Method and kit for regulating and controlling generation of extracellular vesicles, recombinant cell and application

Abstract

The invention discloses a method and a kit for regulating and controlling generation of extracellular vesicles, a recombinant cell and application. The method comprises the following steps: overexpressing at least one adhesion G-like protein coupled receptor in a host cell; the adhesion-like G protein coupled receptor is selected from the group consisting of an ADGRA subgroup, an ADGRG subgroup, an ADGRB subgroup, an ADGRD subgroup, an ADGRE subgroup, an ADGRF subgroup, or an ADGRL subgroup. The recombinant cell at least overexpresses one of the adhesion-like G-protein coupled receptors. The application comprises application of the agonist of the adhesion type G-protein coupled receptor in preparation of a preparation for promoting generation of extracellular vesicles. According to the method provided by the invention, the generation of the extracellular vesicles is regulated and controlled by overexpressing the adhesion type G protein coupled receptor, a new thought is provided for producing the extracellular vesicles through cell engineering, and the method has a relatively high application prospect.

Description

Technical Field

[0001] This invention belongs to the field of extracellular vesicle technology, specifically relating to a method and kit for regulating extracellular vesicle generation, recombinant cells, and their applications. Background Technology

[0002] Extracellular vesicles (EVs) are lipid bilayer-structured vesicles released by cells and play a crucial role in intercellular communication. Based on their biogenic pathway and size, EVs are mainly divided into two categories: exosomes and extracellular bodies. Exosomes originate from multivesicular bodies (MVBs) formed during endocytosis, subsequently fusing with the plasma membrane and released extracellularly; their diameter is typically 30-150 nanometers. Extracellular bodies, on the other hand, are formed by direct budding and shearing from the cell membrane, with a diameter typically ranging from 50-1000 nanometers. EVs carry various bioactive molecules, including proteins, lipids, and nucleic acids (such as DNA, mRNA, and miRNA), and can be taken up by recipient cells, thereby regulating their physiological and pathological processes. Given these characteristics, EVs show great potential for applications in disease diagnostic biomarkers, drug delivery vehicles, and regenerative medicine.

[0003] However, the clinical application of extracellular vesicles still faces many challenges, one of which is the efficient and controllable production of vesicles with specific compositions and functions. Currently used methods for producing extracellular vesicles, such as ultracentrifugation, polymer precipitation, and size exclusion chromatography, mainly isolate natural vesicles from cell culture supernatants. These methods suffer from low yields, high heterogeneity, complex processes, and high costs. Furthermore, methods to increase vesicle yield through physical or chemical stimulation (such as acid treatment, hypoxia, or treatment with small molecule drugs) are often toxic to cells and may alter the biological characteristics of vesicles, leading to functional instability or unpredictable side effects.

[0004] G protein-coupled receptors (GPCRs) are the largest family of cell membrane receptors, capable of sensing various extracellular signals (such as light, hormones, and neurotransmitters) and transmitting these signals into the cell via coupled G proteins, triggering corresponding physiological effects. GPCRs are the most important family of drug targets in humans, with over 30% of currently marketed drugs targeting them. Adhesion GPCRs are an important subclass of the GPCR family, characterized by their large extracellular domains. They participate in cell-cell and cell-extracellular matrix adhesion and play crucial roles in development, immunity, and cancer. However, no studies have yet reported a direct link between adhesion GPCRs and the biogenesis of extracellular vesicles, particularly extracellular bodies, nor have any proposed techniques for specifically inducing extracellular body generation by regulating specific GPCRs.

[0005] Therefore, there is an urgent need in this field to develop a new method that can efficiently and specifically regulate the generation of extracellular vesicles in order to meet the demand for high-quality extracellular vesicles in basic research and clinical applications.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] To address the aforementioned problems in the prior art, this invention provides a method and kit for regulating extracellular vesicle generation, recombinant cells, and their applications. The technical problem to be solved by this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for regulating extracellular vesicle generation, comprising overexpressing at least one adhesion-type G protein-coupled receptor in a host cell. The adhesion-type G protein-coupled receptors are selected from the ADGRA, ADDRG, ADDRB, ADDRD, ADGRE, ADGRF, or ADDRL subfamilies.

[0008] In one embodiment of the present invention, the adhesion-type G protein-coupled receptor is selected from any one of ADGRG1, ADGRG6, ADGRA3, ADGRB1, ADGRD1, ADGRE2, ADGRE5, ADGRF1, and ADGRL1.

