Hippocampus-derived exosome-like vesicles, and preparation method and application thereof
By preparing hippocampal exosome-like vesicles, the shortcomings of hippocampal research in male reproductive function have been overcome. This method promotes the proliferation of testicular interstitial cells and testosterone secretion, and has biosafety and high bioavailability, making it suitable for male reproductive health products.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- OCEAN UNIV OF CHINA
- Filing Date
- 2026-03-10
- Publication Date
- 2026-06-30
AI Technical Summary
Existing research on the role of hippocampus in improving male reproductive function mainly focuses on post-injury repair, lacking research on actively enhancing reproductive function. Furthermore, the development and utilization of hippocampal exosome-like vesicles are insufficient, resulting in low bioavailability.
A method for preparing hippocampal exosome-like vesicles is provided, including steps such as washing, stirring, centrifugation, and dialysis, to obtain structurally intact hippocampal exosome-like vesicles with a particle size of 100-150 nm, which can be used to promote the proliferation of testicular interstitial cells and the secretion of testosterone.
Hippocampal exosome-like vesicles can be efficiently taken up by mouse testicular interstitial cells, significantly promoting cell proliferation and testosterone secretion, showing significant application prospects. The simple and environmentally friendly extraction process facilitates industrial promotion.
Smart Images
Figure CN122303130A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a hippocampal exosome-like vesicle, its preparation method, and its application. Background Technology
[0002] Current research on the effects of seahorses on improving male reproductive function mainly focuses on traditional Chinese medicine and seahorse peptides. For example, the peptide sequence disclosed in Chinese patent CN120842316A, "A type of bloated seahorse peptide and its application in the preparation of functional products for repairing damage to the male reproductive system," involves a relatively complex extraction process, and there are potential risks in the artificial synthesis of peptides. Moreover, this patent mainly focuses on the repair of damage, and there is no research on whether seahorses can actively enhance reproductive function.
[0003] Exosome-like vesicles, composed of specific proteins, lipids, nucleic acids, and secondary metabolites, play a crucial role in maintaining cellular homeostasis and intercellular communication. They possess advantages such as good biocompatibility, low immunogenicity, oral safety, and high bioavailability, leading to significant interest in their application in the food and pharmaceutical fields in recent years. Currently, hippocampal exosome-like vesicles have not been developed, and their application in reproductive health research is even more lacking, representing a technological and market gap that needs to be filled. Summary of the Invention
[0004] The technical problem to be solved by this invention is that existing research on products for male reproductive function mainly focuses on repair after injury and improvement after decline, while research on actively enhancing reproductive function is limited and generally uses single methods with side effects; at the same time, the utilization of hippocampus suffers from low bioavailability and a lack of development and research on hippocampal exosome-like vesicles.
[0005] To address the problems of existing technologies, this invention provides a hippocampal exosome-like vesicle (Hip-Exo), its extraction method, and its applications. This hippocampal exosome-like vesicle can be readily taken up by mouse testicular interstitial cells, actively enhancing and promoting the proliferation and testosterone secretion of these cells, demonstrating significant application potential.
[0006] To achieve the above objectives, the present invention provides a method for preparing hippocampal exosome-like vesicles, comprising the following steps: (1) After cleaning, the seahorse is ground into seahorse paste using a sterile ultrafine grinder or homogenizer, mixed with phosphate buffer solution, and the supernatant is collected after standing. (2) The supernatant is centrifuged at low temperature and 2000-100000g speed until there is no bottom precipitate and the supernatant is clear. The centrifugation time is 40-110min each time. The precipitate is collected, resuspended, filtered and dialyzed to obtain a solution containing hippocampal exosome-like vesicles.
[0007] Further, in step (1), before the seahorse is minced, it needs to be washed with sterile water to clean the impurities on the surface of the seahorse and avoid introducing contamination by other bacteria. The seahorse minced meat is mixed with pre-cooled phosphate buffer solution, and the ratio of seahorse minced meat to phosphate buffer solution is 1g:10-15mL.
