal stem cells is lacked is solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a marker composition for evaluating the effect of mesenchymal stem cells in treating inflammatory bowel inflammation and an application thereof. Background Art

[0002] Inflammatory bowel diseases (IBD) are a type of inflammatory disease that occurs in the digestive tract and is prone to recurrence. It is mainly divided into Crohn's disease (CD) and ulcerative colitis (UC). With the development of industrialization, it has become a global disease. Its clinical manifestations include abdominal pain, persistent diarrhea, weight loss, fever, rectal bleeding and other symptoms. The mortality rate is low, but it is difficult to cure, has a high recurrence rate and high treatment costs, which seriously affects the living standards of patients. At present, there is no effective method to prevent and treat IBD in clinical practice.

[0003] Mesenchymal stem cells (MSCs) are adult stem cells derived from the mesoderm and can be obtained from various tissues such as bone marrow, adipose tissue, placenta, and umbilical cord. More and more studies have shown that MSC transplantation can significantly improve the repair of damaged tissues, including bone defects, brain damage, myocardial infarction, acute liver and lung damage, and ulcerative colitis. Studies have shown that MSCs can significantly improve the clinical abnormalities of colitis mice, reduce colitis lesion scores and intestinal inflammation by regulating intestinal microbial flora in colitis. They can also regulate immune cells and secrete regulatory molecules and cytokines to regulate immune responses, reduce inflammatory responses, and repair intestinal epithelial cell damage and intestinal mucosal barrier function. Previous studies have shown that MSCs injection can regulate intestinal metabolism in colitis mice and restore abnormal flora functions to normal levels. MSCs improve inflammatory responses by regulating the proliferation and differentiation of B cells, inhibiting T cell proliferation, and inducing the production of Treg cells; they promote the reconstruction and regeneration of colon epithelial tissue in colitis mice after destruction by regulating the levels of factors such as IGF-1 and PGE2. At present, the efficacy evaluation method of MSCs in the treatment of IBD still needs further study.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a marker composition for evaluating the effect of mesenchymal stem cells in treating inflammatory bowel disease and its application, so as to alleviate the problem that the prior art lacks a method for evaluating the efficacy of mesenchymal stem cells in treating IBD.

[0006] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions: In a first aspect, there is provided a use of a substance for detecting a marker composition in preparing a product for evaluating the therapeutic effect of inflammatory bowel disease in a subject, wherein the marker composition comprises a first marker, a second marker, a third marker and a fourth marker; The first marker includes the Syt8 gene and / or the expression product of the Syt8 gene; The second marker includes the Gabrg2 gene and / or the expression product of the Gabrg2 gene; The third marker includes the Fpr1 gene and / or the expression product of the Fpr1 gene; The fourth marker includes the Hs3st4 gene and / or the expression product of the Hs3st4 gene; The subject is treated with mesenchymal stem cells.

[0007] In a second aspect, a kit for evaluating the therapeutic effect of inflammatory bowel disease in a subject is provided, the kit comprising a substance for detecting the marker composition described in the first aspect, wherein the subject is treated with mesenchymal stem cells.

[0008] In a third aspect, a device for evaluating the therapeutic effect of inflammatory bowel disease in a subject is provided, the device comprising a detection module and a prediction and judgment module, wherein the subject is treated with mesenchymal stem cells; the detection module is used to detect the marker composition described in the first aspect; The prediction and judgment module includes a computer-readable medium having a judgment rule recorded thereon, and when the computer-readable medium is processed and executed, the amount of the marker composition of the subject sample treated with mesenchymal stem cells and the subject sample not treated is compared; if the amount of the first marker and the second marker in the subject sample treated with mesenchymal stem cells is reduced compared to the subject sample not treated, and the amount of the third marker and the fourth marker is increased, it is determined that the treatment of the subject treated with mesenchymal stem cells is effective.

