A method for applying "astaxanthin" chitosan hydrogel to self-renewal of SSCs

Abstract

The application discloses a method for applying "astaxanthin" chitosan hydrogel to SSCs self-renewal, relates to the field of livestock and veterinary scientific research technology, and specifically relates to a method for applying "astaxanthin" chitosan hydrogel to SSCs self-renewal, which comprises preparation of hydrogels loaded with astaxanthin in different concentrations, separation and culture of mouse SSCs and expression analysis of mouse SSCs self-renewal related genes. The method for SSCs self-renewal comprises the following specific steps: S1, inoculating mouse SSCs on chitosan hydrogel loaded with astaxanthin and continuously culturing for 15 days; observing cell morphology once every 3 days; and detecting cell aging markers once every 5 days. The method uses the biological characteristics of astaxanthin and chitosan, simulates the environment required by SSCs, and realizes the goal of resisting aging and promoting proliferation of mouse SSCs. At the levels of cells and molecules, the method comprehensively analyzes the role and mechanism of astaxanthin loaded into chitosan hydrogel in maintaining mouse SSCs self-renewal.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry and veterinary science research technology, specifically to a method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs. Background Technology

[0002] Astaxanthin possesses antioxidant and anti-inflammatory properties. Studies have confirmed that it helps alleviate oxidative damage caused by high fructose intake. Astaxanthin increases testosterone, GST, SOD levels and testicular weight, reduces MDA concentration, and reverses the increase in iNOS in testicular cells. Astaxanthin can effectively regulate the dynamic balance between oxidants and antioxidants, improve testicular heat stress and reproductive toxicology damage, promote sperm capacitation and acrosome reaction, and alleviate lipopolysaccharide-damaged fertilization and embryonic development by activating the Nrf2 / HO-1 antioxidant signaling pathway. Furthermore, it has no biotoxic side effects; rats ingesting astaxanthin at high doses (700-920 mg / kg / day) in a gelatin / carbohydrate formulation showed no adverse effects. ASX regulates the NF-kappaB / SIRT1, SIRT1-p53, and SIRT1 / FOXO3a pathways, reducing kidney damage caused by oxidative stress.

[0003] Chitosan oligomers bind to the cell surface or are internalized, affecting sperm motility in a dose-dependent manner without affecting sperm motility or morphology, nor causing loss of plasma membrane integrity, generation of reactive oxygen species, or DNA damage. By loading vascular endothelial growth factor C (VEGFC) into polyanionic dextran sulfate in polycationic chitosan, and injecting VEGFC sustained-release ultrafine particles into an azoospermia mouse model, spermatogenesis in the seminiferous tubules of CS-DS-VEGFC mice was found to be significantly better than in the control group, with each tubule containing more spermatogonia and spermatocytes, as well as Ki67 and PCNA-positive cells.

[0004] Self-renewal is regulated by multiple intracellular and extracellular signaling pathways, transcription, and the cell cycle, with different cell types exhibiting different regulatory mechanisms. Self-renewal is also influenced by the extracellular environment, which not only provides spatial anchoring sites for cells but also regulates apoptosis, self-renewal, differentiation, and migration through the secretion of various factors. As the in vivo microenvironment changes at different stages of life, the mechanisms maintaining cell self-renewal also change accordingly. Activation of the GDNF / GFRa1 signaling pathway is crucial for the self-renewal of seminal vesicles (SSCs), both in vivo and in vitro. Activation of the GDNF / GFRα1 signaling pathway promotes the expression of key transcription factors for SSC self-renewal. PLZF is widely expressed in SSCs and early-differentiated spermatogonia, playing a crucial role in promoting SSC self-renewal. Previous studies have reported that the testes of PLZF knockout mice exhibit a gradual loss of spermatogonia, increased apoptosis in seminiferous tubules, and gradual disintegration of seminiferous tubule structure with age. Current research suggests that PLZF, as a transcription factor in SSCs that responds to activation of the GDNF signaling pathway, performs the function of maintaining the identity of SSCs.

