Immortalized cell line of human dermal papilla cells as well as construction method and application of immortalized cell line

Abstract

The invention relates to an immortalized cell line of human dermal papilla cells as well as a construction method and application of the immortalized cell line. The human dermal papilla cells are named as DYZ-20, the immortalized cell line is preserved in the China Center for Type Culture Collection on July 2, 2025, survival is detected, and the preservation number is CCTCC NO: C2025204. The immortalized cell line of the human dermal papilla cells can normally express alkaline phosphatase and a dermal papilla characteristic gene, and hair can grow after the immortalized cell line is transplanted into mouse skin.

Description

Technical Field

[0001] This invention relates to the field of cell biology, and in particular to an immortalized cell line of human dermal papilla cells, its construction method, and its applications. Background Technology

[0002] Dermal papilla cells (DPCs) play a crucial role in hair follicle development, hair cycle regulation, and hair regeneration, making them a key cell type for studying hair biology and related diseases. However, primary DPCs face numerous challenges in in vitro culture: their proliferative capacity is limited, making long-term maintenance difficult, and they readily lose their unique ability to induce hair follicle formation and distinctive gene expression characteristics during passage. These cultural difficulties not only limit the application of DPCs in basic research but also hinder their potential development in hair regeneration therapies and drug screening. Although some studies have attempted to construct immortalized DPC cell lines using immortalization techniques, and some have shown a response to WNT signaling, there is no direct evidence that these immortalized DPC cell lines possess hair follicle-inducing capabilities after in vivo transplantation. Most existing cell lines express characteristic genes of DPCs, but the expression of these genes does not equate to hair follicle-inducing ability.

[0003] Therefore, developing an immortalized human dermal papilla cell line that can stably maintain the characteristics of dermal papilla cells, retain the ability to induce hair follicle formation, and has high proliferative potential has become an urgent need in the fields of hair biology and regenerative medicine. Summary of the Invention

[0004] The purpose of this invention is to disclose an immortalized cell line of human dermal papillary cells, its construction method and its application, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0005] The first aspect of the present invention is to provide an immortalized cell line of human dermal papilla cells with hair follicle induction capability.

[0006] A second aspect of the present invention is to provide a method for constructing an immortalized cell line of human dermal papillary cells.

[0007] A third aspect of the present invention is to provide applications of the immortalized cell lines described in the first aspect of the present invention.

[0008] The human dermal papilla cells described in the first aspect of this invention are named DYZ-20. This immortalized cell line is deposited at the China Center for Type Culture Collection (CCTCC) on July 2, 2025, with viability confirmed. The accession number is CCTCC NO: C2025204, and the classification name is human immortalized dermal papilla cells DYZ-20. The deposit address is: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province. This immortalized cell line of human dermal papilla cells can normally express alkaline phosphatase and dermal papilla characteristic genes, and can grow hair when transplanted into mouse skin.

[0009] The construction method described in the second method of this invention is as follows: Human dermal papilla cells are transfected with a lentiviral stock solution containing the SV40-Large T gene fragment. Immortalized cells are obtained through monoclonal screening. Cells with hair follicle induction ability are screened through animal transplantation experiments to obtain the immortalized cell line. The immortalized human dermal papilla cells require further screening. Even if some express dermal papilla characteristic genes (SOX2, FN1, VCAN, ITGA8, etc.), it is still necessary to verify their true hair follicle induction ability through animal models.

[0010] In some implementations of the second aspect of the present invention, the monoclonal screening includes the following steps: 1) The lentivirus stock solution contains the anti-purinemycin gene, and cells that have completed transfection treatment are initially screened using a culture medium containing puromycin.

[0011] 2) The cells that have undergone preliminary screening are digested with trypsin and centrifuged. After discarding the supernatant, a single-cell suspension is prepared with culture medium. The cells are counted three times and the average value is taken. The suspension is then diluted to a concentration of 0.5 cells / 100μL.

[0012] 3) Seed the cell suspension into a 96-well plate, adding 100 μL of suspension to each well (theoretically ≤0.5 cells per well), and incubate at 37°C in a 5% CO2 incubator. Starting from the third day, replace the culture medium with fresh complete medium every three days, and observe the formation of monoclonal cells using an inverted microscope.

[0013] 4) Select wells containing only a single cell cluster. When the clones have grown to occupy 10-20% of the bottom of the well, perform the first expansion. Specifically, after trypsin digestion, transfer the cells to individual wells of a 24-well plate and add culture medium for continued culture. When the cells reach 70% confluence in the 24-well plate, continue digestion and expansion to 6-well plates (1 well → 1 well).

[0014] 5) Select single clones that can be expanded to 6-well plates for cryopreservation and backup, and perform continuous passage validation: each generation is passaged in 6-well plates at a ratio of 1:4 (digested when 80% confluence is reached), and the doubling time and passage number of each clone are continuously recorded until more than 40 passages are completed.

[0015] In some application embodiments of the second aspect of the present invention, the lentivirus stock solution may be a commercially available product, such as the pLenti-SV40 Large T lentivirus titer solution with product number "CI0001" provided by the PPL plasmid and protein sharing library.

