Construction method and application of a human immortalized sebaceous gland cell derived from the scalp

Abstract

The application discloses a construction method and application of a scalp-derived human immortalized sebaceous gland cell, and belongs to the field of biomedical technologies. A lentiviral vector is used to introduce an SV40 large T antigen coding sequence into human primary scalp sebaceous gland cells, and positive cells are obtained through puromycin screening, and are continuously passed to more than 50 generations to obtain an immortalized sebaceous gland cell line ihSZ40. The ihSZ40 cells express sebaceous gland cell-specific markers CK7, PLIN2 and MUC-1. Function verification shows that the ihSZ40 cells produce stable dose-dependent lipid synthesis responses to stimulation of oleic acid, androgens and insulin-like growth factor-1, and can construct a 3D sebaceous gland model. The ihSZ40 cell line constructed in the application is self-controllable, has high proliferation efficiency and stable functions, and can be adapted to high-throughput screening and evaluation of oil control and acne-removing functional raw materials for cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for constructing and applying immortalized sebaceous gland cells derived from the scalp. Background Technology

[0002] As the largest organ in the human body, the skin's appendages play a crucial role in maintaining skin homeostasis. Sebaceous glands, as the primary source of sebum, are mainly composed of sebaceous gland cells at different stages of differentiation. Terminally differentiated sebaceous gland cells located in the center of the gland release sebum through holopastic secretion. This sebum provides nutrients for the microorganisms colonizing the skin and forms an acidic sebum film with multiple functions, including water retention, UV protection, hair moisturizing, and regulation of microbial growth. Sebum secretion is finely regulated: insufficient secretion can lead to insufficient skin moisture, resulting in dryness and flaking; excessive secretion can cause seborrheic dermatitis, acne, and other skin problems. To deeply understand the mechanisms by which sebaceous gland cells regulate sebum synthesis and secretion, in vitro culture of sebaceous gland cells has become an indispensable research method. However, due to the limited number of passages of primary sebaceous gland cells in vitro, it is difficult to meet the needs of long-term, large-scale research. Therefore, establishing immortalized sebaceous gland cell lines has become a key technological direction in this field.

[0003] Existing technologies primarily employ two strategies to construct immortalized sebaceous gland cells: one utilizes telomerase reverse transcriptase (hTERT) to maintain telomere length and delay cell senescence; the other introduces Simian virus-40 large T antigen (SV40Tag) to disrupt cell cycle checkpoints. The immortalized cell lines obtained through these strategies are functionally close to primary cells, have a stable genetic background, and possess large-scale expansion capabilities. They provide standardized tools for mechanistic research and drug screening of sebaceous gland-related diseases such as acne, and are crucial for understanding the regulatory and pathological mechanisms of sebaceous gland function and developing targeted intervention strategies. The SZ95 cell line (constructed through SV40Tag transfection) is a commonly used immortalized cell model in basic research related to sebaceous glands. Chinese patent application CN112608947A uses hTERT technology to construct an immortalized human sebaceous gland cell line and validates its biological functions, providing an important tool for in vitro research on sebaceous glands and the treatment of related diseases. However, existing immortalized sebaceous gland cell lines still face the following technical bottlenecks in practical applications: Internationally widely used cell lines such as SZ95 often have commercial licensing restrictions or high acquisition costs, and some cell lines originate from Caucasian populations, differing from the genetic background of the Chinese population. The culture systems and detection conditions of existing cell lines (including those constructed using hTERT) are mostly optimized for basic medical or disease mechanism research, making them difficult to directly adapt to the high-throughput, low-cost active ingredient screening systems and efficacy evaluation standards required in the cosmetics field. Furthermore, existing cell lines lack targeted optimization for the cellular response characteristics required for large-scale screening of active ingredients for oil control and acne treatment in cosmetics, failing to fully meet the needs of large-scale screening and evaluation in the cosmetics industry.