[0009] In one embodiment of the present invention, the overexpression of at least one adhesion-type G protein-coupled receptor in host cells includes the following steps: S1. Load the gene encoding the target adhesion-type G protein-coupled receptor into an expression vector to obtain a recombinant expression vector; the loading of the gene encoding the target adhesion-type G protein-coupled receptor into the expression vector may be to load the nucleic acid molecule encoding the target adhesion-type G protein-coupled receptor into the expression vector. S2. The recombinant expression vector is introduced into host cells to obtain recombinant cells; S3. The recombinant cells are seeded into an extracellular matrix and cultured to induce the host cells to produce extracellular vesicles. The extracellular matrix consists of polyornithine, polylysine, collagen, fibronectin, or laminin.

[0010] In one embodiment of the present invention, the expression vector is a viral vector or a non-viral vector.

[0011] In one embodiment of the present invention, the viral vector is a lentiviral vector or an adeno-associated virus vector; the non-viral vector is a plasmid.

[0012] In one embodiment of the present invention, the host cell is a mammalian cell.

[0013] In one embodiment of the present invention, the host cell is HEK293 cell, HeLa cell, mesenchymal stem cell, fibroblast, or tumor cell.

[0014] In a second aspect, the present invention provides a recombinant cell that overexpresses at least one adhesion-type G protein-coupled receptor; The adhesion-type G protein-coupled receptors are selected from the ADGRA, ADDRG, ADDRB, ADDRD, ADGRE, ADGRF, or ADDRL subfamilies.

[0015] Thirdly, the present invention provides a kit for regulating extracellular vesicle generation, comprising: (a) Nucleic acid molecules encoding adhesion-type G protein-coupled receptors; or (b) An expression vector comprising the nucleic acid molecule, wherein the nucleic acid molecule is configured to be operatively linked in the expression vector; The adhesion-type G protein-coupled receptors are selected from the ADGRA, ADDRG, ADDRB, ADDRD, ADGRE, ADGRF, or ADDRL subfamilies.

[0016] Fourthly, the present invention provides the use of an agonist of an adhesion-type G protein-coupled receptor in the preparation of an agent for promoting extracellular vesicle formation, wherein the adhesion-type G protein-coupled receptor is selected from the ADGRA subfamily, ADDRG subfamily, ADDRB subfamily, ADDRD subfamily, ADGRE subfamily, ADGRF subfamily, or ADDRL subfamily.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. High specificity: This invention provides a method for specifically inducing the generation of extracellular vesicles by overexpressing specific subtypes of adhesion-coupled G protein receptors (such as ADGRG1, ADGRG6, etc.), providing a new approach for targeted regulation of specific types of extracellular vesicles.

[0018] 2. High efficiency: By overexpressing adhesion-type G protein-coupled receptors, the number of extracellular vesicles produced by cells can be significantly increased, providing operability for large-scale production of extracellular vesicles.

[0019] 3. Good controllability: The method provided by this invention is based on gene recombination and can precisely control the generation process of extracellular vesicles by regulating the expression level or activity of adhesion-type G protein-coupled receptors.

[0020] 4. Broad Application Prospects: Extracellular vesicles produced using the method of this invention can be used for drug delivery system development, disease diagnostic biomarker screening, immune modulation, tissue engineering, and as a tool for studying cell communication. In particular, by engineering cells overexpressing specific adhesion-type G protein-coupled receptors, engineered extracellular vesicles carrying specific therapeutic molecules (such as proteins and RNA) can be produced, which has significant medical application value.

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 These are confocal images characterizing cells overexpressing adhesion-type G protein-coupled receptors in Examples 1 (A, B), 3 (C, D), and 4 (E, F) provided in the embodiments of the present invention (scale bar: 10 micrometers). Figure 2 These are confocal images characterizing cells overexpressing adhesion-type G protein-coupled receptors in Examples 5 (A, B), 6 (C, D), and 7 (E, F) provided in the embodiments of the present invention (scale bar: 10 micrometers). Figure 3 These are confocal images characterizing cells overexpressing adhesion-type G protein-coupled receptors in Examples 8 (A, B), 9 (C, D), and 10 (E, F) provided in the embodiments of the present invention (scale bar: 10 micrometers). Figure 4 This is a confocal image characterization of cells in Comparative Example 1 of the present invention (scale bar: 10 micrometers). Figure 5 This is a comparison chart of the quantitative analysis results of extracellular vesicle area in HEK293 cells overexpressing different adhesion-type G protein-coupled receptors, provided in the embodiments of the present invention; Figure 6 These are confocal images characterizing cells overexpressing adhesion-type G protein-coupled receptors in Comparative Examples 2 (A), 3 (B), 4 (C), 11 (D), 12 (E), and 13 (F) provided in the embodiments of the present invention (scale bar: 10 micrometers). Figure 7 This is a comparison chart of the quantitative analysis results of extracellular vesicle area overexpression of adhesion-type G protein-coupled receptor G1 in HeLa cervical cancer cells, A875 melanoma cells and U87 glioma cells, provided by the embodiments of the present invention. Detailed Implementation

[0023] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description, in conjunction with the accompanying drawings and specific embodiments, provides a method and kit for regulating extracellular vesicle generation, recombinant cells, and applications based on the present invention.