[0009] Compared with the prior art, the present invention has the following beneficial effects: Umbilical cord mesenchymal stem cells (HUCMSC) can significantly improve intestinal pathological damage in the inflammatory bowel disease (IBD) model and reduce intestinal damage scores, showing significant therapeutic effects. Through transcriptome sequencing, a group of upregulated genes Fpr1 and Hs3st4 genes, and downregulated genes Syt8 and Gabrg2 genes that are highly correlated with the efficacy of HUCMSC were discovered for the first time, and quantitative PCR was used to verify that the detection of these genes can be used to evaluate the effect of HUCMSC in treating IBD.

[0010] Based on this discovery, the present invention uses the discovered gene combination as a marker composition, which is significantly correlated with mouse IBD lesions and treatments, provides new data support and research ideas for the pathogenesis of IBD, and improves the evaluation system of HUCMSC for the treatment of IBD. At the same time, the detection of the marker composition enriches the existing evaluation methods, and the results are more accurate and sensitive. Compared with the prior art, the present invention simplifies the traditional sampling and evaluation methods of IBD treatment evaluation by verifying the high correlation between the expression of the marker composition and the colon histological score, and provides a more intuitive, accurate and sensitive effect evaluation method. Therefore, the present invention provides methodological support for evaluating the effect of HUCMSC in the treatment of IBD, and provides new ideas for developing accurate and sensitive stem cell therapy efficacy evaluation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the specific implementation methods of the present invention 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 invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 The colon tissue morphology of each group of mice in Example 2; Figure 2 This is a colon histological scoring diagram of each group of mice in Example 2; Figure 3 is the relative mRNA expression level of Syt8 gene in each experimental group of mice in Example 4; Figure 4 is the relative mRNA expression level of Gabrg2 gene in each experimental group of mice in Example 4; Figure 5 is the relative mRNA expression level of the Fprl gene in each experimental group of mice in Example 4; Figure 6 is the relative mRNA expression level of Hs3st4 gene in each experimental group of mice in Example 4; Figure 7 The ROC curve for verifying the correlation between gene combination expression and histological score in Example 6. DETAILED DESCRIPTION

[0013] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] Herein, unless otherwise specified, all embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution. All technical features and preferred features mentioned herein can be combined with each other to form a new technical solution. The components involved or their preferred components can be combined with each other to form a new technical solution.

[0015] Herein, unless otherwise stated, arbitrary numbering is used to distinguish one entity or action from another entity or action, and does not necessarily require or imply any actual such relationship, order, or importance between these entities or actions, such as numbering first, second, third, and fourth.

[0016] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0017] In this article, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense. They can mean that in different sets, the specific options expressed by the same type of elements do not affect each other, or that in the same set, the specific options expressed by the same type of elements do not affect each other.

[0018] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0019] As used herein, the terms "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, or cells, blood, tissues, body fluids, or secretions derived from humans or non-human animals, such as humans, monkeys, mice, rats, rabbits, donkeys, cows, horses, pigs, or dogs.

[0020] Herein, the term "gene expression product" refers to the product produced by a gene through processes such as transcription and translation, which may be a direct product or a product of a direct product after shearing, recombination, replication or metabolism. Exemplary "gene expression products" include but are not limited to RNA or polypeptides.

[0021] Unless otherwise specified, the professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to this article.

[0022] In a first aspect, there is provided a use of a substance of a detection marker composition in the preparation of a product for evaluating the therapeutic effect of inflammatory bowel disease in a subject treated with mesenchymal stem cells. In an optional embodiment, the subject is treated with umbilical cord mesenchymal stem cells.

[0023] The marker composition includes a first marker, a second marker, a third marker and a fourth marker.

[0024] The first marker includes the Syt8 gene and / or the expression product of the Syt8 gene, the Syt8 gene encodes a member of the synaptotagmin family, which is related to neurotransmission, hormone secretion and exocytosis; and is also related to the activity of the human insulin gene promoter and insulin secretion. Optionally, the expression product of the Syt8 gene includes at least one of mRNA and polypeptide, preferably mRNA.

[0025] The second marker includes the Gabrg2 gene and / or the expression product of the Gabrg2 gene, the Gabrg2 gene encodes a γ-aminobutyric acid (GABA) receptor and is involved in important neuromodulatory processes. Optionally, the expression product of the Gabrg2 gene includes at least one of mRNA and a polypeptide, preferably mRNA.

