Application of kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenyl glycol as serum biomarkers for evaluating curative effect of Crohn disease

Abstract

The invention discloses application of kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenyl glycol as serum biomarkers for evaluating the curative effect of Crohn's disease, and relates to the technical field of medical treatment. In particular to application of kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenyl glycol as serum markers in preparation of a kit for evaluating the treatment effect of the mesenchymal stem cells on the Crohn disease. On the basis of an isotope labeled liquid chromatography-mass spectrometry technology, the therapeutic effectiveness biomarker is screened by detecting the change of serum metabolites before and after the placenta-derived mesenchymal stem cells treat a mouse with Crohn disease, and the diagnostic efficiency of the therapeutic effectiveness biomarker is evaluated; and data support is provided for clinical transformation of the mesenchymal stem cells for treating Crohn's disease.

Description

Field of the Invention

[0001] The present invention belongs to the field of biomedicine technology, and relates to the application of kynurenine and formyl-5-hydroxykynurenine as serum biomarkers for evaluating the efficacy of Crohn's disease, and specifically to a method for screening and evaluating serum metabolite markers before and after human placenta-derived mesenchymal stem cells are used to treat Crohn's disease mice. Background Art

[0002] Crohn's disease is a chronic inflammatory nonspecific intestinal disease with the main clinical manifestations of abdominal pain, diarrhea, abdominal mass, perianal lesions, etc. The lesions can affect the entire digestive tract. At present, the cause and specific pathogenesis of Crohn's disease are still unclear, and there is no way to completely cure Crohn's disease. Although traditional treatments such as anti-inflammatory drugs and immunosuppressants can relieve patients' symptoms, they cannot completely solve the underlying intestinal inflammation and tissue damage.

[0003] Cell therapy is a method of injecting a cell suspension into the body to repair the damage that has occurred or is ongoing in order to achieve the purpose of treatment. Stem cells with the potential for continuous replication and multidirectional differentiation are considered to be a good source of cells. Among them, mesenchymal stem cells are a type of pluripotent stem cells located in the mesoderm. They can differentiate into a variety of tissue cells under specific induction conditions in vivo or in vitro. They have the potential to promote regeneration and immunoregulation, and they can still self-renew and multidirectionally differentiate after continuous subculture and cryopreservation. Mesenchymal stem cells have low immunogenicity. Even if they are used between allogeneic cells, they can be introduced into the host through various pathways without causing immune rejection reactions in the host, greatly reducing the probability of graft-versus-host disease caused by allogeneic transplantation. Placental-derived mesenchymal stem cells have the advantages of simple separation methods, rich sources, and no ethical restrictions, and have gradually become one of the advantageous cells for stem cell transplantation therapy. Utilizing the regenerative and immunoregulatory properties of cells, mesenchymal stem cells are expected to become a potential supplementary or alternative treatment strategy for Crohn's disease, especially for patients with poor response to traditional therapies.

[0004] At present, the assessment and diagnosis of Crohn's disease mainly rely on a combination of clinical symptoms, endoscopic examination, tissue biopsy, imaging examination and other methods. Metabolomics is one of the key technologies for exploring disease mechanisms, finding disease diagnosis and judging therapeutic efficacy by qualitatively and quantitatively detecting all small molecule metabolites in biological samples and finding the relative relationship between metabolites and physiological and pathological changes in the body. The present invention combines isotope-labeled liquid chromatography-mass spectrometry technology to find the changes in metabolites before and after mesenchymal stem cell treatment through metabolomics, screen the differential metabolites and evaluate their efficacy. Summary of the invention

[0005] The first object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a kit for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease by using kynurenine and formyl-5-hydroxykynurenamine as serum markers.

[0006] The present invention adopts the following technical solutions:

[0007] The invention discloses the use of kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenylethylene glycol as serum biomarkers in the preparation of a product for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease.

[0008] Preferably, the product comprises a test kit, a chip or a test paper.

[0009] Preferably, the kit uses serum as the detection sample.

[0010] Preferably, the chip comprises a solid phase carrier, and probes attached thereto that specifically recognize kynurenine, formyl-5-hydroxykynurenamine and 3-methoxy-4-hydroxyphenylethylene glycol.

[0011] The second object of the present invention is to provide a kit for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease, wherein the kit comprises reagents for detecting kynurenine, formyl-5-hydroxykynurenamine and 3-methoxy-4-hydroxyphenylethylene glycol.

[0012] The third object of the present invention is to provide a reagent for detecting kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenylethylene glycol for use in preparing a product for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease.