[0004] In summary, there is an urgent need to develop a novel immortalized human sebaceous gland cell line that is independently controllable, cost-friendly, and has a culture system adapted to the needs of cosmetic efficacy screening, so as to provide an ideal cell model for the large-scale screening of raw materials for oil control and acne treatment in cosmetics. Summary of the Invention

[0005] This invention addresses the technical problems of existing immortalized human sebaceous gland cell lines (such as SZ95 and hTERT-constructed cell lines) having high commercial costs and poor compatibility with cosmetic screening systems and high-throughput evaluation requirements, thus failing to fully meet the needs of large-scale screening and evaluation of active ingredients for oil control and acne treatment in cosmetics. The invention aims to provide a method for constructing and applying immortalized human sebaceous gland cells derived from the scalp. This cell line is derived from human scalp sebaceous gland tissue, immortalized using SV40 transfection, and can produce stable responses to typical stimuli such as oleic acid, supporting the construction of 3D sebaceous gland models. It fills the gap in domestically developed and controllable sebaceous gland cell models, and is suitable for the efficient screening and functional evaluation of active ingredients for oil control and acne treatment in cosmetics.

[0006] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention provides a method for constructing a human immortalized sebaceous gland cell line derived from the scalp, comprising the following steps: Introducing the SV40 large T antigen coding sequence into primary sebaceous gland cells; Positive cells were obtained through screening and passaged for more than 50 generations to obtain immortalized sebaceous gland cell lines.

[0007] The coding sequence of the SV40 large T antigen is shown in SEQ ID NO.1.

[0008] A lentiviral vector containing a 5'LTR, a 3'LTR, a packaging signal Ψ, a promoter, and an antibiotic resistance selection gene was used for packaging. The lentiviral vector was packaged using psPAX2 and pMD2.G as packaging plasmids.

[0009] The primary sebaceous gland cells were derived from human scalp sebaceous gland tissue. Scalp sebaceous glands are more closely related to hair follicles and have unique microenvironment adaptability and biological characteristics. At the same time, the scalp, as a commonly discarded tissue in plastic surgery, is abundant, easy to obtain, and meets ethical requirements, providing a stable and accessible tissue source for the large-scale construction of cell lines. This reduces the cost of obtaining commercial cell lines and enables the construction of autonomous and controllable cell models.

[0010] The lentiviral infection process involves the addition of a lentiviral enhancement reagent, which is either polybrene or protamine sulfate, at a working concentration of 5-10 μg / mL. As cationic polymers, polybrene or protamine sulfate neutralizes the electrostatic repulsion between the cell membrane and viral particles, significantly improving the binding efficiency of lentivirus to target cells, thereby enhancing the transduction efficiency of the SV40 large T antigen coding sequence. For difficult-to-infect cells such as primary sebaceous gland cells, the addition of the enhancement reagent is a key technique to ensure successful immortalization, effectively reducing the MOI value required for viral infection, decreasing virus dosage, and lowering experimental costs.

[0011] The screening used puromycin as the screening reagent, with a screening concentration of 1-5 μg / mL and a screening time of 3-7 days.

[0012] It also includes the step of culturing immortalized sebaceous gland cells using an optimized culture medium; the optimized culture medium is based on high-glucose DMEM, supplemented with fetal bovine serum at a final concentration of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, insulin at 2.5-5 μg / mL, epidermal growth factor at 1-10 ng / mL, hydrocortisone at 0.25-0.5 μg / mL, and cholera toxin at 40-100 pM.

[0013] In the optimized culture medium, the final concentrations of insulin, epidermal growth factor, hydrocortisone, and cholera toxin were 5 μg / mL, 5 ng / mL, 0.4 μg / mL, and 60 pM, respectively.

[0014] Secondly, the present invention provides an immortalized sebaceous gland cell line obtained by the construction method described above.

[0015] Thirdly, the present invention provides an in vitro oil-control efficacy evaluation model based on the immortalized sebaceous gland cell line, comprising: the immortalized sebaceous gland cell line, and a lipid synthesis inducer.

[0016] The lipid synthesis inducer is oleic acid, androgen, insulin-like growth factor-1, or a combination thereof.

[0017] The concentration of oleic acid is 25-100 μM; the concentration of androgen is 100-200 nM; and the concentration of insulin-like growth factor-1 is 20-50 ng / mL.

[0018] Fourthly, this invention provides the application of the aforementioned in vitro oil-control efficacy evaluation model in the preparation of a screening kit for oil-control efficacy cosmetics.

[0019] The cosmetic efficacy screening includes oil control efficacy screening and / or acne treatment efficacy screening.

