Marker composition for evaluating the effect of mesenchymal stem cell therapy on inflammatory bowel disease and its application

Abstract

The present invention provides a marker composition for evaluating the effect of mesenchymal stem cell treatment on inflammatory bowel disease and its application, relating to the field of biotechnology. The marker composition includes the Syt8 gene, the Gabrg2 gene, the Fpr1 gene, and the Hs3st4 gene. The marker composition is applied to the preparation of a product for evaluating the treatment effect of inflammatory bowel disease in a subject treated with mesenchymal stem cells, alleviating the problem in the prior art of lacking a method for evaluating the efficacy of mesenchymal stem cell treatment for IBD.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a marker composition for evaluating the effect of mesenchymal stem cell treatment of inflammatory bowel disease and its application. Background Art

[0002] Inflammatory bowel diseases (IBD) are a type of inflammatory diseases that occur in the digestive tract and are prone to recurrence. They are 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 relatively low, but the cure is difficult, the recurrence rate is high, and the treatment cost is high, which seriously affects the living standards of patients. At present, there is no effective method for preventing and treating IBD clinically.

[0003] Mesenchymal stem / stromal cells (MSCs) are adult stem cells derived from the mesoderm and can be obtained from various tissues such as bone marrow, adipose tissue, placenta, umbilical cord, etc. More and more studies have shown that MSC transplantation can significantly improve the repair of damaged tissues, including bone defects, brain injuries, myocardial infarctions, acute liver and lung injuries, ulcerative colitis, etc. Research has shown that MSCs can significantly improve the clinical abnormalities of colitis mice, reduce the colitis lesion score and intestinal inflammation by regulating the intestinal microbiota in colitis, and can also regulate immune cells and secrete regulatory molecules and cytokines to regulate the immune response and reduce the inflammatory response, and repair the damage of intestinal epithelial cells and intestinal mucosal barrier function. As previous studies have shown, MSC injection can regulate the intestinal metabolism of colitis mice and restore the function of abnormal microbiota to normal levels. MSCs improve the inflammatory response by regulating the proliferation and differentiation of B cells, inhibiting the proliferation of T cells, inducing the production of Treg cells, etc.; promote the reconstruction and regeneration of the damaged colonic epithelial tissue of colitis mice by regulating the levels of factors such as IGF-1 and PGE2. At present, the efficacy evaluation method of MSCs in treating IBD still needs further research.

[0004] In view of this, the present invention is specifically 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 cell treatment of inflammatory bowel disease and its application, so as to alleviate the problem that the prior art lacks an efficacy evaluation method for mesenchymal stem cell treatment of IBD.

[0006] To solve the above technical problems, the present invention specifically adopts the following technical solutions:

[0007] In a first aspect, there is provided an application of a substance for detecting a marker composition in the preparation of a product for evaluating the treatment effect of inflammatory bowel disease in a subject, wherein the marker composition includes a first marker, a second marker, a third marker, and a fourth marker;

[0008] The first marker includes the Syt8 gene and / or the expression product of the Syt8 gene;

[0009] The second marker includes the Gabrg2 gene and / or the expression product of the Gabrg2 gene;

[0010] The third marker includes the Fpr1 gene and / or the expression product of the Fpr1 gene;

[0011] The fourth marker includes the Hs3st4 gene and / or the expression product of the Hs3st4 gene;

[0012] The subject is treated with mesenchymal stem cells.

[0013] In a second aspect, there is provided a kit for evaluating the treatment effect of inflammatory bowel disease in a subject, the kit comprising a substance for detecting the marker composition described in the first aspect, and the subject is treated with mesenchymal stem cells.