[0008] Furthermore, in step (1), the seahorse meat paste is mixed and stirred with the pre-cooled phosphate buffer for 10-15 hours, and then left to stand for 20-50 minutes after mixing and stirring, so that the PBS-soluble components in the cells and between cells can fully diffuse from the tissue fragments into the buffer solution, and solid-liquid separation is achieved by gravity sedimentation, thereby maximizing the yield.
[0009] Further, in step (2), the filtrate is centrifuged at 4°C and 1500-2500g for 30-40 min to remove unbroken tissue fragments and other impurities, and the supernatant I is collected; the supernatant I is centrifuged at 4°C and 10000-20000g for 40-75 min to remove organelles and other impurities, and the supernatant II is collected; the supernatant II is centrifuged at 4°C and 100000-120000g for 70-110 min to enrich vesicles and collect the precipitate.
[0010] Furthermore, in step (2), the precipitate is resuspended in phosphate buffer and filtered sequentially through 0.45 μm and 0.22 μm filter membranes to remove protein aggregates, thereby achieving further purification and biological sterilization of exosome-like vesicles.
[0011] Further, in step (2), the dialysis process includes: placing the exosome-like vesicles in a 300kDa dialysis bag for dialysis for 12-20 hours, with a sample to dialysis solution volume ratio of 1:100, in order to remove small molecule impurities such as free proteins.
[0012] A hippocampal exosome-like vesicle prepared by the above method, wherein the hippocampal exosome-like vesicle has a saucer-like structure, a particle size of 100-150 nm, and has a complete structure and good uniformity.
[0013] Furthermore, based on 1 ml of the hippocampal exosome-like vesicles, the protein content is 0.2-0.4 mg, indicating high purity.
[0014] The above-mentioned hippocampal exosome-like vesicles are used in the preparation of products that enhance male reproductive function. The products are preferably those that promote the proliferation of testicular interstitial cells and / or promote the secretion of sex hormones by these cells. The hippocampal exosome-like vesicles prepared by this invention can promote the proliferation of mouse testicular interstitial cells TM3 and promote the secretion of testosterone by these cells, thereby enhancing and repairing male reproductive function.
[0015] The beneficial effects of this invention are as follows: (1) The extraction process of hippocampal exosome-like vesicles (Hip-Exo) of the present invention is simple, safe, environmentally friendly and pollution-free, and easy to promote industrially. At the same time, the raw materials of the present invention are easy to raise and can be obtained on a large scale, providing sufficient material support for subsequent research and application.
[0016] (2) Preliminary research results of this application show that Hip-Exo can be highly taken up by TM3 cells; at the same time, it also has a significant proliferative effect on TM3 cells and can significantly promote the secretion of testosterone by TM3 cells, which can enhance and repair male reproductive function.
[0017] (3) Hip-Exo is an innovative extension of traditional Chinese medicine treatment, possessing nutritional, health-promoting, and medicinal value. It has many excellent characteristics, such as biocompatibility, stability of effective functional components, and high bioavailability. These characteristics make Hip-Exo show extremely promising application prospects in related nutritional, health-promoting, and therapeutic fields. Attached Figure Description
[0018] Figure 1 The flowchart for extracting hippocampal exosome-like vesicles.
[0019] Figure 2 Transmission electron microscopy image of the extracted hippocampal exosome-like vesicles.
[0020] Figure 3 Zeta potential map of the extracted hippocampal exosome-like vesicles.
[0021] Figure 4 NTA size distribution of the extracted hippocampal exosome-like vesicles.
[0022] Figure 5 The uptake of hippocampal exosome-like vesicles by TM3 cells after processing.
[0023] Figure 6 The figure shows the statistical results of the effect of the extracted hippocampal exosome-like vesicles on the viability of TM3 cells. In the figure, *** indicates p < 0.001 and **** indicates p < 0.0001.
[0024] Figure 7 The figure shows the statistical results of the effect of the extracted hippocampal exosome-like vesicles on the testosterone secretion ability of TM3 cells. In the figure, * indicates p < 0.05 and ** indicates p < 0.01. Detailed Implementation
[0025] The following description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
[0026] The following embodiments can be understood as illustrating only a part of the structure or method of the present invention, or as a combination of embodiments explaining the broader structure or method of the present invention. Unless otherwise specified, all raw materials of the present invention are commercially available.