[0024] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a more in-depth and concrete understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the technical solutions of the present invention.

[0025] It should be noted that the terms “comprising,” “including,” or any other variations are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed.

[0026] The term "host cell," as used herein, refers to a cell in which a foreign (e.g., exogenous) nucleic acid or protein is introduced. In this article, the host cell is a mammalian cell.

[0027] Example 1 Step 1: Constructing recombinant expression vectors and extracellular vesicle indicator reporter plasmids Recombinant expression vector: In this embodiment, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor G1, and the corresponding gene is ADGRG1. The expression vector used is PCDNA3.1 plasmid, and the tag protein is Flag-Halo. The ADGRG1 gene, which encodes Flag-Halo, is loaded into the PCDNA3.1 plasmid to construct the recombinant expression vector PCDNA3.1-Flag-Halo-ADGRG1. The gene ADGRG1 can also be written as GPR56.

[0028] Extracellular vesicle indicator reporter plasmid: The extracellular vesicle indicator reporter plasmid PCDNA3.1 was constructed by loading the PLCγ-PH gene, which can encode EGFP, into the PCDNA3.1 plasmid.

[0029] The constructed recombinant expression vector and extracellular vesicle indicator reporter plasmid were both validated by DNA sequencing to ensure that the gene sequences and reading frames were correct.

[0030] Step 2: Overexpression of adhesion-type G protein-coupled receptors induces extracellular vesicle formation. Step 21: Coat the laser confocal culture dishes with a coating solution containing 10 µg / mL poly-L-ornithine and 10 µg / mL collagen I, adding 500 µL to each well and incubating overnight at 4°C. Discard the coating solution, wash the laser confocal culture dishes twice with 1 mL PBS, and then add 1.5 mL of complete culture medium to each laser confocal culture dish for later use.

[0031] Step 22: Transfect HEK293 cells transiently with the recombinant expression vector (PCDNA3.1-Flag-Halo-ADGRG1) and extracellular vesicle indicator reporter plasmid (PCDNA3.1-PH-EGFP) from Step 1. 24 hours after transfection, digest and resuspend the cells, then seed them at approximately 50%-70% density (covering 50%-70% of the bottom area of ​​the culture dish after seeding) into the laser confocal culture dishes coated in Step 21. Incubate overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0032] Step 23: Dilute the HaloTag® ligand with complete culture medium at a volume ratio of 1:1000 to obtain a HaloTag® ligand solution. In this example, the HaloTag® ligand used is HaloTag® 660 dye, i.e., a HaloTag® 660 dye solution.

[0033] After the laser confocal culture dish from step 22 is completed, remove it and discard the culture medium. Add 500 µL of HaloTag® 660 dye solution to the laser confocal culture dish and incubate it in a 37°C incubator in the dark for 1 hour.

[0034] After incubation in the laser confocal culture dishes containing the dye solution from steps 24 and 23, discard the dye and wash the cells twice with 1 mL PBS to remove unbound dye. Add 1 mL of fresh complete culture medium and wrap the laser confocal culture dishes with aluminum foil to protect them from light and maintain temperature. Immediately perform live-cell imaging using a confocal microscope (such as an Olympus FV3000).

[0035] Simultaneously, images of the GFP channel (excitation light 488 nm, representing the cell membrane) and the 660 nm channel (representing the Halo-tagged G protein-coupled receptor) were acquired. The images were analyzed using ImageJ software, such as... Figure 5 and Figure 1 As shown in (A) and (B).

[0036] Example 2 Step 1: Constructing recombinant expression vectors and extracellular vesicle indicator reporter plasmids Recombinant expression vector: In this embodiment, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor G1, and the corresponding gene is ADGRG1. The expression vector used is the lentiviral vector PLV, and the tag protein is mCherry. The ADGRG1 gene, which can encode mCherry, is loaded into the lentiviral vector PLV, and the recombinant expression vector obtained is PLV-mCherry-ADGRG1. The ADGRG1 gene can also be expressed as GPR56.

