Immortalized human mammary gland cell line and its use

Abstract

The application discloses an immortalized human hair papilla cell line and application thereof, and belongs to the technical field of immortalized cells, and particularly relates to a preparation method of an immortalized human hair papilla cell line; the method comprises the following steps: infecting human hair papilla cells by using an SV40 LT overexpression vector combined with a dyeing aid, treating the human hair papilla cells by using a puromycin screening solution, picking single clone cells by using a limited dilution method, and expanding the culture of the single clone cells to obtain the immortalized human hair papilla cell line; the dyeing aid 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, and can form 3D cell balls when cultured in vitro in a 3D mode, and the immortalized human hair papilla cell line 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 organoid construction and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immortalized cell, in particular to an immortalized human dermal papilla cell line and application thereof. BACKGROUND

[0002] Dermal papilla cells (DPCs) are specialized mesenchymal components in the hair follicle that play a key role in the morphogenesis and regeneration of hair. The dermal papilla regulates hair follicle development and growth by acting as a reservoir of pluripotent stem cells, nutrients and growth factors. In recent years, dermal papilla cells have become the focus of attention for hair biologists. This cell provides an ideal model for exploring the mechanism of compounds on human hair follicle function.

[0003] Dermal papilla cells have been widely used as an in vitro screening model for evaluating the effects of hair modulators at the cellular and molecular levels for a long time. However, primary dermal papilla cells have low proliferative activity and fast senescence, and the isolation of fresh cells is both time-consuming and labor-intensive. Moreover, the characteristics of inducing hair follicle formation are gradually lost in the dermal papilla cells cultured in vitro as the number of passages increases. How to restore the hair-inducing potential of human dermal papilla cells (hDPCs) in in vitro culture is a great challenge for hair regeneration.

[0004] The mechanisms of cell immortalization mainly include: activation of proto-oncogenes / inactivation of tumor suppressor genes caused by physical and chemical factors; stable activation of telomerase; tumor viruses such as SV40 virus, Epstein-Barr virus, human papillomavirus HPV, etc. Commonly, exogenous immortalization genes are artificially introduced into target cells by transfection technology, or tumor suppressor genes are knocked out, or immortalization-related gene mutations are induced to increase the incidence of immortalization, so as to establish a stable immortalized cell line. At present, the most widely used method for immortalizing normal cells is to integrate the SV40 large T antigen fragment into the nucleus of the target cell to express it to construct an immortalized cell line. Therefore, it is of great scientific value and clinical significance to develop a preparation method for an immortalized human dermal papilla cell line. SUMMARY

[0005] The present application aims to provide an immortalized human dermal papilla cell line and application thereof, which has higher cell viability and cell proliferation capacity compared with human dermal papilla cells, can be stably passaged to P30 or more, and forms 3D cell spheres in in vitro 3D culture, retaining the ability to induce hair follicle formation.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows:

[0007] A method for preparing an immortalized human hair papilla cell line, comprising: infecting human hair papilla cells with SV40 LT overexpression lentivirus combined with a dyeing aid, treating the human hair papilla cells with a puromycin screening solution, picking single clone cells by limited dilution method and expanding the culture to obtain the immortalized human hair papilla cell line; the dyeing aid at least comprises polybrene; in the preparation of the puromycin screening solution, DMEM / F12 culture solution is used to dilute puromycin to a final concentration of 1-10 μg / mL to obtain the puromycin screening solution.

[0008] The present application uses SV40 LT overexpression lentivirus to infect human hair papilla cells to obtain an immortalized human hair papilla cell line which can be stably passaged, has relatively stable proliferation characteristics and functional state, has no obvious cell aging phenomenon and can be stably passaged to P30 or more; and the immortalized human hair papilla cell line can form 3D cell spheres in an ultra-low adsorption culture plate and maintain hair follicle induction capacity. The immortalized human hair papilla cell line established by the present application can replace limited passage human hair papilla cells and be used in application fields such as hair follicle tissue engineering, 3D cell culture and hair follicle organoid construction.