[0013] Preferably, the product includes a test kit, a chip and a test paper.

[0014] The present invention is achieved by detecting serum metabolites of Crohn's disease mice based on isotope-labeled liquid chromatography-mass spectrometry technology, and comprises the following steps:

[0015] (1) Mesenchymal stem cell transplantation for the treatment of Crohn's disease in mice

[0016] In this invention, 200 μL phosphate buffered saline (PBS) was intraperitoneally injected into 24-week-old female SAMP1 / Yit mice as the model group, and 2×10 6 MSCs / 200 μL PBS was used as the treatment group, and AKR / J mice of the same age were intraperitoneally injected with 200 μL PBS as the normal control group; peripheral serum of mice was collected on the 0th, 3rd, 7th and 14th days of the experiment for later use;

[0017] (2) Extraction of serum metabolites

[0018] The collected serum was divided into two parts, one as an independent sample, and the other was mixed evenly as a mixed sample; serum: methanol = 1:3 (volume ratio, v / v) was mixed to extract metabolites; metabolites in the supernatant were collected by centrifugation, and freeze-dried in a freeze dryer for later use;

[0019] (3) 12 C / 13 C-isotope labeling

[0020] Take out the lyophilized metabolites, add pure water to dissolve, add acetonitrile and sodium carbonate / sodium bicarbonate buffer; add 12 Independent serum samples were labeled with C-dansyl marker. 13 The mixed serum sample is labeled with a C-dansyl marker; sodium hydroxide and formic acid / water solution are then added to neutralize the reaction and prepare for quantification;

[0021] (4) Liquid chromatography-ultraviolet spectroscopy (LC-UV) quantification

[0022] In order to ensure that the total concentration of each sample was the same during liquid chromatography-mass spectrometry analysis, the metabolites were quantitatively operated based on liquid chromatography-ultraviolet spectroscopy, and the temperature, injection volume, composition and ratio of the mobile phase, and the steepness of the elution gradient of the sample manager were set to achieve optimal separation between the various chromatographic peaks;

[0023] (5) Liquid chromatography-mass spectrometry (LC-MS) analysis

[0024] According to the LC-UV quantitative results, 13 C-labeled mixed samples were used as unified internal standard reference samples, so that each 12 The C-labeled samples were mixed with the samples in equal molar amounts, and the final volume and concentration of each test sample were set to be the same; the test samples were randomly arranged and loaded onto the machine;

[0025] (6) Data processing and screening of differential metabolites

[0026] The raw data generated by the LC-MS run were preprocessed using IsoMS software to obtain a matrix table containing retention time, mass-to-charge ratio, and peak intensity ratio;

[0027] The matrix table was imported into SIMCA 14.1 software and Metaboanalyst metabolomics online analysis platform for multivariate statistical analysis. Metabolic data were analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Differential metabolites were screened by metabolite difference fold analysis. Metabolites were identified based on the DNS-dansyl standard database and MycompoundID database, and differential metabolic pathways were analyzed based on the KEGG database.

[0028] Furthermore, biomarkers related to the effectiveness of MSCs in treating Crohn's disease mice were screened out, and the receiver operating characteristic (ROC) curve was drawn using Graphpad 9.0 software, and the area under the curve (AUC) of the markers was calculated to evaluate the diagnostic efficacy of the biomarkers.

[0029] Description of the invention principle:

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention discloses the use of kynurenine, formyl-5-hydroxykynurenamine and 3-methoxy-4-hydroxyphenylglycol as serum biomarkers in the preparation of a product for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease, and a method based on isotope-labeled liquid chromatography-mass spectrometry to evaluate the changes in serum metabolites of mice with Crohn's disease treated with mesenchymal stem cells. The serum metabolites are successfully extracted by adopting a new technical means, and the changes in serum metabolites before and after the mesenchymal stem cells are treated with SAMP1 / Yit mice are detected, serum metabolites related to the effectiveness of the mesenchymal stem cells in treating Crohn's disease are screened out and their value is evaluated, thereby providing data support for the clinical transformation of the mesenchymal stem cells in treating Crohn's disease.

[0032] The present invention screens serum differential metabolites related to the effectiveness of MSCs in treating Crohn's disease from the perspective of metabolomics, provides new potential markers for evaluating the effectiveness of mesenchymal stem cell transplantation in treating Crohn's disease, and helps to deeply understand the metabolic changes of Crohn's disease and the effects of mesenchymal stem cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the PCA score plot of serum metabolic profile analysis.

[0034] Figure 2 OPLS-DA score diagrams of model group and model group, treatment group and control group, where (a) is the model group and control group, and (b) is the model group and treatment group.

