Application of vascular cell adhesion molecule 1 in preparation of product for regulating and controlling formation of cell internalization structure
By using VCAM-1 to regulate the formation of cell internalized structure, the problem of lack of cell internalized structure regulation in ITP treatment is solved, and the potential therapeutic effect of ITP is improved and the side effects are reduced.
Patent Information
- Application Number
- CN202510104081.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-05-30
AI Technical Summary
There are currently no effective products or methods that can regulate the formation of internalized cellular structures, especially in the treatment of immune thrombocytopenia (ITP), where existing treatments have problems with bleeding risks and long-term side effects.
Products that regulate the formation of cell internalized structures were developed by using vascular cell adhesion molecule 1 (VCAM-1) as a key molecule to regulate the formation of cell internalized structures. The product includes agents that reduce or overexpress VCAM-1 to inhibit or promote the formation of internalized structures of cells.
By regulating the expression level of VCAM-1, the process of internalizing platelets in macrophages can be effectively regulated, thereby potentially improving the therapeutic effect of ITP, reducing the risk of bleeding, and reducing the side effects of long-term use of hormones and immunosuppressants.
Smart Images
Figure CN120053600A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of biomedical technology, and particularly relates to the application of vascular cell adhesion molecule 1 in the preparation of products for regulating the formation of cell internalization structures. Background Art
[0002] Immune Thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disease in children. The incidence of childhood ITP is about 5-10 per 100,000. The main clinical manifestations are: a decrease in peripheral blood platelet count (<100×10 9 / L), petechiae and ecchymoses on the skin and mucous membranes, and gingival bleeding, etc. In severe cases, visceral or intracranial hemorrhage may occur, even endangering life. Most children with ITP will spontaneously remit within 12 months after diagnosis. However, more than 25% of children with ITP will develop into chronic refractory ITP and show obvious bleeding symptoms, requiring continuous treatment. Even in recent years, with the application of thrombopoietin receptor agonists (TPO-RA) eltrombopag, rituximab, oral immunosuppressants, and splenectomy, there is still a bleeding risk, and the side effects caused by long-term use of hormones and immunosuppressants reduce the quality of life of these children, causing serious social problems and economic burdens. Therefore, it is particularly important to explore the pathogenesis of ITP and develop effective diagnosis and treatment plans to improve the curative effect of children with chronic refractory ITP and improve the quality of life.
[0003] The pathophysiology of ITP has not been fully understood. Currently, it is considered that the imbalance of immune tolerance, the binding of anti-platelet autoantibodies to specific antigens on the platelet surface, and the phagocytosis of platelets by monocytes / macrophages leading to increased platelet destruction are the main causes of ITP. And the internalization of platelets by macrophages is a process of forming cell internalization structures, which may be an important link in the pathogenesis of ITP.
[0004] Currently, there is no record of products for regulating the formation of cell internalization structures. Summary of the Invention
[0005] In view of this, the purpose of this application is to provide the application of vascular cell adhesion molecule 1 in the preparation of products for regulating the formation of cell internalization structures.
[0006] This application provides the application of vascular cell adhesion molecule 1 in the preparation of products for regulating the formation of cell internalization structures.
[0007] Preferably, the application includes the application of a reagent for reducing the expression level of vascular cell adhesion molecule 1 in the preparation of products for inhibiting the formation of cell internalization structures.
[0008] Preferably, the application includes the use of a reagent overexpressing vascular cell adhesion molecule 1 in the preparation of a product for promoting the formation of cell internalization structures.
[0009] The present application also provides the use of vascular cell adhesion molecule 1 as a detection target in the preparation of a product for detecting the formation of cell internalization structures.
[0010] Preferably, the cell internalization structures include heterogeneous cell internalization structures.
[0011] Preferably, the formation of the heterogeneous cell internalization structures is caused by macrophage internalization of platelets.
[0012] The present application also provides the use of a reagent for reducing the expression level of vascular cell adhesion molecule 1 in the preparation of a drug for treating immune thrombocytopenia.
[0013] The present application also provides the use of vascular cell adhesion molecule 1 as a detection target in the preparation of a product for diagnosing immune thrombocytopenia.
[0014] Preferably, the immune thrombocytopenia is caused by the formation of heterogeneous cell internalization structures.
[0015] Preferably, the immune thrombocytopenia is caused by macrophage internalization of platelets.