[0014] In a third aspect, there is provided a device for evaluating the treatment effect of inflammatory bowel disease in a subject, the device including a detection module and a prediction and judgment module, and the subject is treated with mesenchymal stem cells; the detection module is used for detecting the marker composition described in the first aspect;

[0015] The prediction and judgment module includes a computer-readable medium storing judgment rules, and when the computer-readable medium is processed and executed, it realizes comparing the amounts of the marker composition in a sample of a subject treated with mesenchymal stem cells and a sample of an untreated subject; if the amounts of the first marker and the second marker in the sample of the subject treated with mesenchymal stem cells are reduced compared with those in the sample of the untreated subject, and the amounts of the third marker and the fourth marker are increased, it is determined that the treatment of the subject treated with mesenchymal stem cells is effective.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Human umbilical cord mesenchymal stem cells (HUCMSCs) can significantly improve the intestinal pathological damage in the inflammatory bowel disease (IBD) model and reduce the intestinal injury score, showing obvious therapeutic effects. Through transcriptome sequencing, a group of up-regulated genes, namely the Fpr1 gene and the Hs3st4 gene, and down-regulated genes, namely the Syt8 gene and the Gabrg2 gene, which are highly related to the efficacy of HUCMSCs, were first discovered, and the detection of these genes was verified by quantitative PCR to be used for evaluating the effect of HUCMSCs in treating IBD.

[0018] Based on this discovery, the present invention uses the discovered gene combination as a biomarker composition, which is significantly related to the IBD lesions and treatment in mice, provides new data support and research ideas for the pathogenesis of IBD, and improves the evaluation system for HUCMSCs to treat IBD. At the same time, the detection of this biomarker composition enriches the existing evaluation methods, and the results are more accurate and sensitive. Compared with the prior art, the present invention simplifies the sampling and evaluation methods of traditional IBD treatment evaluation by verifying the high correlation between the expression of the biomarker composition and the histological score of the colon, and provides a more intuitive, accurate and sensitive effect evaluation method. Therefore, the present invention provides methodological support for evaluating the effect of HUCMSCs in the treatment of IBD and provides new ideas for developing precise and sensitive stem cell therapy efficacy evaluation technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is the morphological diagram of the colon tissue of each group of mice in Example 2;

[0021] Figure 2 It is the histological score diagram of the colon tissue of each group of mice in Example 2;

[0022] Figure 3 It is the relative mRNA expression level of the Syt8 gene in each experimental group of mice in Example 4;

[0023] Figure 4 It is the relative mRNA expression level of the Gabrg2 gene in each experimental group of mice in Example 4;

[0024] Figure 5 It is the relative mRNA expression level of the Fprl gene in each experimental group of mice in Example 4;

[0025] Figure 6 The relative mRNA expression levels of the Hs3st4 gene in mice of each experimental group in Example 4;

[0026] Figure 7 ROC curve for verifying the correlation between gene composition expression and histological score in Example 6. Detailed implementation manners

[0027] The technical solutions 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0028] In this article, unless otherwise specified, all the implementation manners and preferred implementation methods mentioned in this article can be combined with each other to form new technical solutions. All the technical features and preferred features mentioned in this article can be combined with each other to form new technical solutions. The various components or their preferred components involved can be combined with each other to form new technical solutions.

[0029] In this article, unless otherwise specified, any numbering is used to distinguish one entity or action from another entity or action, rather than necessarily requiring or implying any actual such relationship, order or importance between these entities or actions, such as numbered first, second, third, and fourth.

[0030] In this article, unless otherwise specified, "optionally", "optional", "optional", or "optional" means that the subsequent described event or circumstance can but does not have to occur, and this description includes the occasions where the event or circumstance occurs or does not occur.

[0031] In this article, the terms "each... independently selected from", "each... independently selected from", and "... independently selected from" can be interchanged and should be understood in a broad sense. It can either mean that among different sets, the specific options expressed among the same type of elements do not affect each other, or it can also mean that among the same set, the specific options expressed among the same type of elements do not affect each other.

[0032] In this article, the term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps.

[0033] In this article, the terms "patient", "subject", or "individual" can be used interchangeably and include human or non-human animals, or cells, blood, tissues, body fluids, or secretions derived from human or non-human animals. The mammals include, for example, humans, monkeys, mice, rats, rabbits, donkeys, cows, horses, pigs, or dogs.

