Immortalized human hair papilla cell line and application thereof

Abstract

The invention discloses an immortalized human hair papilla cell line and application thereof, belongs to the technical field of immortalized cells, and particularly relates to a preparation method of the immortalized human hair papilla cell line. The method comprises the following steps: infecting human hair papilla cells by adopting an SV40LT overexpression vector combined with a dyeing assistant, treating the human hair papilla cells by using a puromycin screening solution, selecting monoclonal cells by using a limited dilution method, and performing amplification culture to obtain an immortalized human hair papilla cell line; the dyeing assistant comprises polybrene; compared with human hair papilla cells, the immortalized human hair papilla cell line prepared by the preparation method has higher cell activity and cell proliferation capacity, forms 3D cell balls during in-vitro 3D culture, retains the ability of inducing hair follicle formation, and can be applied to the fields of hair follicle tissue engineering, 3D cell culture or hair follicle organ construction and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immortalized cells, and in particular to an immortalized human dermal papilla cell line and applications thereof. Background Art

[0002] Dermal papilla cells (DPCs), specialized mesenchymal components of hair follicles, play a key role in hair morphogenesis and regeneration. They regulate hair follicle development and growth by serving as a reservoir of multipotent stem cells, nutrients, and growth factors. In recent years, DPCs have become a key focus of hair biology researchers. They provide an ideal model for investigating the mechanistic effects of compounds on human hair follicle function.

[0003] Dermal papilla cells have long been widely used as an in vitro screening model to evaluate the effects of hair conditioning agents at the cellular and molecular levels. However, primary dermal papilla cells have low proliferative activity and rapid senescence, and isolation of fresh cells is time-consuming and laborious. Furthermore, in vitro cultured dermal papilla cells gradually lose their ability to induce hair follicle formation with increasing passage number. Restoring the hair-inducing potential of human dermal papilla cells (hDPCs) in vitro is a major challenge in hair regeneration.

[0004] The mechanisms of cell immortalization primarily include activation of oncogenes and inactivation of tumor suppressor genes due to physical and chemical factors; stable activation of telomerase; and the introduction of oncogenic viruses such as SV40, Epstein-Barr virus, and human papillomavirus (HPV). Transfection is often used to artificially introduce exogenous immortalizing genes into target cells, knock out tumor suppressor genes, or induce mutations in immortalization-related genes to increase the incidence of immortalization, thereby establishing stable immortalized cell lines. Currently, the most widely used method for immortalizing normal cells is to integrate the SV40 large T antigen fragment into the target cell nucleus and enable its expression to create an immortalized cell line. Therefore, the development of a method for generating an immortalized human dermal papilla cell line is of great scientific and clinical significance. Summary of the Invention

[0005] The purpose of the present invention is to provide an immortalized human dermal papilla cell line and its application. Compared with human dermal papilla cells, the immortalized human dermal papilla cell line has higher cell viability and cell proliferation ability, can be stably passaged to P30 or above, and forms 3D cell spheres during in vitro 3D culture, retaining the ability to induce hair follicle formation.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are: A method for preparing an immortalized human dermal papilla cell line comprises: infecting human dermal papilla cells with an SV40 LT overexpression lentivirus in combination with a dyeing aid; treating the cells with a puromycin screening solution; then picking monoclonal cells using a limiting dilution method and expanding the culture to obtain an immortalized human dermal papilla cell line; the dyeing aid comprises at least polybrene; and in preparing the puromycin screening solution, diluting puromycin with DMEM / F12 culture medium to a final concentration of 1-10 μg / mL to obtain the puromycin screening solution.

[0007] This study uses SV40 LT-overexpressing lentivirus to infect human dermal papilla cells, generating a stably passaged immortalized human dermal papilla cell line. This immortalized human dermal papilla cell line exhibits relatively stable proliferation and functional status, exhibits no significant cellular senescence, and can be stably passaged to passages P30 and beyond. Furthermore, the immortalized human dermal papilla cell line can form 3D cell spheres in ultra-low attachment culture plates, maintaining its ability to induce hair follicles. This immortalized human dermal papilla cell line can replace limited-passage human dermal papilla cells for applications such as hair follicle tissue engineering, 3D cell culture, and hair follicle organoid construction.

[0008] Preferably, the SV40 LT overexpression lentivirus is added at a multiplicity of infection of 5-20.

[0009] Preferably, the puromycin screening solution is treated at least twice.

[0010] Preferably, the dyeing aid further comprises a quaternary ammonium surfactant equivalent to 10-30% of the mass of polybrene. The quaternary ammonium surfactant used in the present invention may significantly improve the lentiviral transfection efficiency and significantly enhance the immortalized human dermal papilla cell line by regulating the stability of the cell membrane, promoting lipid exchange, and enhancing the fusion ability of the lentivirus with the cell membrane. SV40T The relative expression level was significantly decreased and the expression of β-actin was effectively maintained in the immortalized human dermal papilla cell line.

[0011] More preferably, in the preparation of the quaternary ammonium surfactant, coconut oil-based dimethyl tertiary amine and anhydrous ethanol are mixed, first reacted with epichlorohydrin, and then reacted with oleyl dimethyl tertiary amine at a high temperature to obtain the quaternary ammonium surfactant.

[0012] More preferably, the usage ratio of coconut dimethyl tertiary amine and anhydrous ethanol is 1 g: 5-20 mL.

[0013] More preferably, the mass ratio of cocodimethyl tertiary amine to epichlorohydrin is 1:1-3.

[0014] More preferably, the mass ratio of coconut dimethyl tertiary amine to oleyl dimethyl tertiary amine is 1:1-5.

