Application of POSTN protein in the preparation of diagnostic and therapeutic products for Treg cell disorder diseases

Abstract

The present invention relates to the field of biomedicine technology, and in particular to the use of POSTN protein in the preparation of diagnostic and therapeutic products for Treg cell disorder diseases, which include one or more diseases of tumors, autoimmune diseases, chronic infections and chronic inflammation. Cell experiments have shown that POSTN protein can regulate the differentiation of initial CD4 cells into Treg cells, and the results of bioinformatics analysis show that POSTN protein is highly expressed in the tumor microenvironment. The above results suggest that the secretion of Treg cells can be stimulated by POSTN protein, or a pharmaceutical preparation can be prepared using POSTN protein in the tumor microenvironment as an identification target, or a diagnostic product can be prepared using POSTN protein biomarkers in the tumor microenvironment. POSTN protein is expected to be used in the preparation of diagnostic and therapeutic products for Treg cell disorder diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of POSTN protein in preparing diagnostic and therapeutic products for Treg cell disorder diseases. Background Art

[0002] Treg cells are a type of T cell subset that controls the body's autoimmune reactivity. They are produced by the thymus and exported to the periphery. They inhibit the activation and proliferation of potential autoreactive T cells through active regulation, thereby maintaining the body's immune tolerance. An increase or decrease in Treg cell secretion usually means an increase or decrease in the number of Treg cells in the immune system. Changes in their secretion may be related to a variety of factors, including autoimmune diseases, chronic infections, tumors, and chronic inflammation. In autoimmune diseases, the increase in Treg cells may be an attempt by the body to suppress excessive immune responses; in chronic infections or tumor environments, Treg cells may be activated to inhibit attacks on pathogens or tumor cells, thereby contributing to the survival of pathogens or tumor cells; some chronic inflammations may lead to an increase in the number of these cells, such as rheumatoid arthritis, rheumatoid arthritis, etc.; certain drugs or treatments may also affect the number of Treg cells, and long-term use of immunosuppressive drugs or certain hormone treatments may also lead to an increase in the number of Treg cells. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the present invention aims to provide a use of POSTN protein in the preparation of a diagnostic and therapeutic product for Treg cell disorder diseases.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] Application of POSTN protein in the preparation of diagnostic and therapeutic products for Treg cell disorder diseases, Treg cell disorder diseases include one or more diseases among tumors, autoimmune diseases, chronic infections and chronic inflammations, and Treg cell disorder refers to increased secretion of Treg cells or decreased secretion of Treg cells. Treg cells are one of the important factors in maintaining the body's immune tolerance. They are produced by the thymus and then exported to the periphery, and inhibit the activation and proliferation of potential autoreactive T cells in the normal body through active regulation, thereby regulating the body's immunity. The reduction or abnormal function of CD4, CD25 and FOXP3 cytokines may lead to the occurrence of autoimmune diseases. The number and function of regulatory T cells in some autoimmune diseases such as multiple sclerosis, active rheumatoid arthritis, type I diabetes, etc. will change. In many malignant diseases such as lung, pancreatic and breast invasive cancer, Treg cells have been found to be significantly increased. Treg cells can also block anti-tumor immune responses, thereby increasing mortality, and can also be used for tracking before and after bone marrow transplantation.

[0006] Furthermore, the POSTN protein is derived from artificial synthesis and / or CAFs in the microenvironment;

[0007] Among them, CAF is a tumor-associated fibroblast.

[0008] Furthermore, CAFs are derived from one or more of iCAFs, pericytes, smooth muscle cells, meCAFs, mCAFs and pCAFs.

[0009] Furthermore, the tumor includes one or more of invasive breast cancer, colorectal cancer, ovarian cancer and liver cancer.

[0010] Furthermore, diagnostic products include diagnostic products and / or therapeutic products.

[0011] Furthermore, the diagnostic product uses POSTN protein as a biomarker.

[0012] Furthermore, the diagnostic product includes a diagnostic reagent and / or a diagnostic kit.

