Method for reprogramming melanocytes into fibroblast-like cells and application

Abstract

The invention discloses a method for reprogramming melanocytes into fibroblast-like cells and application, and the method comprises the following steps: inoculating melanocytes into a pore plate, and generating melanin under the induction of a conditioned medium; s2, respectively adding an astragalus extract, astragaloside, astragalus polysaccharide and astragalus flavone into the pore plate in S1, and observing the cell morphology after 48 hours; optimizing and screening conditions for growing into fibroblast-like cells, and performing high-throughput sequencing and screening on astragalus membranaceus small molecular compounds for transforming cells; the optimized astragalus membranaceus small molecule compound is used for directly reprogramming melanocytes into fibroblast-like cells, and the expression of the melanocytes and fibroblast related markers is identified through immunofluorescence. According to the method disclosed by the invention, a workshop for producing melanin is changed into a factory for producing collagen, and the safety problem caused by introducing exogenous gene editing is not involved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, particularly to the technology of chemical-induced cell reprogramming and the field of regulating cell fate using small molecule substances, and specifically relates to a method and application for reprogramming melanocytes into fibroblast-like cells. Background Art

[0002] The reprogramming of melanocytes is a frontier research field in regenerative medicine. Through chemical reprogramming technology, scientists have been able to transform skin cells into melanocytes, opening up new paths for the treatment of melanocyte dysfunction. For example, some studies have shown that a specific combination of small molecules can efficiently induce skin cells to transform into skeletal muscle cells, and Pax3 (paired box 3) has been found to be a key factor in the transformation of skin melanocytes into skeletal muscle cells during this process.

[0003] In addition, the reprogramming of melanocytes is closely related to their developmental process. These cells are derived from neural crest cells, which exhibit significant cell plasticity during embryonic development. Research has pointed out that even after melanocytes have differentiated, they can still transform into other types of neural crest cells under specific conditions, which provides a scientific basis for the reprogramming of melanocytes.

[0004] In the research of melanocyte reprogramming, the epigenetic regulation mechanism plays a crucial role. Epigenetic changes can affect the developmental fate and function of melanocytes. For example, DNA methylation and histone modification play key roles in melanin production, and the regulation of these mechanisms provides potential new targets for the reprogramming of melanocytes. In addition, the team of Li Yumei from Jiangsu University directly reprogrammed fibroblasts into melanocytes through lentiviral transfection. In-depth understanding of these epigenetic mechanisms may lead to the development of more effective treatment strategies in the future to address skin diseases caused by melanocyte dysfunction, such as vitiligo.

[0005] iPS reprogramming is to reverse cells from a terminal state to a pluripotent state using different transcription factors. However, this technology has faced some doubts in applications due to certain oncogenicity and long production cycle. At the same time, avoid transdifferentiation, that is, to transform one terminal cell into another terminal cell through appropriate transcription factors. Generally speaking, melanocyte reprogramming not only involves the process of cell transformation but is also closely related to developmental biology and epigenetics. However, there is currently no research or application on reprogramming melanocytes into fibroblasts using any small molecule compounds.

[0006] In the global skincare field, whitening and anti-aging have always been the topics that consumers are most concerned about. In the first 10 months of 2024, the number of filings for new cosmetic raw materials has exceeded the total for the whole year of 2023. However, only one new whitening raw material has been successfully registered and put into market use, and no anti-aging raw materials have been successfully registered and actually applied to products. The new whitening raw material is Thiamidol, whose chemical name is "isobutyrylaminothiazolyl resorcinol". It was developed by Beiersdorf AG in Germany with the assistance of AI technology. It mimics the process of skin whitening and melanin fading and is manufactured in the laboratory using synthetic biotechnology. Its principle is to inhibit tyrosine synthesis. Summary of the Invention

[0007] The purpose of the present invention is to provide a method and application for reprogramming melanocytes into fibroblast-like cells, so as to solve the technical problem that there is no research and application of reprogramming melanocytes into fibroblasts in the prior art.