[0034] As used herein, the term "gene expression product" refers to the product generated by a gene through processes such as transcription and translation, which can be a direct product or a product after cleavage, recombination, replication, or metabolism of the direct product. Exemplary "gene expression products" include, but are not limited to, RNA or polypeptides.

[0035] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to persons skilled in the art. In addition, any methods or materials similar or equivalent to the described content can also be applied herein.

[0036] In a first aspect, provided is the use of a substance for detecting a biomarker 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.

[0037] The biomarker composition includes a first biomarker, a second biomarker, a third biomarker, and a fourth biomarker.

[0038] The first biomarker 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; it 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.

[0039] The second biomarker includes the Gabrg2 gene and / or the expression product of the Gabrg2 gene. The Gabrg2 gene encodes the 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 polypeptide, preferably mRNA.

[0040] The third biomarker includes the Fpr1 gene and / or the expression product of the Fpr1 gene. The Fpr1 gene encodes a G protein-coupled receptor of mammalian phagocytes, which mediates the response of phagocytes to microbial invasion of 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.

[0041] The fourth biomarker 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, and the expression of this gene is considered 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.

[0042] In an alternative embodiment, the substances of the detection marker 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. Depending on the specific detection method, those skilled in the art can select the substances of the detection marker composition according to the methods described in general and more specific textbooks, reference documents, process manuals, product descriptions, and standard documents, etc. 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, buffer components, metal ions, salts, fluorescent dyes, surfactants, dNTPs, primers for amplifying genes, probes, positive controls, and negative controls.

[0043] In an alternative embodiment, the marker composition includes the mRNA of the Syt8 gene, the mRNA of the Gabrg2 gene, the mRNA of the Fpr1 gene, and the mRNA of the Hs3st4 gene.

[0044] In an alternative embodiment, the substances of the detection marker composition include the following primers:

[0045] The upstream and downstream primers for detecting the first marker, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM.

[0046] The upstream and downstream primers for detecting the second marker, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.3, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.4, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM.

[0047] The upstream and downstream primers for detecting the third marker, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.5, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.6, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM.

[0048] The upstream and downstream primers for detecting the fourth biomarker, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.7, the working concentration is preferably 8-12 μM, more preferably 10 μM; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8, the working concentration is preferably 8-12 μM, more preferably 10 μM.

[0049] In an alternative embodiment, it further includes upstream and downstream primers for detecting the internal reference.

[0050] In an alternative embodiment, the internal reference is GAPDH, the nucleotide sequence of the upstream primer for detecting GAPDH is as shown in SEQ ID NO.9, the working concentration is preferably 8-12 μM, more preferably 10 μM; the nucleotide sequence of the downstream primer for detecting GAPDH is as shown in SEQ ID NO.10, the working concentration is preferably 8-12 μM, more preferably 10 μM.

[0051] In a second aspect, a kit for evaluating the treatment effect of inflammatory bowel disease in a subject is provided. The kit contains substances for detecting the biomarker composition described in the first aspect, and the subject is treated with mesenchymal stem cells.

[0052] In an alternative embodiment, the kit includes one or more of a probe, reagents for nucleic acid amplification, reagents for detecting nucleic acid amplification products, total RNA extraction reagents, and reverse transcription reagents. Depending on the specific detection method, those skilled in the art can select the composition of the reagents in the kit according to the methods described in general and more specific textbooks, references, process manuals, product descriptions, and standard documents, etc. 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, buffer components, metal ions, salts, fluorescent dyes, surfactants, dNTPs, primers for amplifying genes, probes, positive controls, and negative controls.

[0053] In an alternative embodiment, the kit includes the upstream and downstream primers for amplifying the first to fourth biomarkers respectively with the nucleotide sequences as shown in SEQ ID NOs.1-8.

[0054] In an alternative embodiment, in the kit, the working concentrations of the upstream primers with the nucleotide sequences as shown in SEQ ID NOs.1, 3, 5, and 7 are each independently preferably 8-12 μM, more preferably 10 μM.

[0055] In an alternative embodiment, in the kit, the working concentrations of the downstream primers with nucleotide sequences as shown in SEQ ID NO.2, 4, 6 or 8 are each independently preferably 8 - 12 μM, and more preferably 10 μM.