[0013] Furthermore, the therapeutic product includes a pharmaceutical preparation that uses the POSTN protein in the microenvironment as a recognition target or a pharmaceutical preparation that uses the POSTN protein as an active ingredient.

[0014] Furthermore, the pharmaceutical preparation using POSTN protein as the recognition target is an anti-tumor pharmaceutical preparation; the pharmaceutical preparation using POSTN protein as the active ingredient is for autoimmune diseases, chronic infections or chronic inflammatory diseases.

[0015] Furthermore, the pharmaceutical preparation includes pharmaceutically acceptable adjuvants, excipients or carriers.

[0016] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0017] The present invention provides the use of POSTN protein in the preparation of diagnostic and therapeutic products for Treg cell disorder diseases, including one or more diseases of tumors, autoimmune diseases, chronic infections and chronic inflammation. Cell experiments have shown that POSTN protein can regulate the differentiation of initial CD4 cells into Treg cells, and bioinformatics analysis results show that POSTN protein is highly expressed in the tumor microenvironment. The above results suggest that the secretion of Treg cells can be stimulated by POSTN protein, or diagnostic products can be prepared by using POSTN protein as a biomarker for the diagnosis of diseases related to Treg cell disorder.

[0018] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The correlation between POSTN protein expression and TIL cell types in the tumor microenvironment was analyzed by Spearman correlation as provided in Example 1 of the present invention.

[0021] Figure 2 This is a result diagram of analyzing the expression of POSTN protein in pan-cancer tissues and normal tissues using TCGA data and gepia2 online software provided in Example 2 of the present invention.

[0022] Figure 3 This is the expression of POSTN protein in different cancers provided in Example 3 of the present invention.

[0023] Figure 4 This is the expression of POSTN protein in different subpopulations of CAF cells provided in Example 3 of the present invention.

[0024] Figure 5 This is a flow cytometric graph of the regulation of Treg cells by the POSTN protein provided in Example 4 of the present invention.

[0025] Figure 6This is a statistical chart showing the effect of the POSTN protein provided in Example 4 of the present invention on the secretion of cytokines by Treg cells. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention. Some terms and abbreviations used in the present invention are listed below.

[0027] Regulatory T cells: regulatory cell, Treg cells;

[0028] Cancer associated fibroblast: cancer associated fibroblast, CAF;

[0029] Adrenocortical carcinoma: adrenocortical carcinoma, ACC;

[0030] Bladder urothelial carcinoma: BLCA;

[0031] Breast invasive carcinoma: BRCA;

[0032] Cervical squamous cell carcinoma and endocervical adenocarcinoma: CESC;

[0033] Cholangiocarcinoma: CHOL;

[0034] Colon adenocarcinoma: Colon adenocarcinoma, COAD;

[0035] Esophageal carcinoma: ESCA;

[0036] Glioblastoma multiforme: GBM;

[0037] Head and Neck squamous cell carcinoma: Head and Neck squamous cell carcinoma, HNSC;

[0038] Kidney Chromophobe, KICH;

[0039] Kidney renal clear cell carcinoma: Kidney renal clear cell carcinoma, KIRC;

[0040] Kidney renal papillary cell carcinoma: KIRP;

[0041] Brain Lower Grade Glioma: LGG;

[0042] Liver cancer: Liver hepatocellular carcinoma, LIHC;

[0043] Lung adenocarcinoma: LUAD;

[0044] Lung squamous cell carcinoma: LUSC;

[0045] Mesothelioma: Mesothelioma, MESO;

[0046] Ovarian serous cystadenocarcinoma: OV or OVCA;

[0047] Pancreatic adenocarcinoma, PAAD;

[0048] Pheochromocytoma and Paraganglioma: PCPG;

[0049] Prostate adenocarcinoma, PRAD;

[0050] Rectal adenocarcinoma: Rectum adenocarcinoma, READ;

[0051] Sarcoma: Sarcoma, SARC;

[0052] Skin Cutaneous Melanoma: Skin Cutaneous Melanoma, SKCM;

[0053] Stomach cancer: Stomach adenocarcinoma, STAD;

[0054] Testicular Germ Cell Tumors, TGCT;