[0008] To achieve the above purpose, the present invention provides the following technical solutions: The method for reprogramming melanocytes into fibroblast-like cells provided by the present invention includes the following steps: S1. Inoculate melanocytes in a well plate and generate melanin under the induction of a medium and insulin. S2. Add astragalus extract, astragaloside IV, astragalus polysaccharide, and astragalus flavonoids to the well plate in S1 respectively, and observe the cell morphology after 48 h. S3. Optimize and screen the conditions for growing into fibroblast-like cells, and use high-throughput sequencing to screen the astragalus small molecule compounds that can be used to transform cells. S4. Use the optimized astragalus small molecule compounds to directly reprogram melanocytes into fibroblast-like cells, and use immunofluorescence to identify the expression of melanocyte and fibroblast related markers.

[0009] Further, the induction conditions in S1 are to induce with α-MSH, high-glucose DMEM medium, DMEM / F-12, and insulin for 24 h.

[0010] Further, the components of the astragalus extract in S2 are: the content of astragaloside IV > 0.259%, calycosin-7-O-β-D-glucoside > 0.032%, and astragalus polysaccharide > 15.86%.

[0011] Further, the conditions for optimizing and screening the conditions for growing into fibroblast-like cells in S3 are specifically to screen out differential genes for gene ontology analysis, including analysis of biological processes, cellular components, and molecular functions.

[0012] Furthermore, the biological processes include DNA replication, signal transduction, and metabolic processes; the cellular components include extracellular matrix, actin filaments, actin filament bundles, stress fibers, and contractile fibers; the molecular functions include catalytic activity, binding, and enzyme regulatory activity.

[0013] Furthermore, the small molecule compounds of Astragalus membranaceus that can be used for screening transformable cells by high-throughput sequencing in S3 include the analysis of markers for Fibroblast [BTO:0000452], Melanocyte [BTO:0000847], and Melanoma cell line [BTO:0000849].

[0014] Furthermore, the characteristic genes for fibroblast differentiation in S4 are FN and VIM, and melanocytes are transformed into fibroblasts after adding Astragalus membranaceus extract and Astragalus membranaceus flavonoids.

[0015] The present invention also provides an application of a method for reprogramming melanocytes into fibroblast-like cells, in aspects of skin whitening and collagen production.

[0016] Furthermore, it has an application in the preparation of drugs or skin care products for skin healing.

[0017] It plays an important role in the skin healing process, can synthesize collagen and extracellular matrix, promote skin regeneration and reconstruction, and is used for the repair after skin injury. In the treatment of keloids, it affects the scar formation process by regulating the effect of α-MSH on fibroblasts.

[0018] Based on the above technical solutions, the embodiments of the present invention can at least produce the following technical effects: (1) The present invention relates to a method for inducing the reprogramming of melanocytes into fibroblast-like cells using small molecule chemical substances, and particularly focuses on using small molecule substances in Astragalus membranaceus to achieve this process. The application of this technology can be extended to multiple fields such as tissue engineering, cell therapy, and drug screening, and has important scientific research value and broad clinical application prospects.

[0019] (2) The present invention transforms the "workshop" for producing melanin into a "factory" for producing collagen without involving the safety issues brought by introducing exogenous gene editing.

[0020] (3) The method and application for reprogramming melanocytes into fibroblast-like cells provided by the present invention fill the gap in the research and application of reprogramming melanocytes into fibroblasts using small molecule compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0022] Figure 1 It is the morphology of different Astragalus extracts and monomers in the embodiments of the present invention on the transformation of melanocytes into fibroblast-like cells.

[0023] Figure 2 It is the sequencing analysis of the Astragalus extract in the embodiments of the present invention on the transformation of melanocytes into fibroblast-like cells.

[0024] Figure 3 It is the sequencing analysis of Astragalus flavonoids in the embodiments of the present invention on the transformation of melanocytes into fibroblast-like cells.

[0025] Figure 4 It is the sequencing analysis of astragaloside IV in the embodiments of the present invention on the transformation of melanocytes into fibroblast-like cells.

[0026] Figure 5 It is the high-throughput screening of the gene expression of fibroblasts under the Astragalus extract in the embodiments of the present invention.

[0027] Figure 6 It is the high-throughput screening of the gene expression of fibroblasts under Astragalus flavonoids in the embodiments of the present invention.

[0028] Figure 7 It is the high-throughput screening of the gene expression of fibroblasts under astragaloside IV in the embodiments of the present invention.

[0029] Figure 8 It is the comparison of classical markers of fibroblasts and melanocytes in the embodiments of the present invention. Detailed implementation manners

[0030] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] 1. Prepare high-concentration Astragalus extract, with astragaloside content >0.259%, astragaloside >0.032%, and astragalus polysaccharide >15.86%.