[0005] Currently, a novel culture system has been developed to load astaxanthin onto chitosan hydrogels, enabling anti-aging and proliferation-promoting effects on spermatogonial stem cells. Aging is characterized by persistent cell cycle arrest, and spermatogonial stem cells consistently face the challenge of cellular senescence during in vitro culture, making it difficult to achieve the goal of long-term in vitro culture.

[0006] This study proposes a scientific hypothesis regarding the anti-aging and proliferation-promoting mechanism of astaxanthin-loaded chitosan hydrogel in mouse spermatogonial stem cells (SSCs): when astaxanthin-loaded chitosan hydrogel acts on cultured mouse SSCs, it affects the expression of certain genes, thereby achieving the goal of anti-aging and proliferation-promoting effects on mouse SSCs. Based on this scientific hypothesis, this study investigates the mechanism of action of astaxanthin-loaded chitosan hydrogel in the in vitro culture of mouse SSCs, focusing on its anti-aging and proliferation-promoting effects. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs, solving the problems mentioned in the background section.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for applying astaxanthin-based chitosan hydrogels to the self-renewal of mouse sclerosis cells (SSCs), comprising the preparation of hydrogels loaded with different concentrations of astaxanthin, the isolation and culture of mouse SSCs, and the expression analysis of genes related to the self-renewal of mouse SSCs. The specific steps of the SSCs self-renewal method are as follows: S1. Mouse SSCs were seeded on chitosan hydrogels loaded with astaxanthin and cultured continuously for 15 days. Cell morphology was observed every 3 days and cell senescence markers were detected every 5 days. Cells were collected 15 days after S2, and the proliferation genes Myc and PCNA of mouse SSCs were detected at the molecular level using quantitative PCR and Western blotting. Changes in the expression levels of S3, marker genes PLZF, and Oct4.

[0009] Optionally, the specific steps for preparing hydrogels loaded with astaxanthin at different concentrations are as follows: (1) Chitosan was placed in citric acid solution, stirred evenly, and then poured into a 48-well plate and freeze-dried for 3 days; (2) The gel was then thawed with 0.5 mol / L NaOH solution. After 4 hours, CC hydrogel was obtained and then freeze-dried after soaking in deionized water for 3 days. (3) Dissolve astaxanthin in dimethyl sulfoxide to prepare an astaxanthin stock solution with a concentration of 10 mmol / L; (4) Then dilute with ethanol to prepare three different concentrations of astaxanthin drug solutions; (5) The CC hydrogel was immersed in three different concentrations of astaxanthin solution and shaken at 100r / min for 12h in a constant temperature water bath at 37℃ to adsorb the drug, and hydrogels loaded with astaxanthin at different concentrations were obtained, namely as1-CC, as2-CC and as3-CC.

[0010] Optionally, the mixing ratio of chitosan to citric acid is 7:3.

[0011] Optionally, the concentrations of the three different concentrations of astaxanthin drug solutions are set to 10 μmol / L, 50 μmol / L and 100 μmol / L, respectively, and named as1, as2 and as3.

[0012] Optionally, the isolation and culture of mouse SSCs involves taking testicular tissue from 5-8 day old male mice, separating seminiferous tubules, and then digesting and culturing the cells with collagenase and trypsin in sequence.

[0013] Optionally, the cells in the initial stage of culture are single, round, non-adherent cells. After the sixth or seventh day of culture, the cells proliferate and the number of cells increases, and clusters of cells appear in the culture dish, shaped like grapes or mulberries. After the sixth day, the number of cells is sufficient for cell experiments.