[0015] Preferably, the dyeing aid includes at least one of polybrene, a quaternary ammonium surfactant, and disodium lauryl sulfosuccinate. The present invention further introduces disodium lauryl sulfosuccinate into the dyeing aid, which may reduce the oxidative stress response generated during the transfection process and alleviate the resulting cell damage. This not only significantly improves the survival rate of transfected cells and further maintains cell proliferation activity, but also further enhances the expression efficiency of exogenous genes by improving the intracellular environment.

[0016] More preferably, the amount of the quaternary ammonium salt surfactant used is equivalent to 10-30% of the mass of the polybrene.

[0017] More preferably, the amount of disodium lauryl sulfosuccinate used is equivalent to 0.5-5% of the mass of polybrene.

[0018] More preferably, the preparation of the quaternary ammonium surfactant is specifically as follows: Coconut oil-based dimethyl tertiary amine and anhydrous ethanol are mixed, concentrated hydrochloric acid is added and stirred for 20-40 minutes, epichlorohydrin is slowly added dropwise, and the reaction is carried out at 50-60°C for 1-5 hours. After the reaction is completed, oleyl dimethyl tertiary amine is added, and the reaction is carried out at 80-100°C for 12-36 hours. After the reaction is completed, the mixture is distilled under reduced pressure, washed with anhydrous ether for 2-5 times, and repeatedly crystallized with a recrystallizing agent for 2-5 times to obtain a quaternary ammonium surfactant.

[0019] More preferably, the usage ratio of coconut dimethyl tertiary amine and anhydrous ethanol is 1 g: 5-20 mL.

[0020] More preferably, the mass concentration of concentrated hydrochloric acid is 36-38%.

[0021] More preferably, the volume ratio of anhydrous ethanol to concentrated hydrochloric acid is 1:0.02-0.1.

[0022] More preferably, the mass ratio of cocodimethyl tertiary amine to epichlorohydrin is 1:1-3.

[0023] More preferably, the mass ratio of coconut dimethyl tertiary amine to oleyl dimethyl tertiary amine is 1:1-5.

[0024] More preferably, the volume ratio of anhydrous ethanol to anhydrous ether is 1:1-2.

[0025] More preferably, the recrystallization agent comprises acetone and ethyl acetate, and the volume ratio of acetone to ethyl acetate is 1:1-5.

[0026] More preferably, the volume ratio of anhydrous ethanol to the recrystallizing agent is 1:1-2.

[0027] Preferably, a method for preparing an immortalized human dermal papilla cell line comprises: S1. Seed human dermal papilla cells in a six-well plate and add complete culture medium containing FBS. Culture at 35-37°C and 2-7% carbon dioxide for 12-36 hours. When the cell fusion rate reaches 40-50%, discard the culture medium and wash the cells with phosphate-buffered saline (PBS).

[0028] S2. After adding serum-free DMEM / F12 culture medium, add SV40 large T antigen-Lv105 LentifectTM lentiviral particles (SV40 LT overexpressing lentivirus) at a multiplicity of infection (MOI) of 5-20. After adding a dye co-infection agent for 5-6 hours, add complete culture medium containing FBS for the first time and infect for 12-36 hours. Discard the culture medium, add complete culture medium containing FBS for the second time and continue to culture for 36-72 hours. Discard the culture medium, add puromycin selection solution for a first screening for 36-72 hours, discard the culture medium, and repeat the addition of puromycin selection solution for a second screening for 36-72 hours. After the second screening, the surviving cells are collected to obtain successfully infected immortalized human dermal papilla cells.

[0029] S3. After adding trypsin-EDTA for digestion, add complete culture medium containing FBS and resuspend to obtain digested human immortalized dermal papilla cells.

[0030] S4. Dilute the digested immortalized human dermal papilla cells to a cell density of 1 cell / well using the limiting dilution method, select cell lines with good growth status for cell expansion, and observe the growth status of monoclonal cells every day using a high-content imaging system and a fluorescence microscope. When the confluence of the monoclonal cells reaches 80-90%, use trypsin-EDTA to digest the monoclonal cells and continue to culture them, and add puromycin selection solution for maintenance screening and culture; when the confluence of the monoclonal cells in the 24-well plate reaches 80-90%, use trypsin-EDTA to digest the monoclonal cells and continue to culture them, and add puromycin selection solution for maintenance screening and culture, and culture until the cell confluence reaches 80-90% to obtain an immortalized human dermal papilla cell line.

[0031] More preferably, the seeding cell density of human dermal papilla cells in step S1 is 1×10 4 -2×10 4 pieces / cm 2 .

[0032] More preferably, the amount of complete culture medium containing FBS and human dermal papilla cells used in step S1 is 1 mL: 1×10 5 -2×10 5 indivual.

[0033] More preferably, in step S2, the ratio of serum-free DMEM / F12 culture medium to the dyeing aid is 1 mL: 2-10 μg.

[0034] More preferably, in step S2, the volume ratio of the serum-free DMEM / F12 culture medium to the complete culture medium containing FBS added for the first time is 1:0.5-2.

[0035] More preferably, the volume ratio of the serum-free DMEM / F12 culture medium in step S2 to the complete culture medium containing FBS added for the second time is 1:1-4.

[0036] More preferably, in step S2, the volume ratio of serum-free DMEM / F12 culture medium to puromycin screening solution is 1:1-4.

[0037] More preferably, in step S3, the volume ratio of trypsin-EDTA to complete culture medium containing FBS is 1:1-4.

[0038] More preferably, the volume ratio of the puromycin screening solution in step S4 to the puromycin screening solution in step S2 is 1:1-2.

[0039] More preferably, the preparation of the complete culture medium containing FBS is specifically as follows: Use DMEM / F12 culture medium to dilute recombinant human insulin-like growth factor-1 (IGF-1) to a final concentration of 1-5 ng / mL, then add Australian premium fetal bovine serum (FBS) and cell culture antibodies and mix evenly to obtain complete culture medium containing FBS.