[0016] The present application provides the use of vascular cell adhesion molecule 1 (VCAM-1) in the preparation of a product for regulating the formation of cell internalization structures. VCAM-1 is a molecule that regulates cell internalization structures based on the emperitosis model phenotype. In the regulatory role of macrophage internalization of platelets, VCAM-1 can serve as a therapeutic and evaluation target for thrombocytopenia. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is the process of cell internalization structure formation;
[0018] Figure 2 is VCAM-1 regulating the formation of cell internalization structures;
[0019] Figure 3 is macrophage internalization of platelets, granulocytes, and orthochromatic erythroblasts (belonging to red blood cells) to form hemophagocytes; wherein, A is macrophage internalization of platelets, and B is macrophage internalization of orthochromatic erythroblasts, granulocytes, and platelets;
[0020] Figure 4 is the construction of the Dendra2 expression vector;
[0021] Figure 5 is the live cell workstation photographing the calcium flux change during the process of cell internalization to form nested structures. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0022] The present application provides the use of vascular cell adhesion molecule 1 (VCAM-1) in the preparation of a product for regulating the formation of cell internalization structures.
[0023] In the specific implementation process of the present application, the above application includes the use of a reagent for reducing the expression level of vascular cell adhesion molecule 1 in the preparation of a product for inhibiting the formation of cell internalization structures.
[0024] In the specific implementation process of the present application, the above application includes the use of a reagent for overexpressing vascular cell adhesion molecule 1 in the preparation of a product for promoting the formation of cell internalization structures.
[0025] The present application also provides the use of vascular cell adhesion molecule 1 as a detection target in the preparation of a product for detecting the formation of cell internalization structures.
[0026] In the specific implementation process of the present application, it is shown in the VCAM-1 phenotype study that it can regulate the formation of cell internalization structures.
[0027] In the specific implementation process of the present application, the above cell internalization includes cell stacking.
[0028] In the specific implementation process of the present application, the above cell internalization structures include heterogeneous cell internalization structures.
[0029] In the specific implementation process of the present application, the formation of the above heterogeneous cell internalization structures is caused by macrophage internalization of platelets.
[0030] The present application also provides the use of a reagent for reducing the expression level of vascular cell adhesion molecule 1 in the preparation of a drug for treating immune thrombocytopenia.
[0031] The present application also provides the use of vascular cell adhesion molecule 1 as a detection target in the preparation of a product for diagnosing immune thrombocytopenia.
[0032] In the specific implementation process of the present application, the above immune thrombocytopenia is caused by the formation of heterogeneous cell internalization structures.
[0033] In the specific implementation process of the present application, the above immune thrombocytopenia is caused by macrophage internalization of platelets.
[0034] In the specific implementation process of the present application, the above immune thrombocytopenia includes childhood immune thrombocytopenia.
[0035] Next, the technical solutions in the present application will be clearly and completely described in conjunction with the embodiments in the present application.
[0036] Experimental Example 1
[0037] 1. Research content
[0038] 1) To study the mechanism of action of vascular cell adhesion molecule-1 (VCAM-1) in regulating the ability of target cells to internalize effector cells. In the study of VCAM-1 phenotypes, it was shown that VCAM-1 can regulate the formation of cell internalization structures. Further, the dynamic changes in its expression and localization during the formation of cell internalization structures, as well as its regulation of and interaction with the cytoskeleton, are shown in Figure 1 .
[0039] 2) Phenotypic identification of macrophages in patients with idiopathic thrombocytopenic purpura (ITP). Flow cytometry was used to identify the phenotypic characteristics of macrophages in bone marrow mononuclear cells (BMMC) and peripheral blood mononuclear cells (PBMC) of ITP patients. The cells were cultured in vitro under appropriate conditions, and the levels of cytokines secreted into the culture medium were detected by ELISA.
[0040] 3) Mechanism and function study of macrophage internalization of platelets. VCAM-1+ macrophages in BMMC and PBMC of ITP patients were sorted by flow cytometry and cultured in vitro. The process of platelets penetrating into macrophages was photographed using a live cell workstation. The fate of platelets after entering macrophages was observed and analyzed through changes in platelet calcium flux. Target macrophages were sorted by flow cytometry, and the expression level of VCAM-1 was detected. After knocking down / knocking out and overexpressing VCAM-1, the ability of macrophages to internalize platelets was dynamically observed, and small molecule inhibitors were added to analyze the role of VCAM-1 in macrophage internalization of platelets.