[0037] Extracellular vesicle indicator reporter plasmid: The extracellular vesicle indicator reporter plasmid PCDNA3.1 was constructed by loading the PLCγ-PH gene, which can encode EGFP, into the PCDNA3.1 plasmid.

[0038] The constructed recombinant expression vector and extracellular vesicle indicator reporter plasmid were both validated by DNA sequencing to ensure that the gene sequences and reading frames were correct.

[0039] Step 2: Overexpression of adhesion-type G protein-coupled receptors induces extracellular vesicle formation. Step 21: Coat the laser confocal culture dishes with a coating solution containing 10 µg / mL poly-L-ornithine and 10 µg / mL collagen I, adding 500 µL to each well and incubating overnight at 4°C. Discard the coating solution, wash the laser confocal culture dishes twice with 1 mL PBS, and then add 1.5 mL of complete culture medium to each laser confocal culture dish for later use.

[0040] Step 22: Transfect HEK293 cells transiently with the recombinant expression vector (PLV-mCherry-ADGRG1) and extracellular vesicle indicator reporter plasmid (PCDNA3.1-PH-EGFP) from Step 1. 24 hours after transfection, digest and resuspend the cells, then seed them at approximately 50%-70% density (covering 50%-70% of the bottom area of ​​the culture dish after seeding) into the laser confocal culture dishes coated in Step 21. Incubate overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0041] After the laser confocal culture dishes from steps 23 and 22 are completed, remove them and discard the culture medium. Wash the cells twice with 1 mL PBS, add 1 mL of fresh complete culture medium, and wrap the dishes with aluminum foil to protect them from light and keep them warm. Immediately use a confocal microscope (such as an Olympus FV3000) for live cell imaging.

[0042] During detection, images of the GFP channel (excitation light 488 nm, representing the cell membrane) and the mCherry channel (excitation light 561 nm, representing G protein-coupled receptors with mCherry tags) were simultaneously acquired. The images were analyzed using ImageJ software, and the results were similar to those in Example 1.

[0043] Example 3 Step 1: Constructing recombinant expression vectors and extracellular vesicle indicator reporter plasmids Recombinant expression vector: In this embodiment, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor G6, and the corresponding gene is ADGRG6. The expression vector used is PCDNA3.1 plasmid, and the tag protein is HA-SNAP. The ADGRG6 gene, which encodes HA-SNAP, is loaded into the PCDNA3.1 plasmid, and the resulting recombinant expression vector is PCDNA3.1-HA-SNAP-ADGRG6. The gene ADGRG6 can also be written as GPR126.

[0044] Extracellular vesicle indicator reporter plasmid: The extracellular vesicle indicator reporter plasmid PCDNA3.1 was constructed by loading the PLCγ-PH gene, which can encode EGFP, into the PCDNA3.1 plasmid.

[0045] The constructed recombinant expression vector and extracellular vesicle indicator reporter plasmid were both validated by DNA sequencing to ensure that the gene sequences and reading frames were correct.

[0046] Step 2: Overexpression of adhesion-type G protein-coupled receptors induces extracellular vesicle formation. Step 21: Coat the laser confocal culture dishes with a coating solution containing 10 µg / mL poly-L-ornithine and 10 µg / mL collagen I, adding 500 µL to each well and incubating overnight at 4°C. Discard the coating solution, wash the laser confocal culture dishes twice with 1 mL PBS, and then add 1.5 mL of complete culture medium to each laser confocal culture dish for later use.

[0047] Step 22: Transfect HEK293 cells transiently with the recombinant expression vector (PCDNA3.1-HA-SNAP-ADGRG6) and extracellular vesicle indicator reporter plasmid (PCDNA3.1-PH-EGFP) from Step 1. 24 hours after transfection, digest and resuspend the cells, then seed them at approximately 50%-70% density (covering 50%-70% of the bottom area of ​​the culture dish after seeding) into the laser confocal culture dishes coated in Step 21. Incubate overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0048] Step 23: Dilute the SNAP-tag® ligand using complete culture medium at a volume ratio of 1:1000 (SNAP-tag® ligand: complete culture medium) to obtain a SNAP-tag® ligand solution. In this example, the SNAP-tag® ligand used is SNAP-Surface® 647 dye, i.e., a SNAP-Surface® 647 dye solution is obtained by dilution.