[0040] More preferably, the tertiary antibody for cell culture is penicillin-streptomycin-amphotericin B solution (100X).

[0041] More preferably, the volume ratio of Australian premium fetal bovine serum to DMEM / F12 culture medium is 1:5-20.

[0042] More preferably, the volume ratio of the cell culture triple antibody to the DMEM / F12 culture medium is 1:50-200.

[0043] More preferably, the preparation of serum-free DMEM / F12 culture medium is specifically as follows: Use DMEM / F12 culture medium to dilute recombinant human insulin-like growth factor-1 (IGF-1) to a final concentration of 1-5 ng / mL, then add cell culture antibodies and mix evenly to obtain serum-free DMEM / F12 culture medium.

[0044] More preferably, the volume ratio of the cell culture triple antibody to the DMEM / F12 culture medium is 1:50-200.

[0045] More preferably, the preparation of the puromycin screening solution is specifically as follows: Puromycin was diluted with DMEM / F12 culture medium to a final concentration of 1-10 μg / mL to obtain a puromycin screening solution.

[0046] The invention also discloses the immortalized human dermal papilla cell line prepared by the above preparation method.

[0047] The present invention also discloses the use of immortalized human dermal papilla cell lines in hair follicle tissue engineering, 3D cell culture or hair follicle organoid construction.

[0048] By infecting human dermal papilla cells with an SV40 LT-overexpressing lentivirus, the present invention produces stably passaged immortalized human dermal papilla cells. These cells exhibit relatively stable proliferation and functional status, exhibit no apparent cellular senescence, and can be stably passaged to passages P30 and beyond. Furthermore, they can form 3D cell spheres in ultra-low attachment culture plates and maintain their ability to induce hair follicles. The immortalized human dermal papilla cell line established by the present invention can replace limited-passage human dermal papilla cells and be used in research areas such as hair follicle tissue engineering, 3D cell culture, and hair follicle organoid construction.

[0049] The present invention uses polybrene, quaternary ammonium surfactant and disodium lauryl sulfosuccinate as dyeing aids, and uses them for SV40 LT overexpression lentivirus to infect human hair papilla cells, thereby having the following beneficial effects: the present invention optimizes the infection system of SV40 LT overexpression lentivirus to human hair papilla cells by using dyeing aids, significantly improving SV40T The transfection efficiency of antigens not only improves the preparation of immortalized human dermal papilla cell lines SV40T The relative expression level reached 90.1-176.4, and the proliferation rate of immortalized human dermal papilla cells reached 117.4-169.7%. While achieving efficient transfection and proliferation, it can effectively maintain the normal morphology and biological characteristics of the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a diagram of the cell morphology of the immortalized human dermal papilla cell line.

[0051] Figure 2 The wells were screened to contain only one immortalized human dermal papilla cell monoclone.

[0052] Figure 3 Wells containing three immortalized human dermal papilla cell monoclones were discarded.

[0053] Figure 4 The results of qPCR identification of immortalized human dermal papilla cell line.

[0054] Figure 5 These are the results of cell proliferation rate detection of immortalized human dermal papilla cell line and human dermal papilla cells.

[0055] Figure 6 The results are for the cell activity of human hair papilla cells.

[0056] Figure 7 These are the cell activity results of the immortalized human dermal papilla cell line.

[0057] Figure 8 The aging of human hair papilla cells.

[0058] Figure 9 This is the aging status of the immortalized human dermal papilla cell line.

[0059] Figure 10 3D cell spheroids of human hair papilla cells.

[0060] Figure 11 These are 3D cell spheroids of immortalized human dermal papilla cells.

[0061] Figure 12 The results of immunofluorescence identification of immortalized human dermal papilla cells.

[0062] Figure 13 This is the infrared spectrum of quaternary ammonium surfactant. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0064] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0065] Example 1: Preparation of complete culture medium containing FBS, including: Recombinant human insulin-like growth factor-1 (IGF-1) was diluted to a final concentration of 2.5 ng / mL using DMEM / F12 culture medium, and then Australian premium fetal bovine serum (FBS) and cell culture antibodies were added and mixed evenly to prepare a complete culture medium containing FBS. Recombinant human insulin-like growth factor-1 was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., DMEM / F12 culture medium was purchased from Thermo Fisher Scientific, and Australian premium fetal bovine serum was purchased from Gibco. TMThe tertiary antibody for cell culture was penicillin-streptomycin-amphotericin B solution (100X) purchased from Shanghai Beyotime Biotechnology Co., Ltd. The volume ratio of Australian premium fetal bovine serum to DMEM / F12 culture medium was 1:10; the volume ratio of the tertiary antibody for cell culture to DMEM / F12 culture medium was 1:100.

[0066] Preparation of serum-free DMEM / F12 culture medium, including: Recombinant human insulin-like growth factor-1 (IGF-1) was diluted in DMEM / F12 medium to a final concentration of 2.5 ng / mL, and then the cell culture antibodies were added and mixed thoroughly to obtain serum-free DMEM / F12 medium. Recombinant human insulin-like growth factor-1 was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., DMEM / F12 medium was purchased from Thermo Fisher Scientific, and the cell culture antibody (100X penicillin-streptomycin-amphotericin B solution) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The volume ratio of the cell culture antibody to DMEM / F12 medium was 1:100.

[0067] Preparation of puromycin screening solution, comprising: Puromycin was diluted in DMEM / F12 culture medium to a final concentration of 2 μg / mL.

[0068] Dyeing aids include polybrene.

[0069] A method for preparing an immortalized human dermal papilla cell line, comprising: S1. Human dermal papilla cells were seeded into a six-well plate and cultured in complete culture medium containing FBS at 37°C and 5% CO2 for 24 h. When the cell confluence rate reached 40%, the culture medium was discarded and the cells were washed with phosphate-buffered saline (PBS). Human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd. The seeding cell density of human dermal papilla cells was 2 × 10 4 pieces / cm 2 The volume of complete culture medium containing FBS and human dermal papilla cells used is 1 mL: 1×10 5 indivual.