[0026] The third marker includes the Fpr1 gene and / or the expression product of the Fpr1 gene. The Fpr1 gene encodes the G protein-coupled receptor of mammalian phagocytes, mediates the response of phagocytes to microorganisms invading the host, and plays an important role in host defense and inflammation. Optionally, the expression product of the Fpr1 gene includes at least one of mRNA and polypeptide, preferably mRNA.

[0027] The fourth marker includes the Hs3st4 gene and / or the expression product of the Hs3st4 gene, the Hs3st4 gene encodes heparan sulfate D-glucosaminyl 3-O-sulfotransferase 4, the expression of which is believed to play a role in the pathogenesis of herpes simplex virus type 1 (HSV-1). Optionally, the expression product of the Hs3st4 gene includes at least one of mRNA and polypeptide, preferably mRNA.

[0028] In an optional embodiment, the substance of the detection marker composition includes one or more of a probe, a reagent for nucleic acid amplification, a reagent for detecting nucleic acid amplification products, a total RNA extraction reagent, and a reverse transcription reagent. Depending on the specific detection means, those skilled in the art can select the substance of the detection marker composition according to the methods recorded in general and more specific teaching materials, references, process manuals, product descriptions, and standard documents, etc., and the present invention does not limit this. Specific examples include, but are not limited to, at least one of an enzyme for amplification reaction, an enzyme for reverse transcription, a buffer component, a metal ion, a salt, a fluorescent dye, a surfactant, a dNTP, a primer for amplifying a gene, a probe, a positive control, and a negative control.

[0029] In an optional embodiment, the marker composition includes mRNA of Syt8 gene, mRNA of Gabrg2 gene, mRNA of Fpr1 gene and mRNA of Hs3st4 gene.

[0030] In an optional embodiment, the substances for detecting the marker composition include the following primers: The upstream primer and the downstream primer of the first marker are detected, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the working concentration is preferably 8-12 μM, and more preferably 10 μM; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the working concentration is preferably 8-12 μM, and more preferably 10 μM.

[0031] The upstream primer and the downstream primer of the second marker are detected. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the working concentration is preferably 8-12 μM, and more preferably 10 μM; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, and the working concentration is preferably 8-12 μM, and more preferably 10 μM.

[0032] The upstream primer and downstream primer of the third marker are detected, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, and the working concentration is preferably 8-12 μM, and more preferably 10 μM; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6, and the working concentration is preferably 8-12 μM, and more preferably 10 μM.

[0033] The upstream primer and downstream primer of the fourth marker are detected. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the working concentration is preferably 8-12 μM, and more preferably 10 μM; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8, and the working concentration is preferably 8-12 μM, and more preferably 10 μM.

[0034] In an optional embodiment, an upstream primer and a downstream primer for detecting an internal reference are also included.

[0035] In an optional embodiment, the internal reference is GAPDH, the nucleotide sequence of the upstream primer for detecting GAPDH is shown as SEQ ID NO.9, and the working concentration is preferably 8~12 μM, and more preferably 10 μM; the nucleotide sequence of the downstream primer for detecting GAPDH is shown as SEQ ID NO.10, and the working concentration is preferably 8~12 μM, and more preferably 10 μM.

[0036] In a second aspect, a kit for evaluating the therapeutic effect of inflammatory bowel disease in a subject is provided, the kit comprising a substance for detecting the marker composition described in the first aspect, and the subject is treated with mesenchymal stem cells.

[0037] In an optional embodiment, the kit includes one or more of a probe, a reagent for nucleic acid amplification, a reagent for detecting nucleic acid amplification products, a total RNA extraction reagent, and a reverse transcription reagent. Depending on the specific detection means, those skilled in the art can select the composition of the reagents in the kit according to the methods recorded in general and more specific teaching materials, references, process manuals, product descriptions, and standard documents, etc., and the present invention does not limit this. Optionally, the kit includes, but is not limited to, at least one of an enzyme for amplification reaction, an enzyme for reverse transcription, a buffer component, a metal ion, a salt, a fluorescent dye, a surfactant, a dNTP, a primer for amplifying a gene, a probe, a positive control, and a negative control.