[0009] Preferably, the SV40 LT overexpression lentivirus is added at a dose with a multiplicity of infection of 5-20.

[0010] Preferably, the puromycin screening solution is treated at least twice.

[0011] Preferably, the dyeing aid further comprises a quaternary ammonium salt surfactant accounting for 10-30% of the mass of polybrene. The quaternary ammonium salt surfactant used in the present application can promote lipid exchange by adjusting the stability of the cell membrane, enhance the fusion ability of the lentivirus with the cell membrane, thereby significantly improving the lentivirus transfection efficiency and significantly enhancing the relative expression level of the obtained immortalized human hair papilla cell line and effectively maintaining the stable proliferation of the immortalized human hair papilla cell line. SV40T

[0012] More preferably, in the preparation of the quaternary ammonium salt surfactant, cocodimethyl tertiary amine and anhydrous ethanol are mixed, then reacted with epichlorohydrin, and then reacted with oleyl dimethyl tertiary amine at high temperature to obtain the quaternary ammonium salt surfactant.

[0013] More preferably, the mass ratio of cocodimethyl tertiary amine to anhydrous ethanol is 1g:5-20mL.

[0014] More preferably, the mass ratio of cocodimethyl tertiary amine to epichlorohydrin is 1:1-3.

[0015] More preferably, the mass ratio of cocodimethyl tertiary amine to oleyl dimethyl tertiary amine is 1:1-5.

[0016] ​Preferably, the dyeing aid includes at least one of polybrene, quaternary ammonium salt surfactant and disodium lauryl sulfosuccinate. The present application further introduces disodium lauryl sulfosuccinate into the dyeing aid, which can reduce the oxidative stress generated during transfection, alleviate the cell damage caused thereby, not only significantly improve the survival rate of cells after transfection, but also further maintain the cell proliferation activity, and further enhance the expression efficiency of exogenous genes by improving the intracellular environment.

[0017] More preferably, the quaternary ammonium salt surfactant is used in an amount equivalent to 10-30% of the mass of polybrene.

[0018] More preferably, the disodium lauryl sulfosuccinate is used in an amount equivalent to 0.5-5% of the mass of polybrene.

[0019] More preferably, the quaternary ammonium salt surfactant is prepared, in particular,

[0020] The cocodimethyl tertiary amine and anhydrous ethanol are mixed, concentrated hydrochloric acid is added and stirred for 20-40 min, epoxy chloropropane is slowly added, and the reaction is carried out at 50-60℃ for 1-5 h. After the reaction is completed, oil-based dimethyl tertiary amine is added, and the reaction is carried out at 80-100℃ for 12-36 h. After the reaction is completed, vacuum distillation is carried out, washed with anhydrous ether for 2-5 times, and recrystallized with a recrystallization agent for 2-5 times to obtain the quaternary ammonium salt surfactant.

[0021] More preferably, the ratio of the amount of cocodimethyl tertiary amine to anhydrous ethanol is 1g:5-20mL.

[0022] More preferably, the mass concentration of the concentrated hydrochloric acid is 36-38%.

[0023] More preferably, the volume ratio of anhydrous ethanol to concentrated hydrochloric acid is 1:0.02-0.1.

[0024] More preferably, the mass ratio of cocodimethyl tertiary amine to epoxy chloropropane is 1:1-3.

[0025] More preferably, the mass ratio of cocodimethyl tertiary amine to oil-based dimethyl tertiary amine is 1:1-5.

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

[0027] More preferably, the recrystallization agent includes acetone and ethyl acetate, and the volume ratio of acetone to ethyl acetate is 1:1-5.

[0028] More preferably, the volume ratio of anhydrous ethanol to recrystallization agent is 1:1-2.

[0029] Preferably, a method for preparing an immortalized human hair papilla cell line, in particular,

[0030] S1, inoculate human hair papilla cells into a six-well plate, add complete culture medium containing FBS, and culture at 35-37℃ and a carbon dioxide concentration of 2-7% for 12-36h, until the cell fusion rate reaches 40-50%, discard the culture medium, and wash the cells with phosphate buffered saline (PBS).