[0016] In some embodiments of the second aspect of the present invention, the human dermal papillary cells are P0-P4 generation cells.

[0017] In some embodiments of the second aspect of the present invention, the density of the human dermal papillary cells is 1×10⁻⁶. 4 ~2×10 4 cells / cm 2 .

[0018] In some implementations of the second aspect of the present invention, the transfection conditions are 37°C and 5% CO2.

[0019] In some implementations of the second aspect of the present invention, the transfection duration is 5 to 12 hours.

[0020] In some implementations of the second aspect of the present invention, after transfection, the lentivirus stock solution is aspirated and replaced with a complete culture medium containing 10% FBS.

[0021] In some implementations of the second aspect of the present invention, the infection multiplicity of the lentivirus stock solution is 10 to 50.

[0022] In some embodiments of the second aspect of the invention, the lentivirus stock solution is diluted with a serum-free culture medium containing polybrene at a concentration of 8 μg / mL.

[0023] The third aspect of the application of this invention refers to the fact that the immortalized cell line of human dermal papilla cells can have hair follicle induction ability after being transplanted into the body, which can provide a more efficient and reliable experimental tool for hair regeneration and related research. Therefore, it can be used to study hair regeneration technology or prepare hair regeneration drugs. Attached Figure Description

[0024] Figure 1 This is a map of the lentiviral vector containing the SV40-Large T fragment in Example 1; Figure 2 This is a bar chart showing the relative expression levels of key genes in DYZ-20 in Example 2; Figure 3 This is a bar chart showing the relative expression levels of key genes in DYZ-7 in Example 2; Figure 4 These are photographs of the AP staining results of DYZ-20 in Example 2; Figure 5 These are photographs of the AP staining results of DYZ-7 in Example 2; Figure 6 These are photos of the transplantation experiment of DYZ-20 in Example 2. Detailed Implementation

[0025] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0027] Unless otherwise specified, the molecular biology experimental methods described in the following examples were performed in accordance with Molecular Cloning: A Laboratory Manual (3rd Edition) or the kit and product instructions. Unless otherwise specified, the biological materials used in the kits are commercially available.

[0028] Example 1: Construction of immortalized human dermal papillary cells 24 hours before the experiment, well-grown P2 generation human dermal papillary cells were injected at a rate of 1×10⁻⁶. 4 cells / cm 2 The samples were densely seeded in 6-well plates and cultured in DMEM medium containing 10% FBS.

[0029] On the day of infection, the lentiviral stock solution containing the SV40-Large T gene fragment, stored at -80℃, was slowly thawed on ice. The cell culture supernatant was discarded, and the cells were gently washed once with DPBS. The required viral volume was calculated based on MOI=10, and the virus was diluted with serum-free medium containing 8 μg / mL polybrene, with 1 mL of diluent added to each well. The cell culture plates were incubated at 37℃ in a 5% CO2 incubator for 8 hours. After incubation, the viral solution was discarded, and the culture was replaced with complete medium containing 10% FBS for further incubation.

[0030] Due to the use of lentivirus vectors such as Figure 1 As shown.

[0031] Cells that have completed transfection are screened for immortalization using the following steps: First, cells transfected with the SV40 LT fragment and preliminarily screened with puromycin were trypsinized and centrifuged. The supernatant was discarded, and a single-cell suspension was prepared using culture medium. Cells were counted three times, and the average value was taken, then diluted to a concentration of 0.5 cells / 100 μL. The cell suspension was seeded into 96-well plates, with 100 μL of suspension added to each well (theoretically ≤0.5 cells per well). The plates were incubated at 37°C in a 5% CO2 incubator. From day 3 onwards, the culture medium was replaced every three days with fresh complete medium, and the formation of single colonies was observed using an inverted microscope. Wells containing only a single cell cluster were selected, and initial expansion was performed when the colonies occupied 10-20% of the bottom of the well. Specifically, after trypsinization, the cells were transferred to individual wells of a 24-well plate, and culture medium was added for further incubation. When the cells reached 70% confluence in the 24-well plates, further digestion and expansion were performed to 6-well plates (1 well → 1 well). Single-clonal cells capable of expanding to 6-well plates were selected for cryopreservation and backup, followed by continuous passage validation: each generation was passaged in 6-well plates at a ratio of 1:4 (digested when 80% confluence was reached), and the doubling time and passage number of each clone were continuously recorded until more than 40 passages were completed. Ultimately, 46 human immortalized dermal papilla cell lines with clearly defined single-clonal origin and stable proliferative capacity were obtained.

[0032] Example 2: Screening for immortalized human dermal papilla cells with hair follicle induction capabilities (1) PCR detection verification Following the construction method provided in Example 1, 46 lines of immortalized human dermal papilla cells were used. To verify their hair follicle induction ability, RNA yield was extracted using TRIzol and quantified using Nanodrop. After synthesizing cDNA using reverse transcriptase, real-time quantitative PCR was performed using the SYBR Green method. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous marker gene, the relative expression level of each target gene was determined by 2... The ΔΔCt method was used for detection. The detection results are shown in Table 1.