[0036] 2. Prepare high concentrations of astragaloside Ⅳ (>99%), astragalus flavonoids (>99%), and astragalus polysaccharides (>95%).

[0037] Example 1. Inoculate mouse melanocytes or human melanocytes in a well plate and induce them with α-MSH, DMEM high-glucose medium, DMEM / F-12, and insulin for 24 hours to allow melanin to be produced; 2. Take out astragalus extract, astragaloside IV, astragalus polysaccharide, and astragalus flavonoids and add them to each well according to the amount, and observe the cell morphology after 48 hours; 2.1 The groups were divided into control group (no astragalus component added), astragalus extract (0.5% and 1%), astragaloside IV (0.05 mg / ml and 0.25 mg / ml), astragalus polysaccharide (0.1 mg / ml and 0.5 mg / ml) and astragaloflavone (0.1 mg / ml and 0.5 mg / ml); 2.2 As Figure 1 As shown, melanocytes typically exhibit a dendritic morphology with multiple extended processes that help deliver melanin to surrounding keratinocytes, whereas fibroblasts typically have a spindle- or stellate-shaped morphology with an elongated cell body and cellular processes; 2.3 Morphology of different Astragalus membranaceus extracts and monomers in the transformation of melanocytes into fibroblast-like cells, Figure 1 As shown, Astragalus membranaceus extract, astragaloside IV, and astragalus flavonoids can all transform melanocytes from a dendritic morphology into a spindle shape, among which 1% Astragalus membranaceus extract, 0.25 mg / ml astragaloside IV, and 0.5 mg / ml astragalus flavonoids are more obvious; 3. Optimize and screen the conditions for growing into fibroblast-like cells, and use high-throughput sequencing to screen for small molecules of Astragalus membranaceus that can transform cells; 3.1 Perform mRNA sequencing, compare the control group (without adding Astragalus membranaceus components) with Astragalus membranaceus extract (1%), astragaloside IV (0.25 mg / ml), and astragalus flavonoids (0.5 mg / ml) respectively, screen for differential genes and perform GO (Gene Ontology) analysis, which are BP (Biological Process), CC (Cellular Component), and MF (Molecular Function) respectively, as Figures 2-4 shown; 3.2 Comparing Astragalus membranaceus extract with the control group, those with relatively large changes include DNA replication, signal transduction, metabolic processes, etc. in BP, extracellular matrix, actin filaments, actin filament bundles, stress fibers, contractile fibers, etc. in CC, and enzyme activity, structural molecular activity, transporter protein activity, etc. in MF; 3.3 Comparing astragalus flavonoids with the control group, those with relatively large changes include transcriptional regulation, DNA template, cell processes, etc. in BP, integral components of membranes, myofibrils, contractile fibers, stress fibers, contractile fiber parts, etc. in CC, and catalytic activity, binding, and enzyme regulatory activity, etc. in MF; 3.4 Comparing astragaloside IV with the control group, those with relatively large changes include response to low oxygen levels, neuronal death, regulation of epithelial cell differentiation, etc. in BP, contractile fibers, stress fibers, contractile fiber parts, etc. in CC, and ion channel inhibitory activity, receptor regulatory activity, signal adapter activity, etc. in MF; 3.5 Astragalus membranaceus extract, astragalus flavonoids, and astragaloside IV all have obvious effects on parts such as myofibrils, contractile fibers, stress fibers, and contractile fibers in CC; 3.6 Combine the differentially expressed genes in the high-throughput sequencing results with Fibroblast [BTO:0000452], Melanocyte [BTO:0000847], and Melanoma cell line [BTO:0000849], and analyze the gene expression of fibroblasts, melanin / melanoma cells; 3.7 As Figure 5As shown, after treatment with astragalus extract, the gene expression in fibroblasts [BTO:0000452] increased significantly and the number was large, while the markers of some melanocytes [BTO:0000847] and melanoma cell lines [BTO:0000849] decreased; 3.8 As Figure 6 As shown, after treatment with astragalus flavonoids, the gene expression in some fibroblasts [BTO:0000452] increased significantly, while the markers of melanocytes [BTO:0000847] and melanoma cell lines [BTO:0000849] decreased significantly and the number was large; 3.9 As Figure 7 As shown, after treatment with astragaloside IV, the number of genes with increased expression in fibroblasts [BTO:0000452] was slightly more, but the markers of melanocytes [BTO:0000847] and melanoma cell lines [BTO:0000849] decreased less; 3.10 As Figure 5 As shown, preferably select the astragalus extract with more increased fibroblast [BTO:0000452] markers, and astragalus flavonoids with more decreased markers of melanocytes [BTO:0000847] and melanoma cell lines [BTO:0000849]; 4. Reprogram melanocytes directly into fibroblast-like cells using the optimized astragalus small molecule compounds, and identify the expression of melanocyte and fibroblast related markers by immunofluorescence; 4.1 The characteristic gene expressions of melanocytes are TYR (Tyrosinase) and MITF (Microphthalmia-associated transcription factor), while the characteristic gene expressions of fibroblasts are FN (Fibronectin) and VIM (Vimentin). However, VIM can be detected in many neural crest cells, suggesting the expression of VIM in melanocytes.