[0027] Unless otherwise specified, all raw materials used in the following embodiments were purchased from the market.
[0028] Example 1: Preparation and morphological characterization of hippocampal exosome-like vesicles (Hip-Exo): (I) Preparation of hippocampal exosome-like vesicles (Hip-Exo), specific procedures (e.g.) Figure 1 The steps (as shown) are as follows: After thawing frozen seahorses at 4°C, rinse them thoroughly with sterile water, cut them into small pieces, and grind them into a paste using an ultrafine grinder. Take 20g of the seahorse paste and mix it with 200mL of PBS solution (pre-cooled at 4°C), and stir using a magnetic stirrer at 4°C for 15 hours (speed: 750rpm). After stirring, let it stand for 30 minutes and collect the supernatant.
[0029] Place the supernatant in a 50 mL centrifuge tube, centrifuge at 2000 g for 35 min at 4 °C, and collect supernatant I; Place supernatant I into a 50 mL centrifuge tube, centrifuge at 10,000 rpm for 45 min at 4 °C, and collect supernatant II. Place the supernatant II in an ultracentrifuge tube, centrifuge at 100,000g for 90 min at 4°C, and collect the precipitate; Resuspend each precipitate in 1 mL of pre-cooled PBS, filter it through a 0.45 μm filter membrane, and then filter it through a 0.22 μm filter membrane.
[0030] The filtrate was placed in a 300 kDa dialysis bag and dialyzed for 15 h, with a sample-to-dialysis buffer (PBS) volume ratio of 1:100. After dialysis, the collected liquid was the solution of hippocampal exosome-like vesicles (Hip-Exo) and stored at 4 °C for later use.
[0031] (II) The morphology of the hippocampal exosome-like vesicles extracted in Example 1 was observed using transmission electron microscopy. The transmission electron microscopy results are as follows: Figure 2 As shown.
[0032] The testing process included: taking 10 μL of the hippocampal exosome-like vesicles extracted in Example 1 and dropping it onto a 300-mesh copper grid with a support membrane, letting it stand for 15 min, then adding 2% phosphotungstic acid for negative staining for 1 min, absorbing excess liquid from the edge of the droplet with a filter paper strip, letting it stand to dry, and imaging it with an HT-7800 transmission electron microscope.
[0033] As Figure 2 As can be seen, tea saucer-like membrane vesicle structures with a particle size of less than 200 nm are clearly visible in the electron microscope field of view.
[0034] (III) The zeta potential of the hippocampal exosome-like vesicles extracted in Example 1 was tested, and the results are as follows: Figure 3 As shown.
[0035] The testing procedure included: diluting hippocampal exosome-like vesicles 10-fold with PBS, slowly injecting the diluted sample into the potential sample cell, and detecting the potential using a nanoparticle size analyzer. Each sample was measured three times, and the average value was taken. The zeta potential results are shown below. Figure 3 As shown.
[0036] from Figure 3 The results show that the average potential of the hippocampal exosome-like vesicles is -15mV, indicating that the above vesicles have strong electrostatic repulsion and the system is stable.
[0037] (iv) The protein concentration of the hippocampal exosome-like vesicles extracted in Example 1 was tested.
[0038] The testing process included: using the BCA kit, following the instructions, to test the protein concentration of hippocampal exosome-like vesicles. Each sample was measured three times, and the average value was taken. The results are shown in Table 1.
[0039] As can be seen from the results in Table 1, the protein concentration of hippocampal exosome-like vesicles was 0.21 mg / ml.
[0040] (V) The NTA size and extraction rate of the hippocampal exosome-like vesicles extracted in Example 1 were tested, and the size distribution results are as follows: Figure 4 As shown.
[0041] The testing procedure included: diluting hippocampal exosome-like vesicles 1000 times with PBS, then slowly injecting 1 mL of the diluted sample into the sample cell using a syringe. Particle size and concentration were measured using a nanoparticle size analyzer. Each sample was measured three times, and the average value was taken. The results are as follows: Figure 4 As shown.
[0042] from Figure 4The results show that the average particle size of the hippocampal exosome-like vesicles is 143.1 nm, which falls within the size range of exosomes (50-200 nm).