[0020] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a method for constructing immortalized sebaceous gland cell lines from the scalp. This method uses a lentiviral vector instead of traditional retroviruses, significantly improving the infection efficiency of primary sebaceous gland cells (lentiviruses can infect non-dividing cells). Simultaneously, the SV40 large T antigen effectively disrupts cell cycle checkpoints by binding to and inhibiting p53 and Rb proteins, enabling cells to acquire unlimited proliferative capacity. Validation through more than 50 passages demonstrates that this method can stably obtain immortalized sebaceous gland cell lines, solving the technical challenge of limited in vitro passages and difficulty in large-scale expansion of primary sebaceous gland cells. This provides a stable and sustainable cell source for cosmetic efficacy screening.

[0021] The immortalized sebaceous gland cell line obtained in this invention expresses sebaceous gland cell-specific markers CK7, PLIN2, and MUC-1; it exhibits a dose-dependent lipid synthesis response to stimulation with 25-100 μM oleic acid; and it also exhibits a lipid synthesis response to combined stimulation with 100-200 nM androgens and 20-50 ng / mL IGF-1; its viability after cryopreservation and thawing is >90%. This cell line provides a foundation for exploring the pathophysiology and therapeutic development of human sebaceous gland-related diseases at the molecular and cellular level.

[0022] The application provided by this invention utilizes ihSZ40 cells in the preparation of a cosmetic efficacy screening model, fully leveraging the advantages of this cell line's high proliferation efficiency and stable response to stimuli. Compared to existing cell lines (SZ95, hTERT-constructed cell lines), the culture system and detection conditions of ihSZ40 cells are specifically optimized for high-throughput screening of cosmetics. It exhibits higher tolerance (>90%) to commonly used cosmetic solvents (DMSO, ethanol) and is compatible with high-throughput screening platforms such as 96-well and 384-well plates, providing an ideal cell model for the large-scale screening of active ingredients for oil control and acne treatment in cosmetics.

[0023] Furthermore, the screening for oil-control efficacy was achieved by detecting the inhibitory effect of the test active ingredient on lipid synthesis in ihSZ40 cells, which can evaluate the regulatory ability of the active ingredient on sebum secretion; the screening for acne-reducing efficacy was achieved by detecting the inhibitory effect of the test active ingredient on lipid synthesis in ihSZ40 cells under androgen and / or IGF-1 stimulation, which can evaluate the intervention effect of the active ingredient on hormone-induced excessive sebum secretion. The ihSZ40 cells were validated to have a stable response to oleic acid, androgens, and IGF-1, providing a sensitive, reliable, and quantifiable detection system for evaluating oil-control and acne-reducing efficacy, solving the technical problem of poor compatibility between existing cell lines and cosmetic screening systems. The establishment of a 3D organoid structure of human sebaceous gland cells further expands the research and application value of this invention. Attached Figure Description

[0024] Figure 1 The images shown are bright-field morphological images of the ihSZ40 immortalized human sebaceous gland cell line of the present invention, wherein a is an image taken under a 10x objective lens and b is an image taken under a 20x objective lens. Figure 2 This is an immunofluorescence identification image of the ihSZ40 immortalized human sebaceous gland cell line of the present invention; Figure 3 The images show the Oil Red O staining results and quantitative lipid synthesis analysis of the ihSZ40 immortalized human sebaceous gland cell line of this invention after stimulation with different concentrations of oleic acid. In the images, a is the control group, b is the 25 μM oleic acid stimulation group, c is the 50 μM oleic acid stimulation group, d is the 100 μM oleic acid stimulation group, and e is the quantitative lipid synthesis analysis. Figure 4 Oil Red O staining results and quantitative analysis of lipid synthesis in ihSZ40 cells stimulated by androgens and IGF-1 are shown in the bar chart. a represents the control group, b represents the androgen + IGF-1 stimulation group, and c represents the quantitative analysis of lipid synthesis. Figure 5 Oil Red O staining results for 3D sebaceous gland globules stimulated by oleic acid and DHT; Figure 6The results of lipid droplet green staining in cells after stimulation with oleic acid and the addition of two oil-controlling ingredients are shown. A represents lipid droplet staining images of ihSZ40 cells in different treatment groups (including microscopic staining images of BC, oleic acid, oleic acid + isotretinoin (10 / 50 μM), and oleic acid + WY14643 (25 / 50 μM)). B represents the statistical graph of the relative lipid droplet content in ihSZ40 cells in different treatment groups (the amount of lipid droplet synthesis is expressed as fluorescence intensity or OD value per 100 cells). Data are expressed as mean ± standard deviation (n=3). Compared with the oleic acid-only treatment group, p<0.05, p<0.01, p<0.001. BC: blank control; OA: oleic acid. Detailed Implementation

