A method for liver cancer cell-derived exosome SNORD52 to mediate M2 polarization of macrophages and application thereof

Abstract

The application belongs to the technical field of targeted therapy drug research and development, and particularly relates to a method for mediating macrophage M2 polarization by liver cancer cell-derived exosomes SNORD52 and application thereof, comprising the following steps: S1: obtaining liver cancer cell-derived exosomes; S2: preparing macrophages to be polarized; S3: co-culturing the exosomes and the macrophages to induce M2-type polarization; and S4: detecting M2 polarization marker molecule expression and confirming successful polarization. The application constructs a set of standardized and specific macrophage M2 polarization method through the core steps of obtaining liver cancer cell-derived exosomes, preparing macrophages, co-culturing to induce polarization and detecting and confirming, effectively solves the problems of poor specificity and insufficient targeting of traditional methods, simultaneously uses liver cancer cell-derived exosomes as a core mediation carrier, utilizes the natural cell targeting delivery characteristics of the exosomes, can precisely act on macrophages, avoids damage to normal tissues caused by chemical reagents or non-tumor-derived factors, and lays a foundation for subsequent clinical conversion application.

Description

Technical Field

[0001] This invention belongs to the field of targeted therapy drug development technology, specifically relating to a method for macrophage M2 polarization mediated by SNORD52 exosomes derived from liver cancer cells and its application. Background Technology

[0002] Liver cancer (HCC) is one of the most prevalent malignant tumors worldwide, with both high incidence and mortality rates. While treatment options include surgical resection, radiotherapy, chemotherapy, and targeted therapy, the outcomes for patients with advanced HCC remain unsatisfactory. Abnormal regulation of the tumor microenvironment is a key factor contributing to HCC progression and treatment resistance. Macrophages, as abundant immune cells in the tumor microenvironment, possess strong plasticity and are mainly divided into pro-inflammatory M1 and anti-inflammatory M2 types. M2 macrophages can accelerate the development of HCC by secreting anti-inflammatory cytokines, promoting angiogenesis, and inhibiting anti-tumor immune responses. Therefore, regulating macrophage polarization has become an important research target for HCC treatment.

[0003] Currently, studies have attempted to induce macrophage polarization through various methods, but existing technologies generally suffer from several drawbacks: traditional induction methods often employ chemical reagents or non-tumor-derived biological factors, resulting in poor specificity and insufficient targeting, and are prone to adverse effects on normal tissues; furthermore, existing methods lack precise control over the induction process and have not established standardized validation procedures, leading to unstable polarization efficiency and difficulty in closely integrating with the specific treatment needs of liver cancer. In addition, research on macrophage polarization mediated by biomolecules derived from liver cancer cells themselves is still incomplete, particularly lacking effective technical solutions targeting specific non-coding RNAs in liver cancer cell exosomes to regulate macrophage M2 polarization, thus limiting the development of targeted therapies for liver cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a method and its application for macrophage M2 polarization mediated by SNORD52 exosomes derived from liver cancer cells, thereby solving the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells includes the following steps:

[0007] S1: Obtaining exosomes derived from liver cancer cells;

[0008] S2: Preparation of macrophages to be polarized;

[0009] S3: Co-culture of exosomes with macrophages to induce M2 polarization;

[0010] S4: Detect the expression of M2 polarization marker molecules to confirm successful polarization.

[0011] As a preferred embodiment of the present invention, step S1 is followed by step S11: verifying the presence and enrichment of SNORD52 in the exosomes.

[0012] As a preferred embodiment of the present invention, in step S11, qRT-PCR technology is used to verify the presence and enrichment level of SNORD52 in exosomes.

[0013] As a preferred embodiment of the present invention, in step S1, the exosomes are obtained by ultracentrifugation, reagent kit extraction, or density gradient centrifugation.

[0014] As a preferred embodiment of the present invention, in step S2, the macrophages are derived from peripheral blood mononuclear cell-induced differentiation or macrophage cell lines.

[0015] In a preferred embodiment of the present invention, in step S3, the co-culture conditions are 37°C and 5% CO2, and the co-culture ratio of exosomes to macrophages is 10:1. 5 ~10 7 Exosomes: 1×10 4 One macrophage.

[0016] As a preferred embodiment of the present invention, in step S4, the M2 polarization marker molecule includes at least one of CD206, Arg-1, and IL-10.

[0017] Application of any of the methods described above in the preparation of macrophage M2 polarization inducers and drugs related to the treatment of liver cancer.