[0014] This invention provides a method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs, which has the following beneficial effects: This study proposes a method for applying astaxanthin-loaded chitosan hydrogels to the self-renewal of spermatogonial stem cells (SSCs). This method establishes a novel culture system for anti-aging and proliferation-promoting SSCs by loading astaxanthin-loaded chitosan hydrogels. Aging is characterized by persistent cell cycle arrest, and SSCs consistently face the challenge of cell senescence during in vitro culture, making long-term in vitro culture difficult. Studies have confirmed that astaxanthin possesses multiple biological functions, including anti-inflammatory, antioxidant, anti-aging, and anti-apoptotic effects. Chitosan exhibits good biocompatibility, biosafety, low toxicity, and low immunostimulation, and is commonly used as a matrix material for bone / cartilage tissue engineering scaffolds, such as in the synthesis of hydrogels. Therefore, this study explores whether astaxanthin-loaded chitosan hydrogels can play a role in the in vitro culture of SSCs, aiming to achieve the research objective of anti-aging and proliferation-promoting long-term in vitro culture of SSCs, thus overcoming the challenges of in vitro culture and widespread application of SSCs due to senescence. By utilizing the biological properties of astaxanthin and chitosan, we simulated the environment required for the construction of mouse schizocarpine stem cells (SSCs) to achieve the goals of anti-aging and proliferation promotion of mouse SSCs. At the cellular and molecular levels, we comprehensively analyzed the role and mechanism of astaxanthin loaded into chitosan hydrogel in maintaining the self-renewal of mouse SSCs. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the process for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs. Figure 2 This is a schematic diagram of the process for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs. Figure 3 This is a schematic diagram of the gel in the experimental steps of the method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs; Figure 4 This is a schematic diagram of the cell pores in the experimental steps of the method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs. Figure 5 This is a schematic diagram of a 96-well plate used in the experimental steps of the method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0017] In the description of this invention, unless otherwise stated, "a plurality of" means two or more; the terms "upper," "lower," "left," "right," "inner," "outer," "front end," "rear end," "head," "tail," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the 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, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0018] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "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. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0019] Example 1 Please see Figure 1 This invention provides a technical solution: a method for applying astaxanthin-based chitosan hydrogels to the self-renewal of mouse stem cells (SSCs), including the preparation of hydrogels loaded with different concentrations of astaxanthin, the isolation and culture of mouse SSCs, and the expression analysis of genes related to the self-renewal of mouse SSCs. The specific steps of the SSCs self-renewal method are as follows: S1. Mouse SSCs were seeded on chitosan hydrogels loaded with astaxanthin and cultured continuously for 15 days. Cell morphology was observed every 3 days and cell senescence markers were detected every 5 days. The specific steps for preparing hydrogels loaded with astaxanthin at different concentrations are as follows: (1) Chitosan was placed in citric acid solution, stirred evenly, and then poured into a 48-well plate and freeze-dried for 3 days. The mixing ratio of chitosan to citric acid was 7:3. (2) The gel was then thawed with 0.5 mol / L NaOH solution. After 4 hours, CC hydrogel was obtained and then freeze-dried after soaking in deionized water for 3 days. (3) Dissolve astaxanthin in dimethyl sulfoxide to prepare an astaxanthin stock solution with a concentration of 10 mmol / L; (4) Then dilute with ethanol to prepare an astaxanthin drug solution with a concentration of 10 μmol / L, named as1; (5) The CC hydrogel was immersed in three different concentrations of astaxanthin solution and shaken at 100r / min for 12h in a constant temperature water bath at 37℃ to adsorb the drug and obtain hydrogels loaded with astaxanthin at different concentrations, which were designated as as1-CC. Cells were collected 15 days after S2, and the proliferation genes Myc and PCNA of mouse SSCs were detected at the molecular level using quantitative PCR and Western blotting. Changes in the expression levels of S3, marker genes PLZF, and Oct4.