[0035] Figure 3 This is the bubble diagram of metabolic pathway enrichment of differential metabolism between the model group and the treatment group.

[0036] Figure 4 The scatter plots show the concentration changes of two differential metabolites in different groups, where (a) is Kynurenine, (b) is Formyl-5-hydroxykynurenamine, and (c) is 3-Methoxy-4-hydroxyphenylethyleneglycol.

[0037] Figure 5 This is the ROC curve of kynurenine.

[0038] Figure 6 This is the ROC curve of formyl-5-hydroxykynurenamine.

[0039] Figure 7 This is the ROC curve of 3-methoxy-4-hydroxyphenylethylene glycol.

[0040] Figure 8 The combined ROC curve of the three differential metabolites. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0042] First of all, it should be noted that the placenta-derived mesenchymal stem cells used in the present invention are derived from the sample bank of the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases of the First Affiliated Hospital of Zhejiang University. The present invention does not involve the operation of removing or resecting placental tissue.

[0043] The technical solution of the present invention is described in detail below in conjunction with specific embodiments:

[0044] (I) Mesenchymal stem cell transplantation for the treatment of Crohn's disease mice:

[0045] Ten 24-week-old female SAMP1 / Yit mice were injected intraperitoneally with 200 μL PBS as the model group, and 20 mice were injected intraperitoneally with 2×10 6 MSCs / 200 μL PBS was used as the treatment group, and 10 AKR / J mice of the same age were intraperitoneally injected with 200 μL PBS as the normal control group and observed for 2 weeks;

[0046] On day 0, day 3, day 7, and day 14, 300 μL of peripheral blood was collected from mice through the orbital venous plexus, allowed to stand at room temperature for 1 hour, and then centrifuged at 3500 rpm for 15 minutes at 4°C to obtain serum for later use;

[0047] (II) Serum metabolomics analysis

[0048] (1) Serum metabolite extraction

[0049] The serum was divided into two parts, 30 μL of one part was taken as an independent sample (Individual sample), 90 μL of methanol was added, vortexed for 30 seconds to mix, and placed in a -80°C environment for reaction for 30 minutes; 10 μL of serum was taken from the other part, mixed as a mixed sample, and the corresponding volume of methanol was added according to the ratio of serum: methanol = 1:3 (v / v), vortexed for 30 seconds to mix the solution, and then placed in a -80°C environment for reaction for 30 minutes; after the reaction, the solution was centrifuged at 15,000 rpm at 4°C for 30 minutes, and the supernatant was collected; the supernatant was freeze-dried in a freeze dryer to obtain a freeze-dried sample containing serum metabolites.

[0050] (2) 12 C / 13 C-isotope labeling

[0051] Take out the freeze-dried sample, add 25 μL of pure water to resuspend, add 12.5 μL of sodium carbonate / sodium bicarbonate buffer to the resuspended sample solution, vortex mix; then add 37.5 μL of acetonitrile and dansyl marker mixed solution, vortex mix, and put the reaction system in an oven at 40°C for 45 minutes. 12 C-dansyl marker was used to label independent samples. 13 C-dansyl marker was used to label the mixed sample; after the incubation, the sample was taken out of the oven and cooled to room temperature, 7.5 μL of sodium hydroxide was added, and then placed in an oven at 40°C for 10 minutes, and finally 30 μL of 5% formic acid / water solution was added to neutralize the reaction;

[0052] (3) Liquid chromatography-ultraviolet spectroscopy (LC-UV) quantification

[0053] In this experiment, an ultra-high performance liquid chromatograph equipped with a photodiode array detector and a 338 nm ultraviolet detector was used, and the chromatographic column used was an ACQUITY UPLC BEH C18 reversed-phase column (2.1 mm × 5 cm, particle size: 1.7 μm; pore size: ), set the liquid chromatography conditions as follows:

[0054] Sample manager temperature: 4°C;

[0055] Injection volume: 1 μL;

[0056] Flow rate: 500 μL / min;

[0057] Composition of mobile phase:

[0058] Mobile phase A: formic acid / water (0.1:99.9, v / v);

[0059] Mobile phase B: formic acid / acetonitrile (0.1:99.9, v / v);

[0060] Gradient elution program:

[0061] The initial setting is 95% A, 5% B, which is maintained for 1 minute and then changed to 2% A, 98% B within 0.01 minute; after maintaining for 1 minute, it is changed to 95% A, 5% B within 30 seconds and maintained for 3.5 minutes.