[0070] S2. After adding serum-free DMEM / F12 culture medium, add SV40 large T antigen-Lv105 Lentifect at a multiplicity of infection (MOI) of 10. TMLentiviral particles (SV40 LT overexpressing lentivirus) were added to cells 5 hours after infection with a dye-coating agent. The cells were then inoculated with complete FBS-containing medium for a first time for 19 hours. The medium was then discarded. The cells were then inoculated with complete FBS-containing medium for a second time for another 48 hours. The medium was then discarded, and puromycin selection was added for a first selection for 48 hours. The medium was then discarded, and puromycin selection was repeated for a second selection for 48 hours. Surviving cells were harvested after the second selection to obtain successfully infected immortalized human dermal papilla cells. SV40 LT overexpressing lentivirus was purchased from Guangzhou Yijin Biotechnology Co., Ltd. The ratio of complete FBS-containing medium to dye-coating agent was 1 mL:4 μg. The volume ratio of serum-free DMEM / F12 medium to the first addition of complete FBS-containing medium was 1:1. The volume ratio of serum-free DMEM / F12 medium to the second addition of complete FBS-containing medium was 1:2. The volume ratio of serum-free DMEM / F12 culture medium and puromycin selection solution is 1:2.

[0071] S3. After digestion with trypsin-EDTA, resuspend the cells in complete culture medium containing FBS to obtain digested human dermal papilla cells. The volume ratio of trypsin-EDTA to complete culture medium containing FBS is 1:2.

[0072] S4. Dilute the digested immortalized human dermal papilla cells to a cell density of 1 cell / well using the limiting dilution method. Select well-growing cell lines for cell expansion. Observe the growth of monoclonal cells daily using a high-content imaging system and fluorescence microscopy. When the confluence of the monoclonal cells reaches 90%, digest the monoclonal cells using trypsin-EDTA and continue culturing. Add puromycin selection solution for maintenance selection. When the confluence of the monoclonal cells in the 24-well plate reaches 90%, digest the monoclonal cells using trypsin-EDTA and continue culturing. Add puromycin selection solution for maintenance selection. Cultivate until the cell confluence reaches 90%, thereby obtaining an immortalized human dermal papilla cell line. The volume ratio of puromycin selection solution to the puromycin selection solution in step S2 is 1:1.

[0073] Example 2: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0074] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0075] The preparation of the puromycin screening solution is the same as in Example 1.

[0076] The dyeing auxiliary agent is the same as that in Example 1.

[0077] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method comprises the following steps: in step S4, the immortalized human dermal papilla cells are diluted and digested to a cell density of 3 cells / well; other conditions are the same as those in Example 1.

[0078] Example 3: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0079] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0080] The preparation of the puromycin screening solution is the same as in Example 1.

[0081] The preparation of quaternary ammonium salt surfactant comprises: Coconut dimethyl tertiary amine and anhydrous ethanol were mixed, concentrated hydrochloric acid was added, and stirred for 25 minutes. Epichlorohydrin was slowly added dropwise, and the mixture was reacted at 60°C for 2.5 hours. After the reaction, oleyl dimethyl tertiary amine was added, and the mixture was reacted at 90°C for 24 hours. After the reaction, the mixture was distilled under reduced pressure, washed three times with anhydrous ether, and repeatedly crystallized three times with a recrystallizer to obtain a quaternary ammonium surfactant. The ratio of coconut dimethyl tertiary amine to anhydrous ethanol was 1g:10mL; the mass concentration of concentrated hydrochloric acid was 37%, and the volume ratio of anhydrous ethanol to concentrated hydrochloric acid was 1:0.05; the mass ratio of coconut dimethyl tertiary amine to epichlorohydrin was 1:1.5; the mass ratio of coconut dimethyl tertiary amine to oleyl dimethyl tertiary amine was 1:3; the volume ratio of anhydrous ethanol to anhydrous ether was 1:1; the recrystallizers included acetone and ethyl acetate, with the volume ratio of acetone to ethyl acetate being 1:3; and the volume ratio of anhydrous ethanol to recrystallizer was 1:1.

[0082] The dyeing auxiliary agent is the same as that in Example 1 except that a quaternary ammonium surfactant equivalent to 30% of the mass of polybrene is additionally added. Other conditions are the same as those in Example 1.

[0083] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method is the same as Example 1 except that in step S2, the dyeing aid is replaced with the dyeing aid prepared in this example. Other conditions are the same as Example 1.

[0084] Example 4: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0085] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0086] The preparation of the puromycin screening solution is the same as in Example 1.

[0087] The preparation of quaternary ammonium salt surfactant is the same as that in Example 3.

[0088] The dyeing auxiliary agent is the same as that in Example 1 except that a quaternary ammonium surfactant equivalent to 10% of the mass of polybrene is additionally added. Other conditions are the same as those in Example 1.

[0089] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method is the same as Example 1 except that in step S2, the dyeing aid is replaced with the dyeing aid prepared in this example. Other conditions are the same as Example 1.

[0090] Example 5: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0091] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0092] The preparation of the puromycin screening solution is the same as in Example 1.

[0093] The preparation of quaternary ammonium salt surfactant is the same as that in Example 3.

[0094] The dyeing auxiliary agent is the same as that in Example 1 except that a quaternary ammonium surfactant equivalent to 30% of the mass of polybrene and disodium lauryl sulfosuccinate equivalent to 5% of the mass of polybrene are additionally added. Other conditions are the same as those in Example 1.

[0095] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method is the same as Example 1 except that in step S2, the dyeing aid is replaced with the dyeing aid prepared in this example. Other conditions are the same as Example 1.