[0033] Table 1. Molecular characterization of 46 human immortalized dermal papillary cells detected by qPCR

[0034] Cell lines numbered DYZ-4, DYZ-7, DYZ-11, DYZ-16, DYZ-20, DYZ-26, DYZ-27, DYZ-31, DYZ-32, DYZ-33, DYZ-37, DYZ-44, and DYZ-46 all showed detectable mRNA expression of Sox2, FN1, VCAN, and ITGA8. The detection results for the DYZ-20 and DYZ-7 human immortalized dermal papillary cell lines are as follows: Figure 2 and Figure 3As shown, this suggests that it may have the potential to induce hair follicle regeneration. Figure 2 As shown, compared with fibroblasts, the mRNA expression levels of Sox2, FN1, VCAN, and ITGA8 in DYZ-20 were significantly upregulated, reaching approximately 4, 3, 12, and 5 times that of fibroblasts, respectively. Figure 3 As shown, compared with fibroblasts, the mRNA expression levels of Sox2, FN1, VCAN and ITGA8 in DYZ-7 were significantly upregulated, reaching approximately 5, 1.5, 4.5 and 15 times that of fibroblasts, respectively.

[0035] (2) Validation of immune AP In normal human bodies, alkaline phosphatase is significantly expressed in the dermal papilla cells of hair follicles during the growth phase. This staining method is often used to indicate the location of the hair follicle papilla.

[0036] The 13 cell lines from step (1) were digested and collected, and cultured in hanging drops at a density of 1000 cells / drop. After 24 hours, the microspheres were collected and washed twice with DPBS. The cells were stained using an alkaline phosphatase (AP) staining kit. After standing at room temperature for half an hour, the cells expressing alkaline phosphatase turned purple.

[0037] The test results are shown in Table 2.

[0038] Table 2. Immunohistochemical staining results

[0039] Cell lines numbered DYZ-16, DYZ-26, DYZ-27, and DYZ-37 failed to stain successfully. DYZ-20 and DYZ-7 showed better staining results, as shown in the figure. Figure 4 and Figure 5 As shown in the figure. The results showed that both DYZ-20 and DYZ-7 expressed alkaline phosphatase normally, suggesting that they have the potential to form hair follicle papillae.

[0040] (3) Mouse model transplantation experiment Using BALB / c-nude mice as an animal model, the nine cell lines that were successfully stained in step (2) were transplanted into the skin wounds of nude mice to verify whether they had the ability to induce hair follicle formation.

[0041] 1) After anesthetizing with sodium pentobarbital, a full-thickness skin wound with a diameter of 4 mm was created on the back of 4-5 week old BALB / c-nude mice using a skin biopsy needle.

[0042] 2) The graft consists of 1×10 6 One immortalized dermal papillary cell and 1×10 6A mixture of epidermal stem cells from newborn mice was inoculated into Matrigel, incubated in a 37°C, 5% CO2 cell culture incubator for 30 minutes, and then implanted into the wounds of mice.

[0043] 3) Cover the wound with a transparent Tegaderm dressing, and then wrap it tightly with a self-adhesive elastic bandage.

[0044] 4) After 4 weeks, the mice were sacrificed and the hair growth at the wound site was observed under a stereomicroscope.

[0045] Of the nine animal models, only the mouse model implanted with DYZ-20 grew hair; the other mouse models only experienced wound healing without hair growth. The results for the DYZ-20 group are as follows: Figure 4 As shown, black hair grew at the site of wound healing. This indicates that DYZ-20 is an immortalized human dermal papillary cell with the ability to induce hair follicle formation.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. An immortalized cell line of human dermal papilla cells with hair follicle induction capability, characterized in that, The immortalized cell line is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C2025204.

2. The method for constructing the immortalized cell line according to claim 1, characterized in that, Human dermal papillary cells were transfected with a lentiviral stock solution containing the SV40-Large T gene fragment. Immortalized cells were obtained through monoclonal screening. Cells with hair follicle induction ability were screened through animal transplantation experiments to obtain the immortalized cell line.

3. The construction method according to claim 2, characterized in that, The human dermal papillary cells mentioned are P0 to P4 generation cells.

4. The construction method according to claim 2, characterized in that, The density of the human dermal papillary cells is 1×10⁻⁶. 4 ~2×10 4 cells / cm 2 .

5. The construction method according to claim 2, characterized in that, The transfection conditions were 37°C and 5% CO2.

6. The construction method according to claim 5, characterized in that, The transfection process takes 5 to 12 hours.

7. The construction method according to claim 6, characterized in that, After transfection, the lentivirus stock solution was aspirated and replaced with complete culture medium containing 10% FBS.

8. The construction method according to claim 2, characterized in that, The multiplicity of infection of the lentivirus stock solution is 10 to 50.

9. The construction method according to claim 8, characterized in that, The lentivirus stock solution was diluted with serum-free culture medium containing 8 μg / mL polybrene.

10. The use of the immortalized human dermal papillary cell line of claim 1 in the study of hair regeneration technology or the preparation of hair regeneration drugs.

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