[0038] In an optional embodiment, the kit includes the aforementioned nucleotide sequences as shown in SEQ ID NOs. 1 to 8, which are upstream primers and downstream primers for amplifying the first to fourth markers respectively.

[0039] In an optional embodiment, in the kit, the working concentrations of the upstream primers having nucleotide sequences such as those shown in SEQ ID NO. 1, 3, 5 and 7 are independently preferably 8 to 12 μM, and more preferably 10 μM.

[0040] In an optional embodiment, in the kit, the working concentration of the downstream primers having a nucleotide sequence such as SEQ ID NO. 2, 4, 6 or 8 is independently preferably 8 to 12 μM, and more preferably 10 μM.

[0041] In an optional embodiment, the kit further comprises an upstream primer and a downstream primer for detecting an internal reference.

[0042] In an optional embodiment, the internal reference detected by the kit is GAPDH, the nucleotide sequence of the upstream primer for detecting GAPDH in the kit is shown as SEQ ID NO.9, and the working concentration is preferably 8~12 μM, and more preferably 10 μM; the nucleotide sequence of the downstream primer for detecting GAPDH in the kit is shown as SEQ ID NO.10, and the working concentration is preferably 8~12 μM, and more preferably 10 μM.

[0043] In a third aspect, a device for evaluating the therapeutic effect of inflammatory bowel disease on a subject is provided, wherein the subject is treated with mesenchymal stem cells, and the device comprises a detection module and a prediction and judgment module.

[0044] The detection module is used to detect the marker composition described in the first aspect.

[0045] The prediction and judgment module includes a computer-readable medium having a judgment rule recorded thereon, and when the computer-readable medium is processed and executed, the amount of the marker composition of the subject sample treated with mesenchymal stem cells and the subject sample not treated is compared; if the amount of the first marker and the second marker in the subject sample treated with mesenchymal stem cells is reduced compared to the subject sample not treated, and the amount of the third marker and the fourth marker is increased, it is determined that the treatment of the subject treated with mesenchymal stem cells is effective.

[0046] In an optional embodiment, the amount of the marker composition includes but is not limited to the amount of gene transcription into mRNA or the amount of gene translation into polypeptide or protein.

[0047] In an optional embodiment, the first marker is the mRNA of the Syt8 gene, the second marker is the mRNA of the Gabrg2 gene, the third marker is the mRNA of the Fpr1 gene, and the fourth marker is the mRNA of the Hs3st4 gene. The detection module includes amplifying mRNA or cDNA obtained by reverse transcription of mRNA using a nucleic acid amplification reagent, and the amount of the marker composition includes the expression amount of mRNA.

[0048] In an optional embodiment, the amplification reagent includes the aforementioned nucleotide sequences as shown in SEQ ID NOs. 1 to 8, which are respectively used to amplify the first to fourth markers as upstream primers and downstream primers.

[0049] In an optional embodiment, the detection module is used to implement a fluorescent quantitative PCR reaction procedure, including the following steps: reverse transcribing the mRNA extracted from the sample to be tested into cDNA, and performing a fluorescent quantitative PCR (qRT-PCR) reaction using the cDNA as a template; collecting the fluorescent signal and Ct value of the qRT-PCR, and combining the two for predictive judgment module result analysis.

[0050] In an optional embodiment, the relative expression amount of mRNA in the sample is calculated as the amount of the marker composition according to the Ct value of the sample to be tested and a conventional calculation method known in the art.

[0051] In an optional embodiment, GAPDH is used as an internal reference for calculating the relative expression level of mRNA, and the GAPDH is amplified by the primers shown in the aforementioned nucleotide sequences such as SEQ ID NOs. 9 and 10.

[0052] In an optional embodiment, the amount of the marker composition in the sample of an untreated subject and the amount of the marker composition in the sample of a subject treated with mesenchymal stem cells are obtained from the same detection process; or, the amount of the marker composition in the sample of the untreated subject is preset in the prediction and judgment module.