[0096] Example 6: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0097] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0098] The preparation of the puromycin screening solution is the same as in Example 1.

[0099] The preparation of quaternary ammonium salt surfactant is the same as that in Example 3.

[0100] The dyeing auxiliary agent is the same as that in Example 1 except that a quaternary ammonium surfactant equivalent to 30% of the mass of polybrene and disodium lauryl sulfosuccinate equivalent to 0.5% of the mass of polybrene are additionally added. Other conditions are the same as those in Example 1.

[0101] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method is the same as Example 1 except that in step S2, the dyeing aid is replaced with the dyeing aid prepared in this example. Other conditions are the same as Example 1.

[0102] Comparative Example 1: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0103] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0104] The preparation of the puromycin screening solution is the same as in Example 1.

[0105] The preparation of the quaternary ammonium salt surfactant was compared with Example 3, except that oleyl dimethyl tertiary amine was not used, and other conditions were the same as Example 3.

[0106] The dyeing auxiliary agent is the same as that in Example 1 except that the quaternary ammonium surfactant prepared in this example is additionally added in an amount equivalent to 30% of the mass of polybrene. Other conditions are the same as those in Example 1.

[0107] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method is the same as Example 1 except that in step S2, the dyeing aid is replaced with the dyeing aid prepared in this example. Other conditions are the same as Example 1.

[0108] Comparative Example 2: The preparation of complete culture medium containing FBS was the same as in Example 1.

[0109] The preparation of serum-free DMEM / F12 culture medium is the same as in Example 1.

[0110] The preparation of the puromycin screening solution is the same as in Example 1.

[0111] The dyeing auxiliary agent is the same as that in Example 1 except that disodium lauryl sulfosuccinate equivalent to 5% of the mass of polybrene is additionally added. Other conditions are the same as those in Example 1.

[0112] A method for preparing an immortalized human dermal papilla cell line is provided. Compared with Example 1, the method is the same as Example 1 except that in step S2, the dyeing aid is replaced with the dyeing aid prepared in this example. Other conditions are the same as Example 1.

[0113] Experimental example: 1. Cell morphology of immortalized human dermal papilla cell line The immortalized human dermal papilla cell line prepared in Example 1 was observed using an electron microscope. Figure 1 The figure is a cell morphology diagram of an immortalized human dermal papilla cell line, with a scale of 150 μm. This indicates that the immortalized human dermal papilla cell line was successfully prepared in the present invention.

[0114] The immortalized human dermal papilla cells prepared in Example 1 and Example 2 were observed using a high-content cell imaging system. Figure 2 For the wells that contain only one immortalized human dermal papilla cell monoclone, Figure 3 The discarded wells contained three immortalized human dermal papilla cell monoclones, indicating that the present invention successfully screened a monoclonal immortalized human dermal papilla cell population.

[0115] 2. qPCR experiment of immortalized human dermal papilla cells monoclonal Referring to the preparation method of Example 1, 11 monoclonal immortalized human dermal papilla cells were obtained, numbered A10, B6, B11, C7, D4, H7, A1, D1, C6, C11 and H11, and the 11 monoclonal cells were detected. SV40T The specific steps for relative expression were as follows: (1) Immortalized human dermal papilla cells were digested with trypsin-EDTA and divided into two groups, designated as group A and group B. 1×10 6 The cells were assigned to group A, and the remaining cells were assigned to group B; (2) S6, serum-free cell freezing solution was added to the cells in group A and resuspended to obtain frozen cells. Serum-free cell freezing solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., and the amount of serum-free cell freezing solution used was 1 mL; (3) RNA was extracted from the cells in group B using an RNA rapid extraction kit. After RNA extraction, 1 μg of RNA was reverse transcribed using a one-step reverse transcription kit to obtain cDNA, and then a qPCR experiment was performed using a dye-based fluorescent quantitative PCR premix to detect SV40T The relative expression level of the target gene was determined by PCR. A blank control group was set up in the qPCR experiment, and no cDNA template was added to the blank control group. Four technical replicates were performed for each group of the qPCR experiment. The RNA rapid extraction kit, one-step reverse transcription kit, and dye-based fluorescent quantitative PCR premix were all purchased from Shanghai Adamas Reagent Co., Ltd. In the qPCR experiment, the target gene was detected. SV40T The forward primer sequence is 5'-AATTTGCCCTTGGACAGGCT-3', and its nucleotide sequence is shown in SEQ ID No. 1. SV40T The reverse primer sequence is 5'-GCCTGAAATGAGCCTTGGGA-3', and its nucleotide sequence is shown in SEQ ID No. 2; it is used to detect housekeeping genes GADPH The forward primer sequence is 5'-TTGGCTACAGCAACAGGGTG-3', and its nucleotide sequence is shown in SEQ ID No. 3. GADPH The reverse primer sequence is 5'-GGGGAGATTCAGTGTGGTGG-3', and its nucleotide sequence is shown in SEQ ID No.4.

[0116] Figure 4 are the qPCR identification results of immortalized human dermal papilla cell lines. A10, B6, B11, C7, D4, H7, A1, D1, C6, C11 and H11 correspond to the qPCR identification results of 11 immortalized human dermal papilla cell monoclones. BC are the qPCR identification results of the blank control group. Figure 4 It can be seen that the immortalized human dermal papilla cell line numbered H11 SV40TThe relative expression level is the highest and can be used for subsequent expansion culture and cell bank construction.

[0117] According to the above qPCR experimental method, the immortalized human dermal papilla cell lines prepared in Examples 3-6 and Comparative Examples 1-2 were measured. SV40T Relative expression level. The immortalized human dermal papilla cell line numbered H11 prepared in Example 1 SV40T Relative expression levels, and the immortalized human dermal papilla cell lines prepared in Examples 3-6 and Comparative Examples 1-2 SV40T The relative expression levels are shown in Table 1. SV40T Relative expression levels.