[0055] Thyroid carcinoma: THCA;

[0056] Endometrial cancer: Uterine Corpus Endometrial Carcinoma, UCEC;

[0057] Uterine Carcinosarcoma: Uterine Carcinosarcoma, UCS;

[0058] Uveal Melanoma: UVM;

[0059] Colorectal Cancer: CRC;

[0060] Adoptive T cell therapy: Adoptive Cell Therapy, ACT;

[0061] Effector memory T Cell: Effector memory T Cell, TEM;

[0062] Central Memory T Cells, Tcm;

[0063] Follicular Helper T Cell: Follicular Helper T Cell, Tfh;

[0064] Tumor infiltrating lymphocyte: tumor infiltrating lymphocyte, TIL;

[0065] T helper cell 1: T helper cell 1, Th1;

[0066] T helper cell 2: T helper cell 2, Th2;

[0067] T helper cell 17: T helper cell 17, Th17;

[0068] Immature B cells: Immature B, imm B;

[0069] Memory B cell: Memory B cell, Mem B;

[0070] CD56 high-expressing cells: CD56bright;

[0071] CD56 low expression cells: CD56dim;

[0072] Myeloid-derived suppressor cells, MDSC;

[0073] Natural killer cell: Natural killer cell, NK;

[0074] Natural killer T cell: Natural killer T cell, NKT;

[0075] Plasmacytoid dendritic cell: Plasmacytoid dendritic cell, pDC;

[0076] Interdigitating Dendritic Cell: iDC;

[0077] Diffuse large B-cell lymphoma: Lymphoid Neoplasm Diffuse Large B-cell lymphoma, DLBC;

[0078] Smooth muscle cell: smooth-muscle-cell, SMC;

[0079] Peripheral blood mononuclear cell: peripheral blood mononuclear cell, PBMC;

[0080] The Cancer Genome Atlas, TCGA;

[0081] Forward scatter: Forward Scatter, FSC;

[0082] Side scatter: Side Scatter, SSC;

[0083] Side scatter area identifies lymphocytes and / or monocytes: Side Scatter Area, SSC-A;

[0084] Forkhead box protein P3: Forkhead box protein P3, FOXP3;

[0085] 2-Hydroxyethylpiperazineethanesulfonic acid: 2-hydroxyethyl, HEPES;

[0086] Tumor microenvironment: The occurrence and development of malignant tumors is a dynamic process in which tumor cells interact and evolve together with their microenvironment. The tumor microenvironment is composed of extracellular matrix, stromal cells (fibroblasts, immune cells, inflammatory cells, endothelial cells and bone marrow-derived cells), cytokines and chemokines. The tumor microenvironment can promote or hinder anti-tumor immune responses.

[0087] CAF: It is the most important component of tumor stromal cells in the tumor microenvironment. It can secrete a variety of growth factors, cytokines and extracellular matrix. These factors play a vital role in promoting the occurrence, proliferation, angiogenesis, invasion and metastasis of tumors. The unique metabolic mode of CAF provides energy for the progression of tumors.

[0088] TISIDB: is an online database on tumor-immune system interactions developed by the University of Hong Kong. The database integrates multiple types of data resources, including literature mining results, high-throughput screening data, exome and RNA sequencing data of patient cohorts, genomics and transcriptomics data from TCGA, and public databases such as UniProt, GO, and DrugBank.

[0089] FSC: represents the size of the cell. The larger the cell volume, the higher the FSC value. Through the FSC value, the size of the cell can be preliminarily compared, and the cell grouping and classification can be performed. For example, the volume difference between lymphocytes, monocytes and granulocytes can be distinguished by the FSC value. ‌FSC-H stands for the height of the forward angle scatter signal (Height) in flow cytometry‌, and ‌FSC-A is the area (Area) parameter of the forward scattered light in flow cytometry, which is proportional to the cell size.