[0025] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0026] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0027] The human scalp skin tissue used in this invention was derived from scalp samples removed during plastic surgery (informed consent obtained). 293A cells were purchased from ATCC; the lentiviral expression vector pLVX-Puro was purchased from Addgene, and the packaging plasmids psPAX2 and pMD2.G were purchased from Addgene; CK7, PLIN2, MUC-1 antibodies and fluorescent secondary antibodies were purchased from ProteinTech or Abcloneal; DMEM medium, fetal bovine serum, trypsin, and other cell culture reagents were purchased from Thermo Fisher Scientific; insulin, EGF, hydrocortisone, cholera toxin, oleic acid, DHT, IGF-1, Oil Red O, hematoxylin, and other chemical reagents were purchased from Sigma or PeproTech; Matrigel was purchased from Corning. All other conventional reagents were domestically produced analytical grade.

[0028] Example 1: Construction of an immortalized human sebaceous gland cell (1) Isolation of human primary sebaceous gland cells The obtained isolated human scalp skin tissue was disinfected with 75% alcohol; rinsed with Hanks solution containing penicillin (100 U / mL) and streptomycin (100 μg / mL); subcutaneous adipose tissue was removed with sterile ophthalmic scissors and forceps; the skin tissue was cut into small pieces of about 0.5 cm × 0.5 cm, placed in a neutral protease solution (concentration of 2.0-2.4 U / mL), and digested overnight at 4°C; the epidermis was separated from the dermis with forceps; the sebaceous gland tissue on the dermal side was dissected under a stereomicroscope using microsurgical instruments; and the separated sebaceous gland tissue was collected for later use.

[0029] (2) Culture of human primary sebaceous gland cells The sebaceous gland tissue isolated in step (1) was seeded into a culture dish coated with type I collagen; 2 mL of primary cell culture medium (high glucose DMEM + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin) was added, and the dish was placed in a 37℃, 5% CO2 incubator for static culture, with half of the culture medium replaced every 3 days. After the cells crawled out from the edge of the sebaceous gland and reached confluence, they were passaged (cells up to the 3rd generation were used to construct immortalized sebaceous gland cells). The 2nd generation primary sebaceous gland cells were taken, and the cell density was adjusted to 30-50%, and then seeded into T25 culture flasks, with 5-10 mL of cell suspension added to each flask. The flasks were incubated at 37℃, 5% CO2, and the cells were incubated until the confluence reached about 80%, at which point lentivirus infection could be performed.

[0030] (3) Construction and infection of SV40 lentivirus Cells were infected with lentiviruses based on SV40 in 293T packaging (titer ≥ 1 × 10⁻⁶). 7

[0031] (4) Stable passage and expansion of cells To improve the proliferation efficiency and long-term culture stability of immortalized sebaceous gland cells, insulin, epidermal growth factor, hydrocortisone, and cholera toxin were added to the basal medium to create an optimized medium. The specific formulation was as follows: high-glucose DMEM as the basal medium, supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 5 μg / mL insulin, 5 ng / mL epidermal growth factor, 0.4 μg / mL hydrocortisone, and 60 pM cholera toxin. All added components were purchased from Sigma-Aldrich, and the medium was sterilized by filtration through a 0.22 μm filter after preparation.

[0032] The cells were cultured continuously in the optimized culture medium described above for more than 50 generations, and the resulting immortalized sebaceous gland cell line was named ihSZ40. (See appendix) Figure 1 The ihSZ40 immortalized human sebaceous gland cell line of this invention has uniform cell morphology, exhibits an epithelial-like morphology, is tightly arranged, and has good uniformity.

[0033] Example 2: Immunofluorescence identification of ihSZ40 cells (1) Sebaceous gland cell plating IhSZ40 cells at passage 20 or less were collected. When the cell confluence reached 90%, the cells were digested with trypsin to prepare a single-cell suspension, and the cell density was adjusted to 5.0 × 10⁶ cells / year. 5 cells / mL ~ 5.0 × 10⁻⁶ 6 Cells were seeded at a density of 1 / mL into 24-well culture dishes, 500 μL of culture medium was added, the culture dishes were gently shaken to disperse the cells evenly, and then cultured at 37°C and 5% CO2.

[0034] (2) Fixation of sebaceous gland cells After culturing the cells for 24 hours, the old culture medium was aspirated, the cells were washed twice with sterile PBS, fixed with 4% paraformaldehyde for 20 minutes, and then washed with PBS.