[0018] The beneficial effects of this invention are as follows:

[0019] 1) This invention constructs a standardized and highly specific macrophage M2 polarization method by obtaining exosomes derived from liver cancer cells, preparing macrophages, co-culturing to induce polarization, and detecting and confirming the core steps. This effectively solves the problems of poor specificity and insufficient targeting of traditional methods. At the same time, by using exosomes derived from liver cancer cells as the core mediating carrier, the natural cell-targeting delivery characteristics of exosomes can be used to precisely act on macrophages, avoiding damage to normal tissues by chemical reagents or non-tumor-derived factors. Meanwhile, the standardized step design ensures the stability of polarization efficiency, laying the foundation for subsequent clinical translation applications.

[0020] 2) By confirming the presence and enrichment of SNORD52 in exosomes, this invention ensures the effectiveness of the core factor mediating polarization and improves the specificity of the method. Moreover, the limitations imposed by this invention on the exosome extraction method, macrophage source, co-culture conditions, and polarization marker molecules enrich the implementation path of the technical solution and enhance the flexibility and operability of the method. At the same time, this invention clarifies its application in the preparation of polarization inducers and liver cancer therapeutic drugs, directly linking the technical solution with clinical needs, effectively filling the gap in the existing technology of liver cancer-specific macrophage polarization regulation technology, and providing a new and effective approach for the development of targeted therapies for liver cancer. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0022] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0024] The following detailed description is exemplary and intended to provide further detailed explanation of the invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention.

[0025] In the description of this invention, it should be understood that the terms "center", "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0026] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more. It should be noted in the description of this invention that, unless otherwise explicitly specified and limited, the terms "installed," "connected," and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0027] Example

[0028] As shown in the figure, a method for macrophage M2 polarization mediated by SNORD52 exosomes derived from liver cancer cells includes the following steps:

[0029] S1: Obtaining exosomes derived from liver cancer cells;

[0030] S2: Preparation of macrophages to be polarized;

[0031] S3: Co-culture of exosomes with macrophages to induce M2 polarization;

[0032] S4: Detect the expression of M2 polarization marker molecules to confirm successful polarization.

[0033] Specifically, in research on immune regulation or drug development related to liver cancer, the process involves first obtaining exosomes derived from liver cancer cells via S1 to provide a specific carrier for polarization-mediated processes; then preparing macrophages to be polarized via S2 to establish the target population; subsequently, co-culturing via S3 allows the exosomes and macrophages to come into full contact, enabling the delivery of SNORD52 from the exosomes to the macrophages and initiating the M2 polarization program; finally, detecting the expression of polarization marker molecules via S4 verifies the polarization effect. These four steps form a standardized regulatory pathway, ensuring that the polarization process is orderly and controllable.

[0034] In this embodiment: after step S1, there is also step S11: verifying the presence and enrichment of SNORD52 in the exosomes.

[0035] Specifically, in experiments requiring high-quality polarization-mediated vectors, after obtaining exosomes in S1, the presence and enrichment of SNORD52 are verified in S11. This avoids subsequent polarization failures due to the absence or insufficient content of core mediators in the exosomes, effectively adding a quality screening step to the exosomes obtained in S1. This enhances the specificity and reliability of the method and ensures that the exosomes entering steps S2 and S3 have the ability to effectively mediate polarization.

[0036] In this embodiment: In step S11, qRT-PCR technology is used to verify the presence and enrichment level of SNORD52 in exosomes.

[0037] Specifically, in experiments requiring precise quantification of core mediator content, S11 explicitly needs to verify the presence and enrichment of SNORD52. qRT-PCR technology can achieve precise quantitative detection of SNORD52. Compared to conventional qualitative verification, it can more accurately determine whether the exosome quality meets the standards, providing a quantitative basis for the quality assessment of exosomes obtained in S1, further enhancing the verification effect of S11, ensuring the efficiency and stability of subsequent S3 co-culture polarization, and avoiding fluctuations in polarization effect due to differences in core factor content.

[0038] In this embodiment: In step S1, the exosomes are obtained by ultracentrifugation, reagent kit extraction, or density gradient centrifugation.

[0039] Specifically, depending on the differences in experimental sample size and equipment conditions, ultracentrifugation, reagent kit extraction, or density gradient centrifugation can be used to obtain exosomes in step S1. These three methods provide diverse implementation paths for step S1, ensuring that qualified exosomes can be stably obtained under different experimental environments. This provides a vector guarantee for the smooth implementation of subsequent steps S2 and S3, effectively improving the universality of the method.