[0056] In an alternative embodiment, the kit further includes an upstream primer and a downstream primer for detecting an internal reference.

[0057] In an alternative embodiment, the internal reference detected by the kit is GAPDH. The nucleotide sequence of the upstream primer for detecting GAPDH in the kit is as shown in SEQ ID NO.9, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM; the nucleotide sequence of the downstream primer for detecting GAPDH in the kit is as shown in SEQ ID NO.10, and the working concentration is preferably 8 - 12 μM, more preferably 10 μM.

[0058] In a third aspect, there is also provided a device for evaluating the treatment effect of inflammatory bowel disease in a subject who has been treated with mesenchymal stem cells. The device includes a detection module and a prediction and judgment module.

[0059] The detection module is used to detect the biomarker composition described in the first aspect.

[0060] The prediction and judgment module includes a computer-readable medium storing judgment rules. When the computer-readable medium is processed and executed, it compares the amounts of the biomarker composition in a sample of a subject treated with mesenchymal stem cells and a sample of an untreated subject; if the amounts of the first biomarker and the second biomarker in the sample of the subject treated with mesenchymal stem cells are reduced compared to those in the sample of the untreated subject, and the amounts of the third biomarker and the fourth biomarker are increased, it is determined that the treatment of the subject treated with mesenchymal stem cells is effective.

[0061] In an alternative embodiment, the amount of the biomarker composition includes, but is not limited to, the amount of gene transcription into mRNA or the amount of gene translation into polypeptide or protein.

[0062] In an alternative embodiment, the first biomarker is the mRNA of the Syt8 gene, the second biomarker is the mRNA of the Gabrg2 gene, the third biomarker is the mRNA of the Fpr1 gene, and the fourth biomarker is the mRNA of the Hs3st4 gene. The detection module includes reagents for nucleic acid amplification to amplify mRNA or cDNA reverse transcribed from mRNA, and the amount of the biomarker composition includes the expression level of mRNA.

[0063] In an alternative embodiment, the amplification reagents include the upstream primers and downstream primers for amplifying the first to fourth biomarkers respectively with nucleotide sequences as shown in SEQ ID NO.1 - 8 described above.

[0064] In an alternative embodiment, the detection module is used to implement a fluorescence quantitative PCR reaction program, including the following steps: reverse transcribing mRNA extracted from a sample to be tested into cDNA, and performing a fluorescence quantitative PCR (qRT-PCR) reaction using the cDNA as a template; collecting the fluorescence signal and Ct value of qRT-PCR, and combining the two for result analysis by the prediction and judgment module.

[0065] In an alternative embodiment, according to the Ct value of the sample to be tested, the relative expression level of mRNA in the sample is calculated using known and conventional calculation methods in the art as the amount of the marker composition.

[0066] In an alternative embodiment, GAPDH is used as an internal reference for calculating the relative expression level of mRNA, and the GAPDH is amplified by primers shown in the aforementioned nucleotide sequences such as SEQ ID NO.9 and 10.

[0067] In an alternative 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.

[0068] In an alternative embodiment, the test sample can be derived from conventional samples used in 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.

[0069] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0070] Example 1

[0071] Establishment of a colitis mouse model and HUCMSC treatment:

[0072] Six SPF-grade C57BL / 6J mice (male, 10 weeks old, production license number: SCXK (Zhe) 2024-0002) were randomly selected for each group and raised under a 12 h light / dark cycle at 22 ± 2 °C, with the humidity maintained at 50% - 60%.

[0073] The model group and the HUCMSC treatment group were respectively fed with a 2% dextran sulfate sodium (DSS) aqueous solution on days D1 - D5, D11 - D15, and D21 - D23 of the experiment to induce the establishment of a colitis model. The HUCMSC treatment group was injected with HUCMSC preparation via the tail vein on D1, D8, D15, and D22. The HUCMSC dose was 0.175×10 7 cells / kg, and the dosing volume was 10 mL / kg for all. On the 25th day, after the animals were sacrificed by intraperitoneal injection of an appropriate amount of pentobarbital (150 mg / kg), colon tissue specimens were taken for further analysis.