[0035] (3) Blocking and staining of sebaceous gland cells After cell fixation, the cells were treated with blocking solution containing 5%–10% donkey serum. After cell blocking, the corresponding fluorescently labeled secondary antibody (such as Alexa Fluor 488 or 594 labeled goat anti-rabbit IgG, diluted 1:500) was added to the primary antibody species and incubated at room temperature in the dark for 1 hour.

[0036] (4) DAPI staining of sebaceous gland cells After the secondary antibody incubation, the cells were counterstained with 1-10 ug / mL DAPI for 5-10 minutes.

[0037] (5) Fluorescent imaging of sebaceous gland cells After cell staining, images are captured using a fluorescence microscope under a 20X or 40X objective lens.

[0038] Immunofluorescence Figure 2 The results showed that CK7 was positively expressed in ihSZ40 cells, with green fluorescence mainly distributed in the cytoplasm in a diffuse and uniform manner. Merge images showed that the green fluorescence of CK7 corresponded to the blue nuclei of DAPI, confirming stable expression of CK7 in ihSZ40 cells. CK7 is a characteristic marker of sebaceous gland ducts and glandular cells, and its positive expression indicates that ihSZ40 cells retain the lineage characteristics of sebaceous gland epithelial cells. MUC-1 was positively expressed in ihSZ40 cells, with red fluorescence mainly distributed in the cell membrane and cytoplasmic septum in a ring or semi-ring pattern. Merge images showed that the red fluorescence of MUC-1 corresponded to the blue nuclei of DAPI, confirming stable expression of MUC-1 in ihSZ40 cells. MUC-1 is a marker of sebaceous gland cell differentiation, involved in cell adhesion and signal transduction, and its positive expression indicates that ihSZ40 cells retain the differentiation characteristics of sebaceous gland cells. PLIN2 was positively expressed in ihSZ40 cells, with green fluorescent signals distributed in the cytoplasm as dots or granules, consistent with the distribution of lipid droplets. Merge fusion maps showed that the PLIN2 positive dotted signals surrounded the DAPI blue nuclei, exhibiting a typical "ring-like" distribution. PLIN2 is a lipid droplet surface coating protein involved in lipid metabolism and the maintenance of lipid droplet stability. Its positive expression indicates that ihSZ40 cells have active lipid synthesis and storage capabilities, preserving the lipid metabolism function of sebaceous gland cells.

[0039] Immunofluorescence identification results showed that ihSZ40 cells positively expressed CK7, PLIN2 and MUC-1, and the cell nuclei were clearly stained with DAPI, confirming that the ihSZ40 cell line constructed in this invention has typical sebaceous gland cell phenotype characteristics, providing a reliable cell model for subsequent cosmetic efficacy screening applications.

[0040] Example 3: Detection of lipid synthesis in ihSZ40 cells stimulated by oleic acid (1) Lamination of immortalized sebaceous gland cell lines ihSZ40 cells at passage 20 or less were collected. When the cell confluence reached 90%, the culture medium was discarded, and the cells were digested with 1 mL of trypsin-EDTA digestion solution (0.05% concentration) to prepare a single-cell suspension. The cell density was adjusted to 5.0 × 10⁻⁶ cells / mL.5 cells / mL ~ 5.0 × 10⁻⁶ 6 Cells were seeded at a density of 1 / mL into 24-well culture dishes. 500 μL of cell suspension was added, and the culture dishes were gently shaken to disperse the cells evenly. The dishes were then incubated at 37°C and 5% CO2 for 24 h.

[0041] (2) Oleic acid stimulates ihSZ40 cells After culturing the cells in step (1) for 24 hours, the medium was changed, and 25-100 μM oleic acid was added to the new medium. The culture dish was gently shaken and cultured for another 24 hours at 37°C and 5% CO2. An equal volume of fatty acid-free BSA solution was added to the control group. The oleic acid stock solution was prepared as a 100 mM stock solution using fatty acid-free BSA and stored at -20°C. When using, it was diluted with medium to a final concentration of 25-100 μM.

[0042] (3) Fixation of sebaceous gland cells After 24 hours of oleic acid stimulation culture, the old culture medium was aspirated, and the cells were washed twice with sterile PBS. The cells were then fixed with 4% paraformaldehyde fixative at room temperature for 20 minutes, the fixative was discarded, and the cells were washed with PBS.