[0049] After the laser confocal culture dish from step 22 is completed, remove it and discard the culture medium. Add 500 µL of SNAP-Surface® 647 dye solution to the laser confocal culture dish and incubate it in a 37°C incubator in the dark for 1 hour.

[0050] After incubation in the laser confocal culture dishes containing the dye solution from steps 24 and 23, discard the dye and wash the cells twice with 1 mL PBS to remove unbound dye. Add 1 mL of fresh complete culture medium and wrap the dishes with aluminum foil to protect them from light and maintain temperature. Immediately perform live-cell imaging using a confocal microscope (such as an Olympus FV3000).

[0051] Simultaneously, images of the GFP channel (excitation light 488 nm, representing the cell membrane) and the 647 nm channel (representing G protein-coupled receptors with SNAP tags) were acquired. The images were analyzed using ImageJ software; the results are shown below. Figure 5 and Figure 1 As shown in (C) and (D).

[0052] Example 4 The difference between this embodiment and Embodiment 3 is that the recombinant expression vector is constructed differently in step 1. In Embodiment 4, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor A3, the corresponding gene is ADGRA3, the expression vector used is PCDNA3.1 plasmid, the tag protein is HA-SNAP, and the ADGRA3 gene carrying HA-SNAP is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-HA-SNAP-ADGRA3. The gene ADGRA3 can also be written as GPR125.

[0053] The remaining operations are the same as in Example 3.

[0054] Images of the GFP channel and the 647nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 1 As shown in (E) and (F).

[0055] Example 5 The difference between this embodiment and Embodiment 3 is that the recombinant expression vector is constructed differently in step 1. In Embodiment 5, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor B1, the corresponding gene is ADGRB1, the expression vector used is PCDNA3.1 plasmid, the tag protein is HA-SNAP, and the ADGRB1 gene carrying HA-SNAP is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-HA-SNAP-ADGRB1. The gene ADGRB1 can also be written as the gene BAI1.

[0056] The remaining operations are the same as in Example 3.

[0057] Images of the GFP channel and the 647nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 2 As shown in (A) and (B).

[0058] Example 6 The difference between this embodiment and Embodiment 1 is that the recombinant expression vector is constructed differently in step 1. In Embodiment 6, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor D1, the corresponding gene is ADDRD1, the expression vector used is PCDNA3.1 plasmid, the tag protein is Flag-Halo, and the ADDRD1 gene carrying the Flag-Halo is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-Flag-Halo-ADGRD1. The gene ADDRD1 can also be written as GPR133.

[0059] The remaining operations are the same as in Example 1.

[0060] Images of the GFP channel and the 660 nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 2 As shown in (C) and (D).

[0061] Example 7 The difference between this embodiment and Embodiment 3 lies in the construction of the recombinant expression vector in step 1. In Embodiment 7, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor E2, the corresponding gene is ADGRE2, the expression vector used is PCDNA3.1 plasmid, the tag protein is HA-SNAP, and the ADGRE2 gene carrying HA-SNAP is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-HA-SNAP-ADGRE2. The gene ADGRE2 can also be written as EMR2.

[0062] The remaining operations are the same as in Example 3.

[0063] Images of the GFP channel and the 647nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 2 As shown in (E) and (F).

[0064] Example 8 The difference between this embodiment and Embodiment 3 lies in the construction of the recombinant expression vector in step 1. In Embodiment 8, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor E5, the corresponding gene is ADGRE5, the expression vector used is PCDNA3.1 plasmid, the tag protein is HA-SNAP, and the ADGRE5 gene carrying HA-SNAP is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-HA-SNAP-ADGRE5. The gene ADGRE5 can also be written as the gene CD97.

[0065] The remaining operations are the same as in Example 3.

[0066] Images of the GFP channel and the 647nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 3 As shown in (A) and (B).

[0067] Example 9 The difference between this embodiment and Embodiment 1 is that the recombinant expression vector is constructed differently in step 1. In Embodiment 9, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor F1, the corresponding gene is ADGRF1, the expression vector used is PCDNA3.1 plasmid, the tag protein is Flag-Halo, and the ADGRF1 gene carrying the Flag-Halo is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-Flag-Halo-ADGRF1. The gene ADGRF1 can also be written as the gene GPR110.

[0068] The remaining operations are the same as in Example 1.

[0069] Images of the GFP channel and the 660 nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 3 As shown in (C) and (D).