[0074] Example 2

[0075] Histological examination and RNA - Seq:

[0076] The colon tissue specimens obtained in Example 1 were fixed in 4% paraformaldehyde solution (Sangon Biotech), embedded in paraffin blocks, cut into 5 - μm - thick sections, then stained with HE, observed and photographed under a microscope (Carl Zeiss), and histological injury scores were performed. The histological injury scoring criteria are as follows:

[0077] Table 1

[0078]

[0079] The results are as Figure 1 and Figure 2 shown, Figure 1 and Figure 2 show that tail vein injection of HUCMSC can effectively improve DSS - induced damage to mouse colon tissue and reduce the histological score.

[0080] Total RNA was extracted from the colon tissues of C57BL / 6J mice (n = 3) using TRIzol reagent (Invitrogen). The purity and integrity of the RNA were analyzed using an Agilent Bioanalyzer 2100 (Agilent Technologies). RNA sequencing was performed using an Illumina HiSeq 4000 sequencing system. The MapSplice program was used for RNA - Seq data mapping. Transcripts per million per kilobase were used to measure the expression level of each gene. The quality of the paired - end data fastq (.gz) files was analyzed using the fastqc second - generation sequencing data quality analysis software. The filtered fastq.qz data files (containing cDNA sequences) were aligned with the corresponding reference genome using hisat2 to generate the corresponding sam file. Subsequently, differential expression gene assembly screening, GO annotation and KEGG analysis, protein - protein interaction network and key gene analysis, etc. were carried out.

[0081] Differential gene analysis was performed on RNA-Seq data, and the results are shown in Table 2. A total of 4 genes with the most significant up-regulation and down-regulation of expression were screened out as candidate genes. In the mice treated with HUCMSC injection, the expression of Syt8 gene and Gabrg2 gene was up-regulated compared with that of normal mice, while the expression of Fpr1 gene and Hs3st4 gene was down-regulated.

[0082] Table 2 Expression levels of differential genes in each experimental group

[0083]

[0084] Example 3

[0085] This example provides a group of gene compositions for evaluating the treatment of IBD with umbilical cord mesenchymal stem cells, including the primer combinations as described in Table 3:

[0086] Table 3 Gene primer sequences

[0087]

[0088] Among them, R represents A or G, Y represents C or T, M represents A or C, and the primers of each sequence in the degenerate primer are mixed in an equimolar ratio.

[0089] Example 4

[0090] RT-PCR verification:

[0091] Repeat the colitis model establishment and HUCMSC treatment experiments in Example 1. After 25 days, collect the colon tissues, extract the total RNA using TRIzol reagent (Invitrogen), and reverse transcribe to obtain cDNA. Use the primers provided in Example 3 and the primers of the internal reference gene to perform fluorescence quantitative PCR (qRT-PCR) to determine the relative mRNA levels of the candidate genes. According to the operation instructions of the kit, perform qPCR amplification with a total volume of 10 μL, and the concentration of each primer is 10 μM.

[0092] The amplification conditions of qRT-PCR are as follows: The first step is pre-denaturation at 95°C for 5 min; the second step is the cycling reaction, 5°C for 10 s, 60°C for 30 s, for 45 cycles; the last step is the melting curve, 95°C for 15 s, 60°C for 60 s, 95°C for 15 s. Use 2 -ΔΔCT method with GAPDH as the internal reference.

[0093] Each sample was evaluated with 3 replicates, and the measurement was repeated at least three times independently. The results are as Figures 3 to 6As shown, the results showed that the expression levels of Syt8 gene mRNA and Gabrg2 gene mRNA in the HUCMSC group were down-regulated compared with the model group, while the expression levels of Fprl gene mRNA and Hs3st4 gene mRNA were up-regulated compared with the model group.