[0043] (4) Staining of sebaceous gland cells After cell fixation, Oil Red O was used for staining.

[0044] (5) Hematoxylin staining of sebaceous gland cell nuclei After incubation with the secondary antibody, counterstain the cells with 0.5%-2.5% hematoxylin for 5-10 minutes. After staining, rinse with tap water for 5 minutes to make the cell nuclei appear blue.

[0045] (6) Photographs of sebaceous gland cells After cell staining, images were taken using a regular microscope under 20X or 40X objectives. The results are shown in the appendix. Figure 3 As shown.

[0046] Oil Red O staining results showed that in the control group (0 μM oleic acid), only a few small red lipid droplets were observed in ihSZ40 cells, evenly distributed in the cytoplasm. These droplets were small in size and few in number. In the 25 μM oleic acid stimulation group, the number of lipid droplets in ihSZ40 cells was significantly increased compared to the control group, and the droplets were larger. Some cells showed aggregation of small lipid droplets. In the 50 μM oleic acid stimulation group, the number and size of lipid droplets in ihSZ40 cells further increased. Most droplets were diffusely distributed, with clear cell edges and well-preserved cell morphology. In the 100 μM oleic acid stimulation group, a large number of lipid droplets accumulated in ihSZ40 cells, merging to form large droplets. These droplets were distributed in a typical "ring-like" pattern around the cell nucleus, and the Oil Red O staining depth was significantly increased, indicating a substantial increase in intracellular neutral lipid content. With the lipid content of the control group (0 μM) normalized to 1.00, the relative lipid contents of each concentration group were as follows: 25 μM oleic acid group: 2.42 ± 0.21; 50 μM oleic acid group: 3.27 ± 0.28; 100 μM oleic acid group: 11.31 ± 0.95. With increasing oleic acid stimulation concentration, the intracellular lipid content of ihSZ40 cells increased in a dose-dependent manner (R0). 2 = 0.98), where the lipid content of the 100 μM oleic acid group was 11.31 times that of the control group, and the difference was statistically significant (p<0.01).

[0047] In summary, ihSZ40 cells exhibit a stable dose-dependent lipid synthesis response to oleic acid stimulation, and the degree of lipid accumulation is positively correlated with oleic acid concentration. This characteristic indicates that ihSZ40 cells retain the responsiveness of sebaceous gland cells to lipid stimulation and can serve as an in vitro cell model for screening active ingredients for oil control: the oil-control efficacy can be evaluated by detecting the inhibitory effect of the test active ingredients on oleic acid-induced lipid synthesis.

[0048] Example 4: Combined stimulation of sebaceous gland cells by androgens and IGF-1 (1) Lamination of immortalized sebaceous gland cell lines IhSZ40 cells at passage 20 or less were collected. When the cell confluence reached 90%, the cells were digested with trypsin to prepare a single-cell suspension, and the cell density was adjusted to 5.0 × 10⁶ cells / year. 5 cells / mL ~ 5.0 × 10⁻⁶ 6 Cells were seeded at a density of 1 / mL into 24-well culture dishes, and 500 μL of cell suspension was added. The culture dishes were gently shaken to disperse the cells evenly, and then cultured at 37°C and 5% CO2 for 24 h.

[0049] (2) Androgens and IGF-1 stimulate ihSZ40 cells After 24 hours of cell culture in step (1), the medium was changed, and 100-200 nM of androgen (dihydrotestosterone, DHT) and 20-50 ng / ml of insulin-like growth factor-1 (IGF-1) were added to the new medium. The culture dish was gently shaken and cultured at 37°C and 5% CO2 for 6 days. The medium containing fresh androgen and IGF-1 was changed daily for 6 consecutive days. An equal volume of solvent was added to the control group. The androgen (dihydrotestosterone, DHT) was prepared as a 10 mM stock solution with anhydrous ethanol, and the IGF-1 was prepared as a 100 μg / mL stock solution with sterile water. An equal volume of ethanol / PBS mixed solvent (final concentration ≤0.1%) was added to the control group.

[0050] (3) Fixation of sebaceous gland cells After 6 days of cell culture, the old culture medium was aspirated, the cells were washed twice with sterile PBS, fixed with 4% paraformaldehyde for 20 minutes, and then washed with PBS.

[0051] (5) Staining of sebaceous gland cells After cell fixation, Oil Red O was used for staining.

[0052] (6) Hematoxylin staining of sebaceous gland cell nuclei After the secondary antibody incubation, the cells were counterstained with 0.5%–2.5% hematoxylin for 5–10 minutes.