[0041] 4) Correlation analysis between VCAM-1 gene expression and different cell internalization processes in tissue specimens from splenectomized ITP patients. The "EML" multi-color immunofluorescence labeling technique was used to classify cell internalization structures in tissue specimens from splenectomized ITP patients, and the expression of target key genes was detected simultaneously.
[0042] 2. Research plan
[0043] 1) Effects and mechanism of action of VCAM-1 on the ability to form cell internalization structures
[0044] Three strategies were adopted to study the effect of VCAM-1 on the formation of cell internalization structures.
[0045] 1) Use RNA interference (RNAi) to knockdown or the CRISPR / Cas9 method to knockout the expression of VCAM-1, and then detect the changes in the cell internalization structures of the corresponding macrophages internalizing platelets;
[0046] 2) Detect changes in the ability to form heterogeneous cell internalization;
[0047] a. Monitoring the fate of effector cells after internalization: Using a live-cell workstation dynamic tracking method to observe whether the fate of effector-target cells changes in response to VCAM-1 gene manipulation (overexpression or knockdown / knockout), and simultaneously analyzing other fates of effector cells, including cell division, escape, redrilling, and no change, etc.; for effector cell death, use Cleaved caspase3 staining to distinguish apoptosis from non-apoptosis. The results are shown in Figure 2 ;
[0048] b. Detecting the subcellular localization and its dynamic changes during the formation of intracellular structures through immunostaining, live-cell dynamic imaging techniques, and methods such as FRET and FRAP, analyzing its relationship with cell membrane surface proteins (E-cadherin, ICAM-1, EGFR, etc.) in intracellular sorting and degradation, and simultaneously analyzing its association and interaction with cytoskeleton remodeling to elucidate the cytological mechanism of its action.
[0049] c. Studying the upstream and downstream relationships, interactions between VCAM-1 and specific candidate molecules during the regulation of intracellular structure formation through genetic manipulation, etc., and their regulation and interrelationships with core components (cytoskeleton and cell-cell junctions).
[0050] 2) Phenotypic identification of macrophages in ITP patients
[0051] a. Collection and processing of specimens. Inclusion criteria: Children with chronic refractory ITP diagnosed in the outpatient and inpatient departments of Xinhua Hospital Affiliated to Shanghai Jiao Tong University. In addition to a thorough medical history and physical examination, all children underwent relevant examinations of bone marrow biopsy and / or aspiration at the time of diagnosis, as well as spleen ultrasound examination and complete blood count. To be included in this study, patients were clearly diagnosed with chronic refractory ITP, and a healthy control group was established. All studies were approved by the ethics committee, and informed consent was obtained from all participants in accordance with the Declaration of Helsinki. The results are shown in Figure 3 .
[0052] b. Phenotypic identification of macrophages in ITP patients. Flow cytometry was used to identify bone marrow mononuclear cells (BMMC) and peripheral blood mononuclear cells (PBMC) in ITP patients, compare them with the normal control group, and sort out different macrophage subtypes for classification and comparison.
[0053] c. Sorting VCAM-1 + macrophages from BMMC and PBMC of ITP patients, culturing them in vitro under appropriate conditions, detecting the changes in the levels of cytokines secreted in the culture medium by ELISA, and labeling VCAM-1 with anti-VCAM-1 647 antibody+ Macrophages, for live cell imaging.
[0054] 3) Mechanism and function study of macrophage internalization of platelets.
[0055] a. Isolation of human and mouse platelets.
[0056] Human whole blood was drawn into a syringe containing 1 / 7 volume of acid - citrate - dextrose (39 mM citric acid, 75 mM sodium citrate, 135 mM dextrose). The whole blood was diluted 1:1 with Tyrode's buffer (pH = 6.5) and centrifuged (RT 70g for 30 min). The supernatant contained platelet - rich plasma (PRP), which was transferred to another centrifuge tube for platelet washing for experiments. Platelets were further diluted and washed with PRP (1:3) and PGI2 buffer (pH = 6.5), and automated cell counting and platelet counting were performed (ES60 ABX Micros, Horiba Medical); Blood was drawn from the hearts of anesthetized (isoflurane), fentanyl (0.05 mg / kg body weight) mice, and the blood was processed according to the method for collecting human platelets. The cells were stained with 4.5 μM CFDA - SE (Molecular Probes, carboxyfluorescein diacetate succinimidyl ester) or 4.5 μM DDAO - SE (CellTrace Far Red, Molecular Probes) for 30 min at room temperature, and then washed with Tyrode's buffer (pH = 6.5).