[0118] Table 1 SV40T Relative expression level

[0119] As shown in Table 1, the immortalized human dermal papilla cell lines prepared in Examples 3-4 of the present invention are SV40T The relative expression level was significantly higher than that of Example 1 because, in the preparation of the immortalized human hair papilla cell line, Examples 3-4 introduced a quaternary ammonium surfactant into the dyeing aid, while Example 1 did not introduce a quaternary ammonium surfactant into the dyeing aid; the immortalized human hair papilla cell line prepared in Example 3 SV40T The relative expression level is significantly higher than that in Example 4, which is due to the different amount of quaternary ammonium surfactant used. This shows that the introduction of quaternary ammonium surfactant into the dyeing aid of the present invention helps to improve the infection effect of SV40 LT overexpression lentivirus and significantly improves the prepared immortalized human dermal papilla cell line. SV40T Relative expression levels.

[0120] Immortalized human dermal papilla cell line prepared in Example 3 of the present invention SV40T The relative expression level is significantly higher than that of Comparative Example 1 because, in the preparation of the quaternary ammonium surfactant, Example 3 uses coconut oil dimethyl tertiary amine to react with epichlorohydrin first and then with oleyl dimethyl tertiary amine to obtain the quaternary ammonium surfactant; while Comparative Example 1 only uses coconut oil dimethyl tertiary amine to react with epichlorohydrin first to obtain the quaternary ammonium surfactant. This shows that the present invention uses coconut oil dimethyl tertiary amine, epichlorohydrin and oleyl dimethyl tertiary amine to react to obtain the quaternary ammonium surfactant, which can effectively improve the infection effect of SV40 LT overexpression lentivirus and significantly improve the prepared immortalized human dermal papilla cell line. SV40T Relative expression levels.

[0121] Immortalized human dermal papilla cell lines prepared in Examples 5-6 of the present invention SV40TThe relative expression level was significantly higher than that in Example 3 because in the preparation of the immortalized human hair papilla cell line, Examples 5-6 further introduced disodium lauryl sulfosuccinate into the dyeing aid; the immortalized human hair papilla cell line prepared in Example 5 SV40T The relative expression level was significantly higher than that in Example 6, which was due to the different amount of disodium lauryl sulfosuccinate used; the immortalized human dermal papilla cell line prepared in Example 5 SV40T The relative expression level is significantly higher than that of Comparative Example 2 because, in the preparation of the immortalized human dermal papilla cell line, Example 5 synergistically introduces a quaternary ammonium surfactant and disodium lauryl sulfosuccinate into the dyeing aid, while Comparative Example 2 only introduces disodium lauryl sulfosuccinate alone. This shows that the synergistic introduction of a quaternary ammonium surfactant and disodium lauryl sulfosuccinate into the dyeing aid of the present invention helps to further improve the infection effect of the SV40 LT overexpression lentivirus, and further significantly improves the prepared immortalized human dermal papilla cell line. SV40T Relative expression levels.

[0122] 3. Cell proliferation and activity detection experiments of immortalized human dermal papilla cell lines The immortalized human dermal papilla cell line and human dermal papilla cells prepared in Example 1 were subjected to cell proliferation and activity detection experiments using a CCK-8 kit. Human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd., and CCK-8 reagents were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The specific steps were as follows: the cells were passaged to P10 and 2×10 5 DMEM / F12 culture medium was added to the cells to prepare a cell suspension; 100 μL of cell suspension was taken, and the number of cells in each well was 2×10 4 After culturing at 37°C for 24 hours, 10 µL of CCK-8 reagent was added, mixed, and incubated at 37°C for 1 hour. After the incubation, the absorbance at 450 nm was measured using a microplate reader, and the number of cells per well after incubation was calculated according to the instructions of the CCK-8 kit. The proliferation ability of the two cells was compared and the cell proliferation rate was calculated. Cell proliferation rate (%) = number of cells per well after incubation / number of cells per well before incubation × 100%, where the number of cells per well before incubation was 2 × 10 4 indivual.

[0123] Figure 5 The results of cell proliferation rate detection of immortalized human dermal papilla cell line and human dermal papilla cells are shown in Table 1. S1 is the cell proliferation rate of human dermal papilla cells, and S2 is the cell proliferation rate of immortalized human dermal papilla cell line prepared in Example 1. Figure 5 It can be seen that the cell proliferation rate of the immortalized human dermal papilla cell line prepared in Example 1 is higher than that of human dermal papilla cells, which indicates that the immortalized human dermal papilla cells prepared in the present invention proliferate faster than dermal papilla cells.

[0124] The cell proliferation rate of the immortalized human dermal papilla cell lines prepared in Examples 3-6 and Comparative Examples 1-2 was measured using the cell proliferation assay described above. The results are shown in Table 2. Table 2 shows the cell proliferation rate (%).

[0125] Table 2 Cell proliferation rate (%)

[0126] As shown in Table 2, the cell proliferation rate of the immortalized human dermal papilla cell lines prepared in Examples 3-4 of the present invention was significantly higher than that of Example 1. This is because, in the preparation of the immortalized human dermal papilla cell lines, Examples 3-4 introduced a quaternary ammonium surfactant into the dyeing auxiliaries, while Example 1 did not. The cell proliferation rate of the immortalized human dermal papilla cell line prepared in Example 3 was significantly higher than that of Example 4 due to the different amounts of quaternary ammonium surfactant used. This indicates that the introduction of a quaternary ammonium surfactant into the dyeing auxiliaries in the present invention significantly improves the cell proliferation rate of the immortalized human dermal papilla cell line prepared.