[0090] SSC: represents the granularity of the cell. The more complex the structure inside the cell, the higher the SSC value. The SSC value is related to the granularity and complexity inside the cell, and is often used in conjunction with FSC to further subdivide the cell population. The internal structural characteristics of the cell can be further analyzed by the SSC value. In flow cytometry, FSC and SSC are often used to analyze the physical properties of cells. Data analysis software can classify cell populations by characteristics and calculate the proportion of each population. For example, CD4 and CD8 markers can be used to distinguish T helper cells and T killer cells, and their proportions in the sample can be calculated. This is important for evaluating immune status or disease progression.

[0091] CD4: The full name is CD4-T lymphocyte, which is a cytokine on the surface of T cells. It is an important immune cell in the human immune system. CD4 is an indicator that directly reflects the body's immunity.

[0092] Tgd: is a new antigen-specific immunoregulatory T lymphocyte.

[0093] CD25: The CD25 molecule is the α chain of IL-2R and is considered to be a characteristic marker molecule on the surface of Treg cells.

[0094] FOXP3: It is a signature molecule of Treg cells. FOXP3 is a transcriptional regulatory factor that is essential for the development and suppressive function of Treg cells. FOXP3 is a nuclear protein specifically expressed by Treg cells, which is essential for the development and immunosuppressive function of Treg cells. FOXP3 mutations can cause severe, fatal multi-organ autoimmune diseases. FOXP3 regulates the stability and suppressive function of Treg cells by interacting with other transcription factors and factors such as histone acetylases and deacetylases.

[0095] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0096] Example 1 Analysis of the correlation between POSTN protein and Treg cells in pan-cancer tissues.

[0097] Using bioinformatics tools (TISIDB, http: / / cis.hku.hk / TISIDB / ), we found that POSTN protein has a strong correlation with Treg cells in cancer tissues, such as Figure 1 As shown, the results showed that Treg cells are correlated with TIL cells in various tumor microenvironments;

[0098] Among them, these tumors include ACC, BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, UCEC, UCS, and UVM.

[0099] Figure 1 In the above, Tgd is γδT cell, Imm B is immune B cell, Mem B is memory B cell, NK is natural killer cell, Macrophage is macrophage, Eosinophil is eosinophil, Mast is mast cell, monocyte is monocyte, and Neutrophil is neutrophil.

[0100] Example 2 Analysis of POSTN protein expression in tumor tissues.

[0101] TCGA data and gepia2 online software were used to analyze the expression of POSTN in pan-cancer tissues and normal tissues. Figure 2 As shown, the results showed that the expression level of POSTN protein in BLCA, BRCA, CHOL, DLBC, ESCA, GBM, HNSC, KIRC, LUAD, LUCS, OV, PAAD, PCPG, PRAD, READ, READ, SARC, STAD, TGCT and UCS tumor tissues was higher than that in normal tissues.

[0102] Figure 2 In , TPM is the abbreviation of Transcripts Per Million, which is a method used to standardize gene expression, indicating the number of specific gene transcripts per million transcripts.

[0103] Example 3 Determination of POSTN protein and CAF cells in tumor tissues.

[0104] Download tumor tissue single-cell sequencing data (GSA-Human: HRA004998), use R's seruat package to integrate the data, and further analyze the expression of POSTN protein in different cancers and the expression of POSTN protein in different subpopulations of CAF cells, such as Figure 3 and Figure 4 As shown, the results showed that POSTN protein was highly expressed in BRCA, CRC, LIHC and OVCA cancers, and POSTN protein was highly expressed in different CAF subpopulations iCAF, MeCAF, mCAF and pCAF.

[0105] Example 4 Determination of the regulation of Treg cells by POSTN protein.

[0106] Use 1μg / ml (diluted with PBS buffer solution) of purified anti-human CD3e and purified anti-human CD28 to pre-plate in 96-well plates, 50μl / well. Use the naïve CD4+ T cells isolation kit to sort the naïve CD4+ T cells in PBMCs, inoculate them into 96-well plates for stimulation and induction, and plate 1×10 per well. 5Naïve CD4+ T cells were cultured in 200 μl of 1640 complete culture medium per well. Cytokines IL-2 (5 ng / ml) and TGFβ (3 ng / ml) were added to induce Treg cell differentiation. POSTN protein (1 μg / ml) was added to the experimental group, and an equal volume of PBS solution was added to the control group. After induction culture for 4 days, cells were collected and the proportion of CD4+CD25+FOXP3+ cells was detected by flow cytometry. The results of flow cytometry are shown in Figure 2. Figure 5 As shown, the statistics of CD4+CD25+FOXP3+ results in cells are as follows Figure 6 As shown, the results show that POSTN protein can regulate the differentiation of initial CD4 into Treg cells. In the figure, CTRL group is the PBS solution blank control group, POSTN group is the POSTN protein test group, and CTRL is the abbreviation of CONTROL.