[0031] S2, after adding serum-free DMEM / F12 culture medium, add SV40 large T antigen-Lv105 LentifectTM lentivirus particles (SV40 LT overexpression lentivirus) at a multiplicity of infection (MOI) value of 5-20, add a staining aid for 5-6h of infection, add complete culture medium containing FBS for the first time for 12-36h of infection, discard the culture medium, add complete culture medium containing FBS for the second time for 36-72h of continuous culture, discard the culture medium, add puromycin screening solution for 36-72h of primary screening, discard the culture medium, repeat the addition of puromycin screening solution for 36-72h of secondary screening, and collect the surviving cells after the secondary screening is completed to obtain immortalized human hair papilla cells that have been successfully infected.

[0032] S3, after adding trypsin-EDTA digestion, resuspend with complete culture medium containing FBS to obtain digested human immortalized hair papilla cells.

[0033] S4, use the limiting dilution method to dilute the digested immortalized human hair papilla cells to a cell density of 1 per well, select cell strains with good growth states for cell expansion, and observe the growth state of the single clone cells every day using a high-content imaging system and a fluorescence microscope. When the confluence of the single clone cells reaches 80-90%, use trypsin-EDTA to digest and subculture the single clone cells for continued culture, and add puromycin screening solution for maintenance screening culture. When the confluence of the single clone cells in the 24-well plate reaches 80-90%, use trypsin-EDTA to digest and subculture the single clone cells for continued culture, and add puromycin screening solution for maintenance screening culture. Culture until the cell confluence reaches 80-90% to obtain an immortalized human hair papilla cell line.

[0034] More preferably, the inoculation cell density of human hair papilla cells in step S1 is 1×10 4 -2×10 4 / cm 2 .

[0035] More preferably, the use amount of complete culture medium containing FBS and human hair papilla cells in step S1 is 1mL: 1×10 5 -2×10 5 .

[0036] More preferably, the volume ratio of the serum-free DMEM / F12 culture solution and the first time adding FBS-containing complete culture solution in step S2 is 1:0.5-2.

[0037] More preferably, the volume ratio of the serum-free DMEM / F12 culture solution and the first time adding FBS-containing complete culture solution in step S2 is 1:0.5-2.

[0038] More preferably, the volume ratio of the serum-free DMEM / F12 culture solution and the second time adding FBS-containing complete culture solution in step S2 is 1:1-4.

[0039] More preferably, the volume ratio of the serum-free DMEM / F12 culture solution and the second time adding FBS-containing complete culture solution in step S2 is 1:1-4.

[0040] More preferably, the volume ratio of the serum-free DMEM / F12 culture solution and the second time adding FBS-containing complete culture solution in step S2 is 1:1-4.

[0041] More preferably, the volume ratio of the serum-free DMEM / F12 culture solution and the second time adding FBS-containing complete culture solution in step S2 is 1:1-4.

[0042] More preferably, the preparation of the FBS-containing complete culture solution is specifically,

[0043] The recombinant human insulin-like cell growth factor-1 (IGF-1) is diluted with DMEM / F12 culture solution to a final concentration of 1-5 ng / mL, and then the Australian special fetal bovine serum (FBS) and cell culture three-antibody solution are added and mixed uniformly to obtain the FBS-containing complete culture solution.

[0044] More preferably, the cell culture three-antibody solution is penicillin-streptomycin-amphotericin B solution (100X).

[0045] More preferably, the volume ratio of the Australian special fetal bovine serum and the DMEM / F12 culture solution is 1:5-20.

[0046] More preferably, the volume ratio of the cell culture three-antibody solution and the DMEM / F12 culture solution is 1:50-200.

[0047] More preferably, the preparation of the serum-free DMEM / F12 culture solution is specifically,

[0048] The recombinant human insulin-like cell growth factor-1 (IGF-1) is diluted with DMEM / F12 culture solution to a final concentration of 1-5 ng / mL, and then the cell culture three-antibody solution is added and mixed uniformly to obtain the serum-free DMEM / F12 culture solution.