[0057] b. Dynamically observe the calcium flux changes and their biological responses in platelets.
[0058] Using the traditional chemical reagent dye, the calcium ion indicator Fluo - 4 AM, to observe the changes in Ca 2+ during the process of cell internalization. Before the experiment, macrophages were treated with Fluo - 4 AM, and then co - cultured with platelet cells. Dynamic observation was carried out under a live cell workstation to verify the changes in Ca 2+ flux in vitro. The results are shown in Figure 5 .
[0059] c. Use flow cytometry to sort out VCAM - 1 in BMMC and PBMC of ITP patients + Macrophages were cultured in vitro, and the process of platelets drilling into macrophages was photographed through a live cell workstation. By observing the calcium flux changes in platelets, the fate of platelets after entering macrophages was analyzed. Calcium sparks are generated when cells die, and green fluorescence is enhanced when calcium flux changes.
[0060] d. Sort out target macrophages by flow cytometry, detect the expression level of VCAM-1. After further knocking down / knocking out and overexpressing VCAM-1, dynamically observe the ability of macrophages to internalize platelets, and add small molecule inhibitors to analyze the role of VCAM-1 in macrophage internalization of platelets. Detect VCAM-1 expression by qPCR and / or Western blot.
[0061] e. Use the irreversible monomeric green-to-red fluorescent protein Dendra2 photoconverter and scan with a confocal microscope. Label with anti-VCAM-1 647 and image with a Zeiss LSM880 confocal microscope at 52 hpf to construct a Dendra2 expression vector ( Figure 4 ) and analyze the changes in related molecules during the formation of macrophage internalization of platelets.
[0062] 4) Analysis of the association between VCAM-1 expression and cell internalization structure typing in ITP spleen tissue specimens.
[0063] a. Analysis of the correlation between VCAM-1 expression and cell internalization structure formation in spleen specimens of ITP patients. Use the "EML" multi-color immunofluorescence technique established by the research group in the past (Huang et al, 2015) to simultaneously detect the relationship between VCAM-1 expression and cell internalization structure formation. The analysis indicators include the expression levels, localization of related upstream and downstream molecules such as VCAM-1, CD68, and CD169, and the typing and frequency of cell internalization structures, and analyze them separately with the clinical information of the specimens to clarify the relationship between VCAM-1 expression, cell internalization structure, and ITP prognosis.
[0064] b. Collect specimens of ITP patients treated clinically, detect VCAM-1 expression and cell internalization structure formation, analyze their correlation with the therapeutic efficacy of ITP, select relevant small molecule inhibitors or monoclonal antibodies of immunosuppressants, etc., to explore the impact of the structure on the occurrence, development, and prognosis of ITP patients, and provide new ideas and strategies for enhancing the immunocyte therapy of ITP by targeting VCAM-1 and the formation of cell internalization structures.
[0065] Although the above embodiments have described the present application in detail, they are only a part of the embodiments of the present application rather than all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present application.
Claims
1. Application of vascular cell adhesion molecule 1 in the preparation of products that regulate the formation of cell internalization structures.
2. The use according to claim 1, characterized in that: The application includes the use of an agent for reducing the expression amount of vascular cell adhesion molecule 1 in preparing a product for inhibiting the formation of cell internalization structure.
3. The use according to claim 1, characterized in that: The application includes the use of a reagent that overexpresses vascular cell adhesion molecule 1 in the preparation of a product that promotes the formation of a cell internalization structure.
4. Application of vascular cell adhesion molecule 1 as a detection target in the preparation of products for detecting the formation of cell internalization structures.
5. The use according to any one of claims 1 to 4, characterized in that: The cell internalization structure includes a heterogeneous cell internalization structure.
6. The use according to claim 5, characterized in that: The formation of the heterogeneous cell internalization structure is caused by the internalization of platelets by macrophages.
7. Use of an agent for reducing the expression of vascular cell adhesion molecule 1 in the preparation of a drug for treating immune thrombocytopenia.
8. Application of vascular cell adhesion molecule 1 as a detection target in the preparation of products for diagnosing immune thrombocytopenia.
9. The use according to claim 7 or 8, characterized in that: The immune thrombocytopenia is caused by the formation of heterogeneous cell-internalized structures.
10. The use according to claim 7 or 8, characterized in that: The immune thrombocytopenia is caused by internalization of platelets by macrophages.