[0107] Among them, the formula composition of 1640 complete culture medium is as follows:

[0108] 1640 culture medium 800ml, calf serum 200ml, 200mmol-glutamine solution 10ml, 1mol HEPES 10ml, penicillin (10000u / ml) 10ml, streptomycin (10000μg / ml) 10ml, 100mmol sodium pyruvate 10ml, 0.01mol 2-mercaptoethanol 1ml.

[0109] Aseptically add the remaining ingredients one by one to the 1640 culture medium, mix well, and adjust the pH to 7.0 with 7% to 10% NaHCO3.

[0110] The above results suggest that POSTN protein can be used as an active ingredient to prepare pharmaceutical preparations to stimulate the increase in secretion of Treg cells for diseases with reduced Treg cell secretion, or the increase and / or decrease in secretion of Treg cells in the microenvironment can be monitored by detecting the level of POSTN protein, or targeted anti-tumor drugs can be prepared using POSTN protein as an identification target. Therefore, POSTN protein is expected to be used in the preparation of diagnostic and therapeutic products for diseases with dysregulated Treg cells.

[0111] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of POSTN protein in the preparation of a diagnostic and therapeutic product for Treg cell disorder diseases, characterized in that: The Treg cell disorder disease is selected from one or more of the following diseases: tumors with abnormal CD4+CD25+FOXP3 ratio in PBMC, autoimmune diseases, chronic infections and chronic inflammations.

2. The use of the POSTN protein according to claim 1 in preparing a product for diagnosing and treating Treg cell disorder diseases, characterized in that: The POSTN protein is derived from artificial synthesis and / or CAF in the microenvironment; Among them, CAF is a tumor-associated fibroblast.

3. The use of the POSTN protein according to claim 2 in the preparation of a diagnostic and therapeutic product for a Treg cell disorder disease, characterized in that: CAFs are derived from one or more of iCAFs, meCAFs, mCAFs and pCAFs.

4. The use of the POSTN protein according to claim 1 in preparing a product for diagnosing and treating a Treg cell disorder disease, characterized in that: The tumor includes one or more of invasive breast cancer, colorectal cancer, ovarian cancer and liver cancer.

5. The use of the POSTN protein according to claim 1 in preparing a product for diagnosing and treating a Treg cell disorder disease, characterized in that: Diagnostic and therapeutic products include diagnostic products and / or therapeutic products.

6. The use of the POSTN protein according to claim 5 in preparing a product for diagnosing and treating a Treg cell disorder disease, characterized in that: The diagnostic product is a biomarker.

7. The use of the POSTN protein according to claim 5 in preparing a product for diagnosing and treating a Treg cell disorder disease, characterized in that: The diagnostic products include diagnostic reagents and / or diagnostic kits.

8. The use of the POSTN protein according to claim 5 in preparing a product for diagnosing and treating a Treg cell disorder disease, characterized in that: The therapeutic product includes a pharmaceutical preparation that uses the POSTN protein in the microenvironment as a recognition target or a pharmaceutical preparation that uses the POSTN protein as an active ingredient; The pharmaceutical preparation using POSTN protein as the recognition target is an anti-tumor pharmaceutical preparation; the pharmaceutical preparation using POSTN protein as the active ingredient is for autoimmune diseases, chronic infections or chronic inflammatory diseases.

9. The use of the POSTN protein according to claim 8 in preparing a product for diagnosing and treating a Treg cell disorder disease, characterized in that: The pharmaceutical preparation includes pharmaceutically acceptable auxiliary materials, excipients or carriers.

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