[0094] Example 5

[0095] Analysis of the correlation between gene expression and histological score of the colon:

[0096] The CORREL function was used to analyze the correlation between the changes in the expression of candidate genes and the histological score of the colon. A correlation coefficient close to 1 or -1 indicates a strong correlation between the two. A value close to 1 indicates a positive correlation, a value close to -1 indicates a negative correlation, a correlation coefficient of 0 indicates no correlation, a value below 0.4 indicates a low correlation, a value between 0.4 and 0.7 indicates a significant correlation, and a value above 0.7 indicates a high correlation. The calculation results showed that the changes in the expression of Syt8 gene and Gabrg2 gene were highly positively correlated with the histological score of the colon, while the changes in the expression of Fpr1 gene and Hs3st4 gene were highly negatively correlated with the histological score of the colon. This indicates that the changes in this gene composition are highly correlated with the histological score.

[0097] Table 4

[0098]

[0099] Example 6

[0100] Verification of the correlation between gene expression and histological score of the colon:

[0101] The colitis model establishment and HUCMSC treatment experiments of Example 1 were repeated, with the number of mice in each group increased to 30. After 25 days, colon tissues were obtained, and total RNA was extracted using TRIzol reagent (Invitrogen). Fluorescent quantitative PCR (qRT-PCR) was performed using the primers provided in Example 3 to determine the relative mRNA levels of candidate genes. qPCR amplification was carried out with a total volume of 10 μL according to the operation instructions of the kit, and the concentration of each primer was 10 μM.

[0102] The amplification conditions for qRT-PCR were as follows: First step, pre-denaturation at 95°C for 5 min; Second step, cyclic reaction, 5°C for 10 s, 60°C for 30 s, for 45 cycles; Finally, melting curve, 95°C for 15 s, 60°C for 60 s, 95°C for 15 s. The 2 -ΔΔCT method was used with GAPDH as the internal reference. Each sample was evaluated with 3 replicates, and the measurement was repeated at least three times independently. Finally, data analysis was performed according to the average threshold (Ct).

[0103] When the Ct value of the sample to be tested is less than 40, 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 level.

[0104] When the expression levels of Syt8 and Gabrg2 genes in the model group are significantly higher than those in the blank control group (the model group without HUCMSC treatment), and the expression levels of Fpr1 and Hs3st4 genes are significantly lower than those in the blank control group, it is determined that the HUCMSC treatment is effective. If at least one of the above four conditions does not meet, it is determined that the HUCMSC treatment is ineffective.

[0105] In the verification experiment, according to the histological score and the results of the PCR experiment, an ROC curve is drawn to verify the gene composition.

[0106] The result analysis shows that the AUC value of the ROC curve = 91.11%, P＜0.0001。 The ROC curve is as Figure 7 shown.

[0107] In summary, 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 the candidate genes shows that the nerve conduction and hormone secretion in the mice after HUCMSC injection decrease, and the inflammation level decreases. The change of the gene combination as a biomarker composition detected by RT-PCR is highly correlated with the histological score of the colon. Therefore, the biomarker 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 efficacy evaluation of IBD.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions 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 ulcerative 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 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; The fourth marker is the mRNA of the Hs3st4 gene; The subject is treated with umbilical cord mesenchymal stem cells.

2. The use according to claim 1, 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.

3. The use according to claim 2, 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 NOs. 1 and 2, respectively; upstream primers and downstream primers for detecting a second marker, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; upstream primers and downstream primers for detecting a third marker, whose nucleotide sequences are shown in SEQ ID NOs. 5 and 6, respectively; and upstream primers and downstream primers for detecting a fourth marker, whose nucleotide sequences are shown in SEQ ID NOs. 7 and 8, respectively.

4. The use according to claim 2, 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.

5. The use according to claim 3 or 4, 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.

6. The use according to claim 1, characterized in that: The substance for detecting the marker composition includes a device for evaluating the treatment effect of inflammatory bowel disease in a subject, the device includes 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; 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.

7. The use according to claim 6, characterized in that: The detection module includes using a nucleic acid amplification reagent to amplify mRNA or cDNA obtained by reverse transcription of mRNA.

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