[0053] (7) Photographs of sebaceous gland cells After cell staining, images were taken using a standard microscope under 20X or 40X objectives. (See Appendix) Figure 4 As shown.

[0054] Oil Red O staining results showed that in the control group, only a small number of tiny red lipid droplets were observed in ihSZ40 cells, evenly distributed in the cytoplasm. These droplets were small in size and number, reflecting the basal lipid level of sebaceous gland cells. In the combined androgen and IGF-1 stimulation group, the number of lipid droplets in ihSZ40 cells was significantly increased compared to the control group, and the droplet volume was significantly larger. Most droplets were diffusely distributed, while some droplets fused to form larger droplets. The Oil Red O staining depth was significantly increased, indicating that combined hormone stimulation induced the synthesis and accumulation of large amounts of neutral lipids within the cells. With the lipid content of the control group normalized to 1.00, the relative lipid content in the combined androgen and IGF-1 stimulation group was significantly increased. This indicates that after combined androgen and IGF-1 stimulation, the lipid content of ihSZ40 cells was 3.85 times that of the control group, a highly statistically significant difference (p<0.01).

[0055] In summary, ihSZ40 cells exhibit a stable lipid synthesis response to combined stimulation by androgens and IGF-1, with a significantly increased lipid accumulation level. This characteristic indicates that ihSZ40 cells retain the responsiveness of sebaceous gland cells to hormone regulation and can serve as an in vitro cell model for screening acne-fighting active ingredients: their acne-fighting efficacy (especially for hormone-dependent acne) can be evaluated by detecting the inhibitory effect of the test active ingredients on androgen and / or IGF-1-induced lipid synthesis.

[0056] Example 5: Construction of a 3D sebaceous gland model using ihSZ40 cells and oleic acid stimulation (1) Preparation of matrix gel culture medium The Matrigel (Corning, catalog number 354230) was diluted to a final concentration of 5-6 μg / mL using the optimized culture medium described in Example 1(4). All operations were performed on ice, using pre-cooled pipette tips and EP tubes to avoid premature solidification of the Matrigel.

[0057] (2) Plating of immortalized sebaceous gland cell lines (3D culture) IhSZ40 cells at passage 20 or less were collected. When the cell confluence reached 90%, the cells were digested with trypsin to prepare a single-cell suspension. 1 mL of 0.25% trypsin-EDTA digestion solution was added, and digestion was continued at room temperature for 2-3 minutes. After the cells became rounded and began to detach, 2 mL of serum-containing culture medium was added to stop the digestion. The cells were then gently pipetted to prepare a single-cell suspension. The cell density was adjusted to 1.0 × 10⁶ cells / year. 6 cells / mL ~ 5.0 × 10⁻⁶ 6 Centrifuge at 1 / mL and discard the supernatant. After centrifugation, resuspend the precipitate in 450 μL of the prepared substrate gel medium from step (1) (note that this should be done on ice, and your hands should only touch the top of the EP tube with your fingers). Use a pre-cooled pipette tip to aspirate 25 μL / well and inoculate it into the center of each well of a 24-well plate. Gently transfer the plate to an incubator and incubate for 30 minutes to allow the substrate gel to solidify. Finally, add 300-400 μL of pre-warmed sebaceous gland growth medium and incubate at 37°C and 5% CO2.

[0058] (3) Oleic acid stimulates sebaceous gland cell spheroids After 24 hours of cell plating and culture in step (2), the cell medium was changed, and 25-100 μM oleic acid (prepared with fatty acid-free BSA) was added to the new medium. The cells were then cultured at 37°C and 5% CO2. The medium was changed every two days, and the cells were cultured for 6 days. An equal volume of fatty acid-free BSA solution was added to the control group.

[0059] (4) Fixation of sebaceous gland cells After culturing the cells for 24 hours, the old culture medium was aspirated, the cells were washed twice with sterile PBS, fixed with 4% paraformaldehyde for 20 minutes, and then washed with PBS.

[0060] (5) Staining of sebaceous gland cells After cell fixation, Oil Red O was used for staining.

[0061] (6) Photographs of sebaceous gland cells After cell staining, images were taken using a regular microscope under 20X or 40X objectives. The results are shown in the appendix. Figure 5 As shown.