[0127] The immortalized human dermal papilla cell line prepared in Example 3 of the present invention exhibited a significantly higher cell proliferation rate than that in Comparative Example 1. This is because, in the preparation of the quaternary ammonium surfactant, Example 3 used cocodimethylamine, which was first reacted with epichlorohydrin and then with oleyldimethylamine to produce the quaternary ammonium surfactant; whereas Comparative Example 1 used only cocodimethylamine, which was first reacted with epichlorohydrin to produce the quaternary ammonium surfactant. This demonstrates that the present invention, using cocodimethylamine, epichlorohydrin, and oleyldimethylamine to produce the quaternary ammonium surfactant, can significantly enhance the cell proliferation rate of the immortalized human dermal papilla cell line.

[0128] The cell proliferation rate of the immortalized human hair papilla cell line prepared in Examples 5-6 of the present invention is significantly higher than that of Example 3 because, in the preparation of the immortalized human hair papilla cell line, Examples 5-6 further introduce disodium lauryl sulfosuccinate into the dyeing auxiliary; the cell proliferation rate of the immortalized human hair papilla cell line prepared in Example 5 is significantly higher than that of Example 6 because the amount of disodium lauryl sulfosuccinate used is different; the cell proliferation rate of the immortalized human hair papilla cell line prepared in Example 5 is significantly higher than that of Comparative Example 2 because, in the preparation of the immortalized human hair papilla cell line, Example 5 synergistically introduces a quaternary ammonium surfactant and disodium lauryl sulfosuccinate into the dyeing auxiliary, while Comparative Example 2 only introduces disodium lauryl sulfosuccinate alone. This shows that the synergistic introduction of a quaternary ammonium surfactant and disodium lauryl sulfosuccinate into the dyeing auxiliary of the present invention helps to further significantly improve the cell proliferation rate of the prepared immortalized human hair papilla cell line.

[0129] 4. Cell viability and cell counting experiments of immortalized human dermal papilla cell lines The immortalized human dermal papilla cell line and human dermal papilla cells prepared in Example 1 were subjected to cell viability and cell counting experiments using a Calcein / PI cell viability and cytotoxicity detection kit. Each experiment was repeated three times. Human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd., and the Calcein / PI cell viability and cytotoxicity detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The specific steps were as follows: the cells were subcultured to P10 in a 96-well plate, the culture medium was discarded, the cells were washed with PBS, 100 μL of Calcein AM / PI detection working solution was added and mixed, and the cells were incubated at 37°C in the dark for 30 min; the stained cells were photographed using a high-content cell imaging system, and the cell count was performed using the analysis function of the system.

[0130] Figure 6 The results of cell activity of human hair papilla cells are as follows. Figure 7 These are the cell activity results of the immortalized human dermal papilla cell line. Figure 6 and Figure 7 In comparison, the green fluorescence intensity of the immortalized human dermal papilla cell line prepared in Example 1 was stronger, indicating that the immortalized human dermal papilla cell line prepared in Example 1 exhibited higher cell viability and a significantly higher cell proliferation rate than finite-passage human dermal papilla cells. Table 3 shows the cell count results for immortalized human dermal papilla cells and human dermal papilla cells.

[0131] Table 3 Cell counting results of immortalized human dermal papilla cell lines and human dermal papilla cells

[0132] As shown in Table 3, the number of live cells in the immortalized human dermal papilla cell line prepared in Example 1 is significantly greater than that of human dermal papilla cells. This indicates that after immortalization, the cell viability of human dermal papilla cells is improved and the cell proliferation rate is also accelerated to a certain extent.

[0133] 5. β-Galactosidase staining of immortalized human dermal papilla cell line for cell senescence The immortalized human dermal papilla cell line and human dermal papilla cells prepared in Example 1 were stained for β-galactosidase using a cell senescence β-galactosidase staining kit. Human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd., and the cell senescence β-galactosidase staining kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The specific steps were as follows: the cells were passaged to P10 and 1×10 5 Add DMEM / F12 culture medium to the cells to prepare a cell suspension; add 500 μL of cell suspension to a 24-well plate to make the number of cells per well 5×10 4, mix well, culture at 37℃ for 48h, discard the culture medium, wash the cells with PBS, add 250μL of 4% paraformaldehyde solution to each well, the paraformaldehyde solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., fix at room temperature for 15min, remove the solution, wash the cells 3 times with PBS, each washing time is 3min; after washing, add 250μL of β-galactosidase staining working solution to each well, mix well, incubate at 37℃ overnight, observe and take pictures under a microscope after the incubation, and evaluate the aging of the cells.

[0134] Figure 8 The aging of human hair papilla cells, Figure 9 The aging status of immortalized human dermal papilla cell line. Figure 8 and Figure 9 It can be seen that human dermal papilla cells showed significantly high positive expression of β-galactosidase, and the positive expression level was significantly higher than that of the immortalized human dermal papilla cell line prepared in Example 1. This shows that the immortalized human dermal papilla cells prepared in Example 1 are less likely to show signs of aging.

[0135] 6. 3D cell culture experiment of immortalized human dermal papilla cell line Human dermal papilla cells were subcultured to P8, and human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd.; the immortalized human dermal papilla cell line prepared in Example 1 was subcultured to P10, and then P8 and P10 immortalized human dermal papilla cells were used for 3D cell culture, respectively. The specific steps are as follows: 1×10 5 Add DMEM / F12 culture medium to the cells to prepare a cell suspension; add 100 μL of cell suspension to a 96-well plate to make the number of cells in each well 1×10 4 The cells were cultured on a shaker at 80 rpm at 37°C for 36 h, and the 3D cell spheroids were observed by taking pictures using a high-content cell imaging system.

[0136] Figure 10 It is a 3D cell sphere of human hair papilla cells. Figure 11 It is a 3D cell spheroid of immortalized human dermal papilla cells. Figure 10 and Figure 11 It can be seen that the immortalized human dermal papilla cell line prepared in Example 1, like the P8 human dermal papilla cells, can form 3D cell spheres in the ultra-low attachment culture plate. This indicates that the immortalized human dermal papilla cell line prepared in Example 1 has not lost its ability to induce hair follicle formation.