[0053] In an optional embodiment, the test sample can be derived from conventional samples used for clinical testing in the art, including but not limited to cells, blood (plasma or serum), cells, tissues, body fluids and secretions, etc., preferably including at least one of tissues and blood.

[0054] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0055] Example 1 Establishment of colitis mouse model and HUCMSC treatment: Six SPF-grade C57BL / 6J mice (male, 10 weeks old, production license number: SCXK (Zhejiang) 2024-0002) were randomly selected from each group and housed at 22 ± 2 °C with a 12-h light / dark cycle and humidity maintained at 50%-60%.

[0056] The model group and HUCMSC treatment group were fed with 2% dextran sulfate sodium (DSS) aqueous solution on D1-D5, D11-D15, and D21-D23 of the experiment to induce colitis model. The HUCMSC treatment group was injected with HUCMSC preparations in the tail vein on D1, D8, D15, and D22. The HUCMSC dose was 0.175×10 7 cells / kg, and the dosage was 10 mL / kg. On the 25th day, the animals were killed by intraperitoneal injection of an appropriate amount of pentobarbital (150 mg / kg), and colon tissue samples were taken for further analysis.

[0057] Example 2 Histology and RNA-Seq: The colon tissue specimens obtained in Example 1 were fixed in 4% paraformaldehyde solution (Sangon Biotechnology), embedded in paraffin blocks, cut into 5 μm thick sections, and then stained with HE. The sections were observed and photographed under a microscope (Carl Zeiss) and scored for histological damage. The histological damage scoring criteria are as follows: Table 1

[0058] The results are as follows Figure 1 and Figure 2 As shown, Figure 1 and Figure 2 The results showed that tail vein injection of HUCMSC could effectively improve DSS-induced colon tissue damage in mice and reduce histological scores.

[0059] Total RNA was extracted from colon tissue of C57BL / 6J mice (n=3) using TRIzol reagent (Invitrogen). The purity and integrity of RNA were analyzed using Agilent Bioanalyzer 2100 (Agilent Technologies). RNA sequencing was performed using the Illumina HiSeq 4000 sequencing system. The MapSplice program was used for RNA-Seq data mapping. Transcripts per kilobase per million were used to measure the expression level of each gene. The quality of paired-end data fastq (.gz) files was analyzed using fastqc second-generation sequencing data quality analysis software. The filtered fastq.qz data files (containing cDNA sequences) were aligned to the corresponding reference genome using hisat2 to generate the corresponding sam files. Subsequently, assembly screening of differentially expressed genes, GO annotation and KEGG analysis, protein-protein interaction network and key gene analysis were performed.

[0060] The RNA-Seq data were subjected to differential gene analysis, and the results are shown in Table 2. A total of four genes with the most significant up- and down-regulation were screened as candidate genes. The Syt8 and Gabrg2 genes of mice treated with HUCMSC injection were up-regulated compared with those of normal mice, while the Fpr1 and Hs3st4 genes were down-regulated.

[0061] Table 2 Expression levels of differentially expressed genes in each experimental group

[0062] Example 3 This example provides a set of gene combinations for evaluating umbilical cord mesenchymal stem cells for the treatment of IBD, including the primer combinations described in Table 3: Table 3 Gene primer sequences

[0063] Wherein, R represents A or G, Y represents C or T, and M represents A or C. The primers of each sequence in the degenerate primer are mixed in an equimolar ratio.

[0064] Example 4 RT-PCR validation: The colitis model establishment and HUCMSC treatment experiment in Example 1 were repeated. Colon tissue was obtained 25 days later, total RNA was extracted using TRIzol reagent (Invitrogen), and cDNA was obtained by reverse transcription. Fluorescence quantitative PCR (qRT-PCR) was performed using the primers provided in Example 3 and the internal reference gene primers to determine the relative mRNA levels of the candidate genes. qPCR amplification was performed in a total volume of 10 μL according to the operating instructions of the kit, and the concentration of each primer was 10 μM.

[0065] The amplification conditions of qRT-PCR were as follows: the first step was pre-denaturation at 95°C for 5 min; the second step was cycle reaction at 5°C for 10 s, 60°C for 30 s, and 45 cycles; the last step was melting curve at 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. -ΔΔCT Methods GAPDH was used as an internal reference.