[0020] Example 2 Please see Figure 1 This invention provides a technical solution: a method for applying astaxanthin-based chitosan hydrogels to the self-renewal of mouse stem cells (SSCs), including the preparation of hydrogels loaded with different concentrations of astaxanthin, the isolation and culture of mouse SSCs, and the expression analysis of genes related to the self-renewal of mouse SSCs. The specific steps of the SSCs self-renewal method are as follows: S1. Mouse SSCs were seeded on chitosan hydrogels loaded with astaxanthin and cultured continuously for 15 days. Cell morphology was observed every 3 days and cell senescence markers were detected every 5 days. The specific steps for preparing hydrogels loaded with astaxanthin at different concentrations are as follows: (1) Chitosan was placed in citric acid solution, stirred evenly, and then poured into a 48-well plate and freeze-dried for 3 days. The mixing ratio of chitosan to citric acid was 7:3. (2) The gel was then thawed with 0.5 mol / L NaOH solution. After 4 hours, CC hydrogel was obtained and then freeze-dried after soaking in deionized water for 3 days. (3) Dissolve astaxanthin in dimethyl sulfoxide to prepare an astaxanthin stock solution with a concentration of 10 mmol / L; (4) Then dilute with ethanol to prepare an astaxanthin drug solution with a concentration of 50 μmol / L, named as2; (5) The CC hydrogel was immersed in three different concentrations of astaxanthin solution and shaken at 100r / min for 12h in a constant temperature water bath at 37℃ to adsorb the drug and obtain hydrogels loaded with astaxanthin at different concentrations, which were designated as as2-CC. Cells were collected 15 days after S2, and the proliferation genes Myc and PCNA of mouse SSCs were detected at the molecular level using quantitative PCR and Western blotting. Changes in the expression levels of S3, marker genes PLZF, and Oct4.

[0021] Example 3 Please see Figure 1 This invention provides a technical solution: a method for applying astaxanthin-based chitosan hydrogels to the self-renewal of mouse stem cells (SSCs), including the preparation of hydrogels loaded with different concentrations of astaxanthin, the isolation and culture of mouse SSCs, and the expression analysis of genes related to the self-renewal of mouse SSCs. The specific steps of the SSCs self-renewal method are as follows: S1. Mouse SSCs were seeded on chitosan hydrogels loaded with astaxanthin and cultured continuously for 15 days. Cell morphology was observed every 3 days and cell senescence markers were detected every 5 days. The specific steps for preparing hydrogels loaded with astaxanthin at different concentrations are as follows: (1) Chitosan was placed in citric acid solution, stirred evenly, and then poured into a 48-well plate and freeze-dried for 3 days. The mixing ratio of chitosan to citric acid was 7:3. (2) The gel was then thawed with 0.5 mol / L NaOH solution. After 4 hours, CC hydrogel was obtained and then freeze-dried after soaking in deionized water for 3 days. (3) Dissolve astaxanthin in dimethyl sulfoxide to prepare an astaxanthin stock solution with a concentration of 10 mmol / L; (4) Then dilute with ethanol to prepare an astaxanthin drug solution with a concentration of 100 μmol / L, named as3; (5) The CC hydrogel was immersed in three different concentrations of astaxanthin solution and shaken at 100r / min for 12h in a constant temperature water bath at 37℃ to adsorb the drug and obtain hydrogels loaded with astaxanthin at different concentrations, which were designated as as3-CC. Cells were collected 15 days after S2, and the proliferation genes Myc and PCNA of mouse SSCs were detected at the molecular level using quantitative PCR and Western blotting. Changes in the expression levels of S3, marker genes PLZF, and Oct4.

[0022] The mouse seminiferous tubules (SSCs) from Examples 1, 2, and 3 were simultaneously monitored, recorded, and compared. The mouse SSCs were isolated and cultured from testicular tissue of 5-8 day old male mice. After separating the seminiferous tubules, the cells were digested and cultured sequentially with collagenase and trypsin. In the early stage of culture, the cells were single, round, non-adherent cells. After the sixth or seventh day of culture, the cells proliferated and the number of cells increased. Clusters of cells appeared in the culture dish, resembling grapes or mulberries. After the sixth day, the number of cells was sufficient for cell experiments.