[0070] Example 10 The difference between this embodiment and Embodiment 1 is that the recombinant expression vector is constructed differently in step 1. In Embodiment 10, when constructing the recombinant expression vector, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor L1, the corresponding gene is ADGRL1, the expression vector used is PCDNA3.1 plasmid, the tag protein is Flag-Halo, and the ADGRL1 gene carrying the Flag-Halo is loaded into the PCDNA3.1 plasmid, resulting in the recombinant expression vector PCDNA3.1-Flag-Halo-ADGRL1. The gene ADGRL1 can also be written as LPHN1.

[0071] The remaining operations are the same as in Example 1.

[0072] Images of the GFP channel and the 660 nm channel were acquired simultaneously. ImageJ software was used to analyze the images; the results are shown below. Figure 5 and Figure 3 As shown in (E) and (F).

[0073] Comparative Example 1 Step 1: Construct extracellular vesicle indicator reporter plasmid Extracellular vesicle indicator reporter plasmid: The extracellular vesicle indicator reporter plasmid PCDNA3.1 was constructed by loading the PLCγ-PH gene, which can encode EGFP, into the PCDNA3.1 plasmid.

[0074] The constructed extracellular vesicle indicator reporter plasmid was validated by DNA sequencing to ensure that the gene sequence and reading frame were correct.

[0075] Step 2: Detect extracellular vesicles Step 21: Coat the laser confocal culture dishes with a coating solution containing 10 µg / mL poly-L-ornithine and 10 µg / mL collagen I, adding 500 µL to each well and incubating overnight at 4°C. Discard the coating solution, wash the laser confocal culture dishes twice with 1 mL PBS, and then add 1.5 mL of complete culture medium to each laser confocal culture dish for later use.

[0076] Step 22: Transfect HEK293 cells by transfecting the extracellular vesicle indicator reporter plasmid (PCDNA3.1-PH-EGFP) from Step 1 into HEK293 cells. 24 hours after transfection, digest and resuspend the cells, and seed them at a density of approximately 50%-70% (covering 50%-70% of the bottom area of ​​the culture dish after seeding) into the laser confocal culture dishes coated in Step 21. Incubate overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0077] After the laser confocal culture dishes from steps 23 and 22 are completed, remove them and discard the culture medium. Add 1 mL of fresh complete culture medium and wrap the dishes with aluminum foil to protect them from light and keep them warm. Immediately use a confocal microscope (such as an Olympus FV3000) for live cell imaging.

[0078] Images of the GFP channels were acquired and analyzed using ImageJ software. See the results below. Figure 4 and Figure 5 .

[0079] Comparative Example 2 The difference from Comparative Example 1 is that in step 22, HeLa cervical cancer cells were used for transfection. The remaining procedures were the same as in Example 1.

[0080] Images of the GFP channels were acquired and analyzed using ImageJ software. See the results below. Figure 7 and Figure 6 (A).

[0081] Comparative Example 3 The difference from Comparative Example 1 is that, in step 22, A875 melanoma cells were used for transfection. The remaining procedures were the same as in Example 1.

[0082] Images of the GFP channels were acquired and analyzed using ImageJ software. See the results below. Figure 7 and Figure 6 (B).

[0083] Comparative Example 4 The difference from Comparative Example 1 is that in step 22, U87 glioma cells were transfected. The remaining procedures were the same as in Example 1.

[0084] Images of the GFP channels were acquired and analyzed using ImageJ software. See the results below. Figure 7 and Figure 6 (C).

[0085] Example 11 Step 1: Constructing a recombinant expression vector Recombinant expression vector: In this embodiment, the target adhesion-type G protein-coupled receptor is adhesion-type G protein-coupled receptor G1, the corresponding gene is ADGRG1, the expression vector used is PCDNA3.1 plasmid, the tag protein is Venus, and the ADGRG1 gene carrying Venus is loaded into PCDNA3.1 plasmid to construct the recombinant expression vector PCDNA3.1-Venus-ADGRG1.

[0086] The constructed recombinant expression vector was validated by DNA sequencing to ensure that the gene sequence and reading frame were correct.

[0087] Step 2: Overexpression of adhesion-type G protein-coupled receptors induces extracellular vesicle formation. Step 21: Coat the laser confocal culture dishes with a coating solution containing 10 µg / mL poly-L-ornithine and 10 µg / mL collagen I, adding 500 µL to each well and incubating overnight at 4°C. Discard the coating solution, wash the laser confocal culture dishes twice with 1 mL PBS, and then add 1.5 mL of complete culture medium to each laser confocal culture dish for later use.

[0088] Step 22: Transfect HeLa cervical cancer cells with the recombinant expression vector (PCDNA3.1-Venus-ADGRG1) from Step 1. 24 hours after transfection, digest and resuspend the cells, then seed them at a density of approximately 50%-70% (covering 50%-70% of the bottom area of ​​the culture dish after seeding) into the laser confocal culture dishes coated in Step 21. Incubate overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion.