[0066] Three replicate wells were evaluated for each sample, and the measurements were repeated at least three times independently. Figure 3~Figure 6 As shown, the results showed that the expression levels of Syt8 gene mRNA and Gabrg2 gene mRNA in the HUCMSC group were downregulated compared with those in the model group, and the expression levels of Fprl gene mRNA and Hs3st4 gene mRNA were upregulated compared with those in the model group.

[0067] Example 5 Correlation analysis between gene expression and colon histological scores: The CORREL function was used to analyze the correlation between the expression changes of candidate genes and colon histology scores. A correlation coefficient close to 1 or -1 indicated a strong correlation between the two, close to 1 indicated a positive correlation, close to -1 indicated a negative correlation, a correlation coefficient of 0 indicated no correlation, below 0.4 indicated a low correlation, 0.4-0.7 indicated a significant correlation, and above 0.7 indicated a high correlation. The calculation results showed that the expression changes of Syt8 and Gabrg2 genes were highly positively correlated with colon histology scores, and the expression changes of Fpr1 and Hs3st4 genes were highly negatively correlated with colon histology scores. This indicates that the changes in the gene composition are highly correlated with the pathological histology score.

[0068] Table 4

[0069] Example 6 Validation of the correlation between gene expression and colon histological scores: The colitis model establishment and HUCMSC treatment experiments in Example 1 were repeated, and the number of mice in each group was increased to 30. Colon tissue was obtained after 25 days, and total RNA was extracted using TRIzol reagent (Invitrogen). Fluorescence quantitative PCR (qRT-PCR) was performed using the primers provided in Example 3 to determine the relative mRNA levels of the candidate genes. qPCR amplification was performed in a total volume of 10 μL according to the operating instructions of the kit, and the concentration of each primer was 10 μM.

[0070] The amplification conditions of qRT-PCR were as follows: the first step was pre-denaturation at 95°C for 5 min; the second step was cycle reaction, 5°C for 10 s, 60°C for 30 s, for 45 cycles; the last step was melting curve, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. -ΔΔCT GAPDH was used as an internal reference. Three replicates of each sample were evaluated, and the measurement was repeated at least three times independently. Finally, data analysis was performed based on the average threshold (Ct).

[0071] When the Ct value of the sample to be tested is less than 40 and there is an obvious amplification curve, and when the Ct value of the blank control is greater than 40 and there is no obvious amplification curve, the fluorescence signal is used for the result analysis of the mRNA expression amount.

[0072] When the expression levels of Syt8 and Gabrg2 genes in the model group were significantly higher than those in the blank control group (the model group not treated with HUCMSCs), and the expression levels of Fpr1 and Hs3st4 genes were significantly lower than those in the blank control group, HUCMSC treatment was determined to be effective. If at least one of the above four conditions was not met, HUCMSC treatment was determined to be ineffective.

[0073] In the validation experiment, a ROC curve for validating the gene combination was drawn based on the histological score and the PCR experimental results.

[0074] The result analysis showed that the ROC curve AUC value = 91.11%, P＜0.0001。 ROC curve Figure 7 shown.

[0075] The above results show that HUCMSC can alleviate the damage of DSS-induced colitis to a certain extent and has a therapeutic effect on IBD. The functional analysis of candidate genes shows that the nerve conduction and hormone secretion in mice injected with HUCMSC decreased, and the level of inflammation decreased. RT-PCR detection verified that the changes in the gene combination as a marker composition were highly correlated with the colon histology score. Therefore, the marker composition and its kit provided by the present invention can reflect the therapeutic effect of HUCMSC on IBD to a certain extent, and can be used for the evaluation of the efficacy of IBD.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention 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 invention.