[0040] In this embodiment: In step S2, the macrophages are derived from peripheral blood mononuclear cell-induced differentiation or macrophage cell lines.

[0041] Specifically, depending on the experimental requirements, peripheral blood mononuclear cells can be induced to differentiate into macrophages via PMA, or macrophage lines such as THP-1 can be used directly. The former is closer to the physiological state, while the latter is simple to operate and batch-stable. The two sources provide S2 with cellular flexibility, ensuring that functionally consistent macrophages to be polarized can be obtained under different research backgrounds, thus guaranteeing the reliability and reproducibility of the S3 co-culture system.

[0042] In this embodiment: In step S3, the co-culture conditions are 37°C and 5% CO2, and the co-culture ratio of exosomes to macrophages is 10:1. 5 ~10 7 Exosomes: 1×104 One macrophage.

[0043] Specifically, the optimized co-culture conditions ensure sufficient interaction between exosomes and macrophages, avoiding excessive or insufficient polarization signals due to ratio imbalance; 10 5 ~10 7 The dynamic range takes into account both low-dose and high-dose saturation effects, adapts to the activity differences of exosomes from different sources, maintains cell activity while ensuring polarization efficiency, thereby achieving precise regulation of M1 or M2 type polarization and improving the stability and comparability of S3 results.

[0044] In this example: in step S4, the M2 polarization marker molecule includes at least one of CD206, Arg-1 and IL-10.

[0045] Specifically, CD206, as a mannose receptor, is highly expressed on the surface of M2 macrophages, Arg-1 participates in urea metabolism and inhibits nitric oxide production, and IL-10 exerts an anti-inflammatory effect. These three factors together reflect the functional status of M2 polarization. By detecting CD206 by flow cytometry, Arg-1 by qRT-PCR or Western blot, and IL-10 by ELISA, the S3 induction effect can be verified from multiple dimensions, ensuring the consistency between phenotype and function, providing reliable criteria for M2 polarization, and supporting the accuracy and scientific validity of subsequent experimental conclusions.

[0046] Application of any of the methods described above in the preparation of macrophage M2 polarization inducers and drugs related to the treatment of liver cancer.

[0047] Specifically, the above method can efficiently induce macrophages to polarize towards the M2 phenotype. The resulting M2 macrophages exhibit typical surface marker expression characteristics and functional activity, and can be used to prepare macrophage M2 polarization inducers for further application in the development of therapeutic drugs that regulate the liver cancer microenvironment. Through exosome-mediated polarization regulation, it is possible to target the phenotypic transformation of tumor-associated macrophages, inhibit inflammatory responses, and promote tissue repair and immune escape, providing a new strategy for liver cancer immunotherapy. This application pathway has been experimentally proven to significantly improve drug targeting and biocompatibility.

[0048] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0049] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A method for macrophage M2 polarization mediated by SNORD52 exosomes derived from liver cancer cells, characterized in that, Includes the following steps: S1: Obtaining exosomes derived from liver cancer cells; S2: Preparation of macrophages to be polarized; S3: Co-culture of exosomes with macrophages to induce M2 polarization; S4: Detect the expression of M2 polarization marker molecules to confirm successful polarization.

2. The method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells according to claim 1, characterized in that, Step S1 is followed by step S11: verifying the presence and enrichment of SNORD52 in the exosomes.

3. The method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells according to claim 222, characterized in that, In step S11, qRT-PCR technology is used to verify the presence and enrichment level of SNORD52 in exosomes.

4. The method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells according to claim 1, characterized in that, In step S1, the exosomes are obtained by ultracentrifugation, reagent kit extraction, or density gradient centrifugation.

5. The method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells according to claim 1, characterized in that, In step S2, the macrophages are derived from peripheral blood mononuclear cell-induced differentiation or macrophage cell lines.

6. The method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells according to claim 1, characterized in that, In step S3, the co-culture conditions are 37°C and 5% CO2, and the co-culture ratio of exosomes to macrophages is 10:

1. 5 ~10 7 Exosomes: 1×10 4 One macrophage.

7. The method for SNORD52-mediated macrophage M2 polarization mediated by exosomes derived from liver cancer cells according to claim 1, characterized in that, In step S4, the M2 polarization marker molecule includes at least one of CD206, Arg-1, and IL-10.

8. The use of the method according to any one of claims 1-7 in the preparation of macrophage M2 polarization inducers and drugs related to the treatment of liver cancer.

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