[0038] 4.2 As Figure 8As shown, the control group basically did not express FN, but weakly expressed VIM, highly expressed TYR and MITF, and the cells were identified as melanocytes; after adding the astragalus extract, FN and VIM were strongly expressed, while TYR and MITF were weakly expressed, and the cells were identified to transform into fibroblasts; after adding astragalus flavonoids, FN was strongly expressed, while TYR and MITF were weakly expressed, and although the characteristic gene VIM was weakly expressed, combined with the sequencing results, the cells were identified to transform into fibroblasts.

[0039] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for reprogramming melanocytes into fibroblast-like cells, characterized in that: The following steps are involved: S1, melanocytes are seeded in a well plate and produce melanin under the induction of conditioned medium; S2, add astragalus extract, astragaloside IV, astragalus polysaccharide, and astragalus flavonoids to the well plate in S1, and observe the cell morphology after 48 hours; S3. Optimize and screen the conditions that can be used to grow fibroblast-like cells, and screen the Astragalus small molecule compounds that can be used to transform cells by high-throughput sequencing; S4. The optimized Astragalus small molecule compound was used to directly reprogram melanocytes into fibroblast-like cells, and the expression of melanocyte and fibroblast-related markers was identified by immunofluorescence.

2. The method for reprogramming melanocytes into fibroblast-like cells according to claim 1, characterized in that: The induction conditions in S1 are α-MSH, DMEM high-glucose medium, DMEM / F-12, and insulin induction for 24 h.

3. The method for reprogramming melanocytes into fibroblast-like cells according to claim 1, characterized in that: The astragalus extract components in S2 include astragaloside Ⅳ content>0.259%, astragalus isoflavone glucoside>0.032%, and astragalus polysaccharide>15.86%.

4. The method for reprogramming melanocytes into fibroblast-like cells according to claim 1, characterized in that: The conditions optimized and screened for growing fibroblast-like cells in S3 specifically include screening differentially expressed genes for gene ontology analysis, including analysis of biological processes, cellular components, and molecular functions.

5. The method for reprogramming melanocytes into fibroblast-like cells according to claim 4, characterized in that: The biological processes include DNA replication, signal transduction, and metabolic processes; the cell components include extracellular matrix, actin filaments, actin filament bundles, stress fibers, and contractile fibers; and the molecular functions include catalytic activity, binding, and enzyme regulatory activity.

6. The method for reprogramming melanocytes into fibroblast-like cells according to claim 1, characterized in that: The high-throughput sequencing screening of Astragalus small molecule compounds that can be used to transform cells in the S3 includes the analysis of fibroblast [BTO:0000452] markers, melanocyte [BTO:0000847] markers and melanoma cell [BTO:0000849] markers.

7. The method for reprogramming melanocytes into fibroblast-like cells according to claim 1, characterized in that: The characteristic genes of fibroblasts in the S4 are fibronectin and vimentin, and after adding astragalus extract and astragalus flavonoids, melanocytes are transformed into fibroblasts.

8. Use of the method for reprogramming melanocytes into fibroblast-like cells according to any one of claims 1 to 7, characterized in that: Application in skin whitening and collagen production.

9. Use of the method for reprogramming melanocytes into fibroblast-like cells according to claim 8, characterized in that: Application in the preparation of medicines or skin care products for healing the skin.

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