[0043] The extraction rate of Hip-Exo was calculated using the following formula, and the results are shown in Table 1.
[0044] Table 1: Summary of characterization data of extracted hippocampal exosome-like vesicles.
[0045] Average particle size (nm) Zeta potential (mV) Protein concentration (mg / ml) Extraction rate (particles / g seahorse meat paste) 143.1 -15 0.21 <![CDATA[4.015×10 11 ]]>
[0046] Extraction rate = number of exosome-like vesicle particles from the hippocampus / weight of the hippocampus mince (g) Formula (1).
[0047] As can be seen from the results in Table 1, the extraction rate of hippocampal exosome-like vesicles prepared in Example 1 was 4.015 × 10⁻⁶. 11 particles / g of seahorse meat paste.
[0048] (vi) Cell uptake experiments, such as Figure 5 As shown, the specific steps are as follows: ①Dil staining experiment: Dil is a lipophilic fluorescent dye.
[0049] The testing procedure included: 450 μL of exosome solution diluted with phosphate-buffered saline (PBS) was thoroughly mixed with 5 μL of 1 mM Dil dye and incubated at 37°C in the dark for 30 min. To remove excess dye, the labeled exosome vesicles were transferred to an 8-14 kDa dialysis bag and dialyzed in 100 times the volume of dialysis buffer (PBS) for 15 h. The resulting solution was then ready for subsequent uptake experiments.
[0050] ②Intake test: The testing process includes: using a density of 1×10 5 TM3 cell suspension at a concentration of [number] cells / mL was seeded in φ20mm glass dishes and incubated overnight. 500 μL of Dil-labeled Hip-Exo solution was added to the cell culture dishes and co-incubated with the cells for 24 h. Cells were washed with PBS, fixed with 4% paraformaldehyde at room temperature in the dark for 10 min, and washed again with PBS. 100 μL of DAPI staining solution was added and incubated at room temperature in the dark for 4 min to allow the staining agent to fully bind to the cells. After mixing, the cells were washed 2-3 times with PBS. Finally, the cells were imaged using a laser confocal imaging system.
[0051] The results are as follows Figure 5 As shown, exosomes stained with Dil appear red ( Figure 5Column 3), cells stained with DAPI appear blue ( Figure 5 (Column 2) The blue and red fluorescent parts in the cells after uptake are almost overlapping. Therefore, based on the position and color of the fluorescence in the figure, it can be determined that hippocampal exosome-like vesicles can be taken up and absorbed by TM3 cells.
[0052] Effect Verification 1: Experiment on Hip-Exo promoting the proliferation of mouse testicular interstitial TM3 cells: Testosterone is one of the main male hormones, and its main source is Leydig cells. It is crucial for the regulation and differentiation of testicular interstitial cells during spermatogenesis, as well as for maintaining male reproductive function and health.
[0053] The testing procedure included: mouse testicular interstitial TM3 cells were cultured in DMEM-F12 medium containing 2.5% fetal bovine serum and 5% horse serum, and incubated at 37°C in a 5% CO2 incubator. Cells were passaged after reaching the logarithmic growth phase. The effect of the synthesized hippocampal exosome-like vesicles on mouse TM3 cell viability was detected using the CCK-8 assay: After passage, TM3 cells (8 × 10⁶ cells / year) were... 4 Cells per mL were seeded into 96-well plates and cultured for 24 h with different concentrations of positive control (5, 10, 20 μM sildenafil) and different concentrations of the above vesicles (0, 1, 5, 10, 20, 50, 100 μg / mL). 10 μL of CCK-8 reagent was added to each well, and the plates were cultured for another 1 h. The OD value of each well was measured at 450 nm, and the average OD value of all parallel wells was taken.
[0054] Cell viability % = ( OD- OD) / ( OD- OD)×100% formula (3).
[0055] The results are as follows Figure 6 As shown, different concentrations of sildenafil significantly promoted the proliferation of TM3 cells, with the best effect observed at 10 μM. Different concentrations of Hip-Exo significantly promoted the proliferation of mouse TM3 cells, with 10 μg / mL Hip-Exo showing the greatest increase in cell viability, significantly better than the control group and similar to the positive control group. This indicates that hippocampal exosome-like vesicles have a significant promoting effect on the proliferation of TM3 cells.