[0062] (4) Liquid chromatography-mass spectrometry (LC-MS) analysis

[0063] According to the LC-UV quantitative results, take 10 μL 13 C-labeled mixed samples were used as unified internal standard reference samples, and each 12 The C-labeled samples were mixed with the samples in equal molar amounts. After mixing, the total molar concentration was set to 0.49 mmol / L, and the final volume of each sample was 25 μL.

[0064] The samples were analyzed by LC-MS using an UltiMate 3000 UHPLC coupled to an Impact II quadrupole time-of-flight mass spectrometer. All MS spectra were acquired in positive ion mode at a spectral acquisition rate of 1 Hz. The liquid chromatography conditions were set as follows:

[0065] Flow rate: 400 μL / min

[0066] Composition of mobile phase:

[0067] Mobile phase A: formic acid / water (0.1:99.9, v / v);

[0068] Mobile phase B: formic acid / acetonitrile (0.1:99.9, v / v);

[0069] Gradient elution program:

[0070] The initial setting was 75% A and 25% B, changed to 1% A and 99% B after 10 minutes, maintained for 3 minutes, changed to 75% A and 25% B, and maintained for 3 minutes.

[0071] Before testing the samples, repeat the quality control samples for at least 5 times to examine the stability of LC-MS chromatographic peak separation and mass spectrometry signal response; during the test, a blank sample and a 12 C-labeled mixed amino acid standard sample and a quality control sample were loaded in duplicate;

[0072] (5) Data analysis and processing and screening of differential metabolites

[0073] A total of 1842 peak pairs were detected in this experiment. The raw data generated by the LC-MS run were preprocessed using IsoMS software. After peak collection, peak pairing, peak filtering, and using a zero-fill program to replace missing values, IsoMS-Quant determined 12 C- / 13 The peak intensity ratio of C- was used to obtain a matrix table containing retention time, mass-to-charge ratio and peak intensity ratio; 1369 metabolites were identified after matching with the DNS-standard database and MycompoundID database based on the precise mass-to-charge ratio and retention time.

[0074] The matrix table was imported into SIMCA 14.1 software and Metaboanalyst metabolomics online analysis platform for multivariate statistical analysis. First, PCA analysis was performed on all sample data including QC samples ( Figure 1 ), and then perform OPLS-DA analysis on the data between different groups ( Figure 2 ), to obtain the overall trend of changes and verify the reliability of LC-MS detection; set the difference multiple>1.2 and P<0.05 to screen the differential metabolites, and match them in the database to obtain 66 differential metabolites between the treatment group and the model group; based on the KEGG database to analyze the differential metabolic pathways, the differential metabolites were mainly enriched in the tryptophan metabolic pathway ( Figure 3 ); after eliminating the differential metabolites not related to treatment, three differential metabolites related to MSCs treatment were obtained, including kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenylethylene glycol ( Figure 4 );

[0075] The ROC curve was used to analyze the diagnostic efficiency of metabolite concentration changes for Crohn's disease. The AUC value of the ROC curve for kynurenine was 0.7526 ( Figure 5 ), the ROC curve AUC value of formyl-5-hydroxykynurenine was 0.8289 ( Figure 6 ), the ROC curve AUC value of 3-methoxy-4-hydroxyphenylethylene glycol was 0.8263 ( Figure 7 ), the joint dataset of the three metabolites was randomly divided into separate training and test sets (70% for training and 30% for testing), and classified using leave-one-out cross-validation. The AUC value of the joint ROC curve of the three metabolites was 0.8646 ( Figure 8 ). These results indicate that these three metabolites have good value in evaluating the effectiveness of MSCs in treating Crohn's disease.

Claims

1. The use of kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenylethylene glycol as serum biomarkers in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease.

2. The use according to claim 1, characterized in that: The product includes a test kit, a chip or a test paper.

3. The use according to claim 2, characterized in that: The kit uses serum as a test sample.

4. The use according to claim 2, characterized in that: The chip comprises a solid phase carrier and probes attached thereto for specifically recognizing kynurenine and formyl-5-hydroxykynurenamine.

5. A kit for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease, characterized in that: The kit comprises reagents for detecting kynurenine, formyl-5-hydroxykynurenamine and 3-methoxy-4-hydroxyphenylethylene glycol.

6. Use of reagents for detecting kynurenine, formyl-5-hydroxykynurenine and 3-methoxy-4-hydroxyphenylethylene glycol in the preparation of products for evaluating the therapeutic effect of mesenchymal stem cells on Crohn's disease.

7. The use according to claim 6, characterized in that: The product includes a test kit, a chip or a test paper.

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