[0089] After the laser confocal culture dishes from steps 23 and 22 are completed, remove them and discard the culture medium. Add 1 mL of fresh complete culture medium and wrap the dishes with aluminum foil to protect them from light and keep them warm. Immediately use a confocal microscope (such as an Olympus FV3000) for live cell imaging.

[0090] Images were acquired in the 528 nm channel (representing the Venus-tagged G protein-coupled receptor) and analyzed using ImageJ software, such as... Figure 7 and Figure 6 As shown in (D).

[0091] Example 12 The difference between this embodiment and Embodiment 11 is that, in step 22, A875 melanoma cells are used for transfection. The remaining operations are the same as in Embodiment 11.

[0092] Images were acquired in the 528 nm channel and analyzed using ImageJ software, such as... Figure 7 and Figure 6 As shown in (E).

[0093] Example 13 The difference between this embodiment and Embodiment 11 is that, in step 22, U87 glioma cells are transfected. The remaining operations are the same as in Embodiment 11.

[0094] Images were acquired in the 528 nm channel and analyzed using ImageJ software, such as... Figure 7 and Figure 6 As shown in (F).

[0095] Results analysis: Examples 1-10 and Comparative Example 1 all used HEK293 cells as host cells.

[0096] Comparing Example 1 and Example 2, the difference being the use of different expression vectors and tag proteins, the image analysis results of the two examples were similar, indicating that the generation of extracellular vesicles is related to the overexpression of adhesion-type G protein-coupled receptors, and has no significant correlation with the expression vectors and tag proteins used.

[0097] Compared with the image analysis results of Comparative Example 1, the image analysis results of Examples 1 and 3-10 clearly show that at the cell edge of cells overexpressing adhesion-type G protein-coupled receptors, there are a large number of vesicle structures that directly "bud" from the plasma membrane and detach. These vesicles simultaneously exhibit PH-EGFP membrane signals and G protein-coupled receptor fluorescence signals, confirming that overexpression of adhesion-type G protein-coupled receptors can promote the generation of extracellular vesicles. Furthermore, by regulating the expression level of adhesion-type G protein-coupled receptors, the formation process of extracellular vesicles can be effectively controlled.

[0098] like Figure 5 The figure shows a quantitative analysis of the extracellular vesicle area of ​​a single cell. Data are expressed as mean ± standard error. Data for each adhesion-type G protein-coupled receptor (GPCR) were obtained from measurements of more than 40 cells in three independent experiments. ****P < 0.0001; ns indicates no significant difference (P > 0.05). Statistical significance was determined using ordinary one-way ANOVA and Tukey's multiple comparison test. The results showed that, compared with the results of Comparative Example 1, the extracellular vesicle area in Examples 1 and 3-10, which overexpressed GPCRs, was significantly increased, further demonstrating that overexpression of GPCRs can promote the generation of extracellular vesicles.

[0099] To further illustrate that overexpression of adhesion-type G protein-coupled receptors can regulate the generation of extracellular vesicles in various mammalian cells, Examples 11-13 and Comparative Examples 2-4 were verified using different cells as host cells.

[0100] like Figure 6 (A) and Figure 6 As shown in (C), compared with Comparative Example 2, overexpression of ADGRG1 in HeLa cervical cancer cells significantly increased the number of vesicle structures clustered at the cell periphery. Figure 7 As shown, the extracellular vesicle area of ​​individual cells was used for quantitative analysis. The experiments in Example 11 and Comparative Example 2 were repeated three times, resulting in 50 cell samples from Example 11 and 41 cell samples from Comparative Example 2. Statistical analysis was performed, and data are expressed as mean ± standard error. ****P < 0.0001. Statistical significance was determined using a two-tailed unpaired t-test. The results showed that the extracellular vesicle area of ​​Example 11 was significantly increased compared to Comparative Example 2 (P < 0.0001).

[0101] Compared with Comparative Example 3, Example 12 shows that... Figure 6 As can be seen in (B) and body 6 (E), overexpression of ADGRG1 in A875 melanoma cells leads to the aggregation of more vesicle structures at the cell edge and the release of a large number of vesicle structures distributed around the cell. Figure 7 As shown, the extracellular vesicle area of ​​individual cells was used for quantitative analysis. The experiments in Example 12 and Comparative Example 3 were repeated three times, resulting in 52 cell samples from Example 12 and 54 cell samples from Comparative Example 3 for statistical analysis. The results showed that, compared with Comparative Example 3, the extracellular vesicle area of ​​A875 melanoma cells in Example 12 was significantly increased (P<0.0001).