[0056] Effect Verification 2: Experiment on Hip-Exo promoting testosterone secretion by TM3 cells in mouse testicular interstitial tissue: Leydig cells in the testes are the main cells in the human body that cause testosterone secretion. TM3 cells are a commonly used cell model in male sex research. TM3 cells can trigger testosterone production through hCG stimulation.
[0057] The test procedure included: mouse testicular interstitial TM3 cells were cultured in DMEM-F12 medium containing 2.5% fetal bovine serum and 5% horse serum, and incubated at 37°C in a 5% CO2 incubator. Cells were passaged after reaching the logarithmic growth phase. The effect of the synthesized hippocampal exosome-like vesicles on testosterone secretion from mouse TM3 cells was detected using an ELISA kit: TM3 cells (8 × 10⁶ cells / year) were... 4 TM3 cells were seeded in 96-well plates with cells per mL and stimulated with hCG (0.1 IU). The cells were then cultured for 24 h with positive control (10 μM silfenafil) and different concentrations of the above vesicles (0, 1, 5, 10, 20, 50, 100 μg / mL). The cell supernatant was collected and the testosterone secretion of TM3 cells was measured using an ELISA kit.
[0058] The results are as follows Figure 7 As shown, 5-20 μg / mL Hip-Exo significantly promoted testosterone secretion from TM3 cells. Among them, 10 μg / mL Hip-Exo showed the greatest effect in promoting testosterone secretion, which was significantly better than the control group and the positive drug group, indicating that hippocampal exosome-like vesicles have a significant promoting effect on testosterone secretion from TM3 cells.
[0059] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.
[0060] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.
[0061] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.
Claims
1. A method for preparing hippocampal exosome-like vesicles, characterized in that... Includes the following steps: (1) After cleaning, the seahorse is ground into seahorse paste using a sterile ultrafine grinder or homogenizer, mixed with phosphate buffer solution, and the supernatant is collected after standing. (2) The supernatant is centrifuged at low temperature and 2000-100000g speed until there is no bottom precipitate and the supernatant is clear. The centrifugation time is 40-110min each time. The precipitate is collected, resuspended, filtered and dialyzed to obtain a solution containing hippocampal exosome-like vesicles.
2. The preparation method according to claim 1, characterized in that: In step (1), the seahorse is washed with sterile water before being minced into seahorse meat. The seahorse meat is mixed with pre-cooled phosphate buffer solution, and the ratio of seahorse meat to phosphate buffer solution is 1g:10-15mL.
3. The preparation method according to claim 1, characterized in that: In step (1), the seahorse meat paste is mixed with the pre-cooled phosphate buffer solution for 10-15 hours, and then left to stand for 20-50 minutes after mixing.
4. The preparation method according to claim 1, characterized in that: In step (2), the filtrate is centrifuged at 4℃ and 1500-2500g for 30-40 min, and the supernatant I is collected; the supernatant I is centrifuged at 4℃ and 10000-20000g for 40-75 min, and the supernatant II is collected; the supernatant II is centrifuged at 4℃ and 100000-120000g for 70-110 min, and the precipitate is collected.
5. The preparation method according to claim 1, characterized in that: In step (2), the precipitate is resuspended in phosphate buffer and filtered sequentially through 0.45 μm and 0.22 μm filter membranes.
6. The preparation method according to claim 1, characterized in that: In step (2), the dialysis process includes: placing the exosome-like vesicles in a 300kDa dialysis bag for dialysis for 12-20 hours, with a sample to dialysis solution volume ratio of 1:
100.
7. A hippocampal exosome-like vesicle prepared by any one of claims 1-6.
8. The hippocampal exosome-like vesicle as described in claim 7, characterized in that: The hippocampal-derived exosome-like vesicles have a saucer-like structure and a particle size of 100-150 nm.
9. The hippocampal exosome-like vesicle as described in claim 7, characterized in that: Based on 1 ml of the hippocampal exosome-like vesicles, the protein content is 0.2-0.4 mg.