[0023] The experimental steps are as follows: Prepare a 96-well plate, first as follows Figure 3 Add gel to the corresponding well as shown, incubate in an incubator for 30 min, remove, aspirate the unsolidified liquid from the well, add cells, incubate at 37℃ for 24 h, and measure cell viability using CCK8.

[0024] ; This time, there were 2000 cells per well.

[0025] Gel plating method: First, prepare 0.1%, 0.2%, and 0.3% chitosan / chitooligosaccharide solutions and 0.1% and 0.2% astaxanthin solutions. Add 20 μL of chitosan / chitooligosaccharide solution to each well, followed by 20 μL of astaxanthin solution. Mix well and incubate in a cell culture incubator at 37°C and 5% CO2 for 330 min. Remove the incubator and aspirate any unsolidified liquid. Cell plating methods: The cell suspension was counted using a hemocytometer, and then diluted to the required concentration with culture medium. 200 μL of cell-containing culture medium was added to each well, and the cck8 value was measured after 24 h and 48 h of incubation.

[0026] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for applying astaxanthin-based chitosan hydrogels to the self-renewal of mouse stem cells (SSCs), comprising the preparation of hydrogels loaded with different concentrations of astaxanthin, isolation and culture of mouse SSCs, and expression analysis of genes related to the self-renewal of mouse SSCs, characterized in that: The specific steps of the SSCs self-update method are as follows: S1. Mouse SSCs were seeded on chitosan hydrogels loaded with astaxanthin and cultured continuously for 15 days. Cell morphology was observed every 3 days and cell senescence markers were detected every 5 days. Cells were collected 15 days after S2, and the proliferation genes Myc and PCNA of mouse SSCs were detected at the molecular level using quantitative PCR and Western blotting. Changes in the expression levels of S3, marker genes PLZF, and Oct4.

2. The method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs according to claim 1, characterized in that: The specific steps for preparing hydrogels loaded with astaxanthin at different concentrations are as follows: (1) Chitosan was placed in citric acid solution, stirred evenly, and then poured into a 48-well plate and freeze-dried for 3 days; (2) The gel was then thawed with 0.5 mol / L NaOH solution. After 4 hours, CC hydrogel was obtained and then freeze-dried after soaking in deionized water for 3 days. (3) Dissolve astaxanthin in dimethyl sulfoxide to prepare an astaxanthin stock solution with a concentration of 10 mmol / L; (4) Then dilute with ethanol to prepare three different concentrations of astaxanthin drug solutions; (5) The CC hydrogel was immersed in three different concentrations of astaxanthin solution and shaken at 100r / min for 12h in a constant temperature water bath at 37℃ to adsorb the drug, and hydrogels loaded with astaxanthin at different concentrations were obtained, namely as1-CC, as2-CC and as3-CC.

3. The method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs according to claim 2, characterized in that: The mixing ratio of chitosan to citric acid is 7:

3.

4. The method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs according to claim 2, characterized in that: The concentrations of the three different astaxanthin drug solutions were set at 10 μmol / L, 50 μmol / L, and 100 μmol / L, respectively, and named as1, as2, and as3.

5. The method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs according to claim 1, characterized in that: The mouse SSCs were isolated and cultured by taking testicular tissue from 5-8 day old male mice, separating seminiferous tubules, and then digesting and culturing the cells with collagenase and trypsin in sequence.

6. The method for applying astaxanthin-based chitosan hydrogel to the self-renewal of SSCs according to claim 5, characterized in that: In the early stages of culture, the cells are single, round, non-adherent cells. After the sixth or seventh day of culture, the cells proliferate and increase in number, and clumps of cells will appear in the culture dish, resembling grapes or mulberries. After the sixth day, there are enough cells to be used for cell experiments.

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