[0062] Optical microscopy revealed that ihSZ40 cells formed three-dimensional spherical structures in matrix gel, with regular cell sphere morphology and clear boundaries. Oil Red O staining showed that the control group cell spheres contained only a few lipid droplets; the oleic acid-stimulated group showed a significant increase and enlargement of lipid droplets within the cell spheres, with the droplets evenly distributed inside the cell spheres. These results indicate that ihSZ40 cells can be used to construct a 3D sebaceous gland model and exhibit a stable lipid synthesis response to oleic acid stimulation, providing a more physiologically accurate in vitro model for sebaceous gland-related research and cosmetic efficacy evaluation.

[0063] Example 6: Application of ihSZ40 cells in cosmetic efficacy screening Take ihSZ40 cells from passage 30, at a concentration of 1×10⁻⁶. 4 Cells / well were seeded into 96-well plates and cultured for 24 hours using the optimized medium from Example 2. Different concentrations of lipid-controlling active ingredients (such as isotretinoin and wy-14643) were added, and the plates were cultured for another 24 hours. Changes in lipid synthesis were detected, and the results are as follows: Figure 6 As shown, lipid droplet synthesis was abundant in the group with only oleic acid added. When oleic acid was added, the synthesis of lipid droplets was inhibited at high concentrations by the use of isotretinoin and wy-14643. The inhibitory effect was significantly weakened at low concentrations, indicating that the two have a concentration-dose dependent relationship on lipid droplet synthesis.

[0064] ihSZ40 cells exhibit a dose-dependent response to active ingredients with oil-controlling and acne-reducing effects, with a sensitive detection window and stable and reliable results. ihSZ40 cells are suitable for high-throughput screening and evaluation of active ingredients for oil-controlling and acne-reducing cosmetics.

[0065] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a human immortalized sebaceous gland cell line derived from the scalp, characterized in that, Includes the following steps: Introducing the SV40 large T antigen coding sequence into primary sebaceous gland cells; Positive cells were obtained through screening and passaged for more than 50 generations to obtain immortalized sebaceous gland cell lines encoded by the SV40 large T antigen.

2. The method for constructing immortalized human sebaceous gland cells derived from the scalp according to claim 1, characterized in that, The coding sequence of the SV40 large T antigen is shown in SEQ ID NO.1; a lentiviral vector is used, which contains a 5'LTR, a 3'LTR, a packaging signal Ψ, a promoter, and an antibiotic resistance selection gene; the lentiviral vector is packaged using psPAX2 and pMD2.G as packaging plasmids.

3. The method for constructing immortalized human sebaceous gland cells derived from the scalp according to claim 1, characterized in that, The primary sebaceous gland cells are derived from human scalp sebaceous gland tissue.

4. The method for constructing immortalized human sebaceous gland cells derived from the scalp according to claim 1, characterized in that, The lentivirus infection process involves the addition of a lentivirus enhancement reagent; the lentivirus enhancement reagent is polyglobulin or protamine sulfate, and the screening uses puromycin as the screening reagent.

5. The method for constructing immortalized human sebaceous gland cells derived from the scalp according to claim 1, characterized in that, It also includes the step of culturing immortalized sebaceous gland cells using an optimized culture medium; the optimized culture medium is based on high-glucose DMEM, supplemented with fetal bovine serum at a final concentration of 10%, penicillin at 100 U / mL, streptomycin at 100 μg / mL, insulin at 2.5-5 μg / mL, epidermal growth factor at 1-10 ng / mL, hydrocortisone at 0.25-0.5 μg / mL, and cholera toxin at 40-100 pM.

6. The method for constructing immortalized human sebaceous gland cells derived from the scalp according to claim 5, characterized in that, In the optimized culture medium, the final concentrations of insulin, epidermal growth factor, hydrocortisone, and cholera toxin were 5 μg / mL, 5 ng / mL, 0.4 μg / mL, and 60 pM, respectively.

7. The immortalized sebaceous gland cell line obtained by the construction method according to any one of claims 1-6.

8. An in vitro oil-control efficacy evaluation model, characterized in that, include: The immortalized sebaceous gland cell line of claim 7, and the lipid synthesis inducer.

9. The in vitro oil-control efficacy evaluation model according to claim 8, characterized in that, The lipid synthesis inducer is oleic acid, androgen, insulin-like growth factor-1, or a combination thereof.

10. The application of the in vitro oil-control efficacy evaluation model according to claim 8 or 9 in the preparation of an oil-control efficacy cosmetic screening kit.

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