[0137] 7. Immunofluorescence identification of immortalized human dermal papilla cell lines Human dermal papilla cells were subcultured to P12, and the human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd.; the immortalized human dermal papilla cell line prepared in Example 1 was subcultured to P8, and then immunofluorescence identification was performed using the P12 and P8 immortalized human dermal papilla cells, respectively. The specific steps were as follows: 1×10 5 Add DMEM / F12 culture medium to the cells to prepare a cell suspension; add 500µL of cell suspension to a 24-well plate to make the number of cells in each well 5x10 4 / well; mix well, place in a 37℃ incubator and culture for 24h. Discard the culture medium, add PBS, wash once, add 500μL of anhydrous methanol and place at 4℃ for 5min; use TBST buffer, TBST buffer purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., rinse three times, add 500μL of 5% goat serum solution to each well, goat serum solution purchased from Shanghai Biyuntian Biotechnology Co., Ltd., block for 1h; add 300μL of primary anti-LEPR polyclonal antibody to each well, primary anti-LEPR polyclonal antibody purchased from Wuhan Sanying Biotechnology Co., Ltd., incubate overnight at 4℃; discard the primary antibody, rinse three times with TBST buffer, add 300μL of 5% goat serum solution to each well, goat serum solution purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Cy3-labeled goat anti-rabbit IgG (H+L) secondary antibody was purchased from Shanghai Beyotime Biotechnology Co., Ltd. and incubated at room temperature for 2 h. The secondary antibody was discarded, and the cells were rinsed three times with TBST buffer. 300 μL of 1.43 μmol / L 4',6-diamidino-2-phenylindole (DAPI) was added to each well. DAPI was purchased from Aibixin (Shanghai) Biotechnology Co., Ltd. and incubated at room temperature for 10 min in the dark to stain the cell nuclei. The DAPI was discarded, and the cells were rinsed three times with TBST buffer. Then, fluorescence microscopy was used to photograph the cells.

[0138] Figure 12 The results of immunofluorescence identification of immortalized human dermal papilla cell lines are shown in Figure 2. DPCs represent dermal papilla cells, SV40T-DPCs represent immortalized dermal papilla cells, the target antibody used is LEPR, red fluorescence represents the CY3 channel, blue fluorescence represents the DAPI channel, and Merged represents the double staining channel. LEPR is mainly expressed in the cell membrane and cytoplasm of dermal papilla cells and is involved in the hair follicle induction signaling pathway. Figure 12 Immunofluorescence results showed that both immortalized and limited-passage human dermal papilla cells expressed the dermal papilla cell-specific protein LEPR, with no difference in fluorescence intensity between the two cell types. This indicates that the immortalization process did not affect the phenotypic maintenance of human dermal papilla cells; immortalized human dermal papilla cells still expressed dermal papilla cell marker proteins and retained their hair follicle-inducing properties.

[0139] 8. Material Characterization The quaternary ammonium salt surfactant prepared in Example 3 of the present invention was collected and detected using a Fourier transform infrared spectrometer.

[0140] Figure 13 This is the infrared spectrum of the quaternary ammonium surfactant, 3410 cm -1 The absorption peak of -OH appears near 2900 cm -1 The absorption peak of -CH3 appears near 2800cm -1 The absorption peak of -CH2 appears near 1720cm -1 The absorption peak of C=O appears near 1630cm -1 There is a C=C absorption peak near 1100 cm -1 The absorption peak of CO appears near

[0141] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0142] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing an immortalized human dermal papilla cell line, comprising: Human dermal papilla cells were infected with SV40 LT overexpression lentivirus in combination with a dye co-agent. After treatment with puromycin selection solution, monoclonal cells were picked by limiting dilution and expanded to obtain an immortalized human dermal papilla cell line. The dye co-agent at least included polybrene. In the preparation of the puromycin selection solution, puromycin was diluted with DMEM / F12 culture medium to a final concentration of 1-10 μg / mL to obtain the puromycin selection solution.

2. The method for preparing an immortalized human dermal papilla cell line according to claim 1, wherein: The SV40 LT overexpression lentivirus was added at a multiplicity of infection of 5-20.

3. The method for preparing an immortalized human dermal papilla cell line according to claim 1, characterized in that: The puromycin screening solution is treated at least twice.

4. The method for preparing an immortalized human dermal papilla cell line according to claim 1, characterized in that: The dyeing assistant further comprises a quaternary ammonium surfactant in an amount equivalent to 10-30% of the mass of the polybrene.

5. The method for preparing an immortalized human dermal papilla cell line according to claim 4, characterized in that: In the preparation of the quaternary ammonium salt surfactant, coconut oil-based dimethyl tertiary amine and anhydrous ethanol are mixed, first reacted with epichlorohydrin, and then reacted with oleyl dimethyl tertiary amine to obtain the quaternary ammonium salt surfactant.

6. The method for preparing an immortalized human dermal papilla cell line according to claim 5, characterized in that: The usage ratio of the coconut oil-based dimethyl tertiary amine and anhydrous ethanol is 1g:5-20mL.

7. The method for preparing an immortalized human dermal papilla cell line according to claim 5, characterized in that: The mass ratio of the coconut oil-based dimethyl tertiary amine to epichlorohydrin is 1:1-3.

8. The method for preparing an immortalized human dermal papilla cell line according to claim 5, characterized in that: The mass ratio of the coconut oil-based dimethyl tertiary amine to the oleyl dimethyl tertiary amine is 1:1-5.

9. The immortalized human dermal papilla cell line prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the immortalized human dermal papilla cell line according to claim 9 in hair follicle tissue engineering, 3D cell culture or hair follicle organoid construction.

Images