Claims

1. Use of a substance of a detection marker composition in the preparation of a product for evaluating the therapeutic effect of inflammatory bowel disease in a subject, wherein the marker composition comprises a first marker, a second marker, a third marker and a fourth marker; The first marker includes the Syt8 gene and / or the expression product of the Syt8 gene; The second marker includes the Gabrg2 gene and / or the expression product of the Gabrg2 gene; The third marker includes the Fpr1 gene and / or the expression product of the Fpr1 gene; The fourth marker includes the Hs3st4 gene and / or the expression product of the Hs3st4 gene; The subject is treated with mesenchymal stem cells.

2. The use according to claim 1, characterized in that: The expression product of the Syt8 gene includes at least one of mRNA and polypeptide; and / or, the expression product of the Gabrg2 gene includes at least one of mRNA and polypeptide; and / or, the expression product of the Fpr1 gene includes at least one of mRNA and polypeptide; And / or, the expression product of the Hs3st4 gene includes at least one of mRNA and polypeptide.

3. The use according to claim 1, characterized in that: The subject is treated with umbilical cord mesenchymal stem cells.

4. A kit for evaluating the therapeutic effect of inflammatory bowel disease in a subject, characterized in that: A method comprising detecting a marker composition according to any one of claims 1 to 3, wherein the subject is treated with mesenchymal stem cells.

5. The kit according to claim 4, characterized in that The substances of the marker detection composition include one or more of a probe, a reagent for nucleic acid amplification, a reagent for detecting nucleic acid amplification products, a total RNA extraction reagent and a reverse transcription reagent.

6. The kit according to claim 5, characterized in that The materials of the marker detection composition include upstream primers and downstream primers for detecting a first marker, whose nucleotide sequences are shown in SEQ ID NO.1 or 2 respectively; upstream primers and downstream primers for detecting a second marker, whose nucleotide sequences are shown in SEQ ID NO.3 or 4 respectively; upstream primers and downstream primers for detecting a third marker, whose nucleotide sequences are shown in SEQ ID NO.5 or 6 respectively; and upstream primers and downstream primers for detecting a fourth marker, whose nucleotide sequences are shown in SEQ ID NO.7 or 8 respectively.

7. The kit according to claim 6, characterized in that The material for detecting the marker composition also includes an upstream primer for detecting the nucleotide sequence of the internal reference as shown in SEQ ID NO.9 and a downstream primer as shown in SEQ ID NO.

10.

8. The kit according to claim 6 or 7, characterized in that The working concentration of each upstream primer is independently 8-12 μM; and / or the working concentration of each downstream primer is independently 8-12 μM.

9. A device for evaluating the therapeutic effect of inflammatory bowel disease on a subject, characterized in that: Comprising a detection module and a prediction and judgment module, the subject is treated with mesenchymal stem cells; the detection module is used to detect the marker composition described in any one of claims 1 to 3; The prediction and judgment module includes a computer-readable medium having a judgment rule recorded thereon, and when the computer-readable medium is processed and executed, the amount of the marker composition of the subject sample treated with mesenchymal stem cells and the subject sample not treated is compared; if the amount of the first marker and the second marker in the subject sample treated with mesenchymal stem cells is reduced compared to the subject sample not treated, and the amount of the third marker and the fourth marker is increased, it is determined that the treatment of the subject treated with mesenchymal stem cells is effective.

10. The device according to claim 9, characterized in that The first marker is the mRNA of the Syt8 gene, the second marker is the mRNA of the Gabrg2 gene, the third marker is the mRNA of the Fpr1 gene, and the fourth marker is the mRNA of the Hs3st4 gene; The detection module includes using a nucleic acid amplification reagent to amplify mRNA or cDNA obtained by reverse transcription of mRNA, and the amount of the marker composition includes the expression amount of mRNA; And / or, the nucleic acid amplification reagent includes an upstream primer and a downstream primer for detecting a first marker, and the nucleotide sequences are shown in SEQ ID NO.1 or 2 respectively; an upstream primer and a downstream primer for detecting a second marker, and the nucleotide sequences are shown in SEQ ID NO.3 or 4 respectively; an upstream primer and a downstream primer for detecting a third marker, and the nucleotide sequences are shown in SEQ ID NO.5 or 6 respectively; an upstream primer and a downstream primer for detecting a fourth marker, and the nucleotide sequences are shown in SEQ ID NO.7 or 8 respectively.

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