[0102] Comparing Example 13 with Comparative Example 4, as follows: Figure 6 (C) and Figure 6 As shown in (F), overexpression of ADGRG1 in U87 glioma cells significantly increased the number of vesicle structures aggregated at the cell periphery, and also showed a greater number of vesicle structures scattered around the cells. Figure 7 As shown, the extracellular vesicle area of ​​a single U87 glioma cell was quantitatively analyzed. The experiments in Example 13 and Comparative Example 4 were repeated three times, resulting in 41 cell samples from Example 12 and 44 cell samples from Comparative Example 3 for statistical analysis. The results showed that, compared with Comparative Example 4, the extracellular vesicle area of ​​U87 glioma cells in Example 12 was significantly increased (P<0.0001). In summary, overexpression of adhesion-like G protein-coupled receptors in cells can significantly increase the yield of extracellular vesicles, effectively induce direct cleavage of the plasma membrane on the extracellular matrix, release a large number of extracellular vesicles, and achieve targeted regulation of vesicle generation pathways. This solves the problems of low extracellular vesicle yield and high heterogeneity in existing technologies, providing an efficient and controllable technical platform for the engineering application of extracellular vesicles in drug delivery systems, diagnostic biomarker development, and other fields.

[0103] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A method of modulating extracellular vesicle production, characterized in that, overexpressing at least one adhesion class G protein-coupled receptor in a host cell; the adhesion class G protein-coupled receptor is selected from the ADGRA subfamily, the ADGRG subfamily, the ADGRB subfamily, the ADGRD subfamily, the ADGRE subfamily, the ADGRF subfamily, or the ADGRL subfamily.

2. The method of modulating extracellular vesicle production according to claim 1, wherein, the adhesion class G protein-coupled receptor is selected from any one of ADGRG1, ADGRG6, ADGRA3, ADGRB1, ADGRD1, ADGRE2, ADGRE5, ADGRF1, and ADGRL1.

3. The method of modulating extracellular vesicle production of claim 1, wherein, The step of overexpressing at least one adhesion class G protein-coupled receptor in a host cell comprises the following steps: S1, loading a gene encoding a target adhesion class G protein-coupled receptor into an expression vector to obtain a recombinant expression vector; S2, introducing the recombinant expression vector into a host cell to obtain a recombinant cell; S3, inoculating the recombinant cell into an extracellular matrix for culture to induce the host cell to produce extracellular vesicles.

4. The method of modulating extracellular vesicle production according to claim 3, wherein, The expression vector is a viral vector or a non-viral vector.

5. The method of modulating extracellular vesicle production according to claim 4, wherein, The viral vector is a lentiviral vector or an adeno-associated viral vector. The non-viral vector is a plasmid.

6. The method of modulating extracellular vesicle production of claim 3, wherein, The host cell is a mammalian cell.

7. The method of modulating extracellular vesicle production according to claim 6, wherein, The host cell is a HEK293 cell, a HeLa cell, a mesenchymal stem cell, a fibroblast cell, or a tumor cell.

8. A recombinant cell, characterized in that, The recombinant cell overexpresses at least one adhesion class G protein-coupled receptor. the adhesion class G protein-coupled receptor is selected from the ADGRA subfamily, the ADGRG subfamily, the ADGRB subfamily, the ADGRD subfamily, the ADGRE subfamily, the ADGRF subfamily, or the ADGRL subfamily.

9. A kit for modulating extracellular vesicle production, characterized in that, comprises: (a) a nucleic acid molecule encoding an adhesion class G protein-coupled receptor; or (b) an expression vector comprising the nucleic acid molecule, wherein the nucleic acid molecule is configured to be operably linked in the expression vector; the adhesion class G protein-coupled receptor is selected from the ADGRA subfamily, the ADGRG subfamily, the ADGRB subfamily, the ADGRD subfamily, the ADGRE subfamily, the ADGRF subfamily, or the ADGRL subfamily.

10. Use of an agonist of a G protein coupled receptor of the adhesion class for the preparation of a formulation for promoting the production of extracellular vesicles, characterized in that, the adhesion class G protein-coupled receptor is selected from the ADGRA subfamily, the ADGRG subfamily, the ADGRB subfamily, the ADGRD subfamily, the ADGRE subfamily, the ADGRF subfamily, or the ADGRL subfamily.

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