[0049] More preferably, the volume ratio of the cell culture tertiary antibody and DMEM / F12 culture solution is 1:50-200.

[0050] More preferably, the preparation of the puromycin screening solution is specifically,

[0051] The puromycin is diluted with DMEM / F12 culture solution to a final concentration of 1-10 μg / mL to obtain the puromycin screening solution.

[0052] The application further discloses the immortalized human hair papilla cell line prepared by the preparation method.

[0053] The application further discloses applications of the immortalized human hair papilla cell line in hair follicle tissue engineering, 3D cell culture or hair follicle organoid construction.

[0054] The application has the following beneficial effects: the immortalized human hair papilla cell line prepared by the application has relatively stable proliferation characteristics and functional states, has no obvious cell aging phenomenon, and can be stably passaged to P30 generations or more; moreover, the immortalized human hair papilla cell line can form 3D cell spheres in an ultra-low adsorption culture plate and maintain hair follicle induction capacity.

[0055] The application has the following beneficial effects: the application optimizes the infection system of the SV40 LT overexpression lentivirus on human hair papilla cells by using the dyeing aid, significantly improves the transfection efficiency of the antigen, not only improves the relative expression level of the prepared immortalized human hair papilla cell line to 90.1-176.4, but also makes the proliferation rate of the immortalized human hair papilla cell line reach 117.4-169.7%, realizes efficient transfection and proliferation, and effectively maintains the normal morphology and biological characteristics of the cells. SV40T SV40T BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 It is a cell morphology diagram of the immortalized human hair papilla cell line.

[0057] Figure 2 It is a well-selected hole containing only one immortalized human hair papilla cell monoclonal.

[0058] Figure 3 It is a discarded hole containing three immortalized human hair papilla cell monoclonals.​​

[0059] Figure 4 qPCR identification results for immortalized human hair papilla cell lines.

[0060] Figure 5 Cell proliferation rate detection results for immortalized human hair papilla cell lines and human hair papilla cells.

[0061] Figure 6 Cell viability results for human hair papilla cells.

[0062] Figure 7 Cell viability results for immortalized human hair papilla cell lines.

[0063] Figure 8 Senescence of human hair papilla cells.

[0064] Figure 9 Senescence of immortalized human hair papilla cell lines.

[0065] Figure 10 3D cell spheroids of human hair papilla cells.

[0066] Figure 11 3D cell spheroids of immortalized human hair papilla cells.

[0067] Figure 12 Immuno fluorescence identification results for immortalized human hair papilla cell lines.

[0068] Figure 13 Infrared spectrogram of quaternary ammonium salt surfactant. DETAILED DESCRIPTION

[0069] The application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0070] The experimental methods in the following examples are all routine methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0071] Example 1:

[0072] Preparation of complete culture solution containing FBS, including,

[0073] The recombinant human insulin-like growth factor-1 (IGF-1) is diluted with DMEM / F12 culture solution to a final concentration of 2.5 ng / mL, and then mixed with FBS and cell culture three-antibiotics to obtain the preparation of complete culture solution containing FBS. The recombinant human insulin-like growth factor-1 is purchased from Yixing Biotech Co., Ltd. (Shanghai), the DMEM / F12 culture solution is purchased from Thermo Fisher Scientific, and the FBS is purchased from Gibco TM Grand Island Biological Company, and the cell culture three-antibiotics are penicillin-streptomycin-amphotericin B solution (100X) purchased from Shanghai Biyun Tian Biological Technology Co., Ltd. The volume ratio of FBS to DMEM / F12 culture solution is 1:10, and the volume ratio of cell culture three-antibiotics to DMEM / F12 culture solution is 1:100.

[0074] Preparation of serum-free DMEM / F12 culture solution, comprising,

[0075] The recombinant human insulin-like growth factor-1 (IGF-1) is diluted with DMEM / F12 culture solution to a final concentration of 2.5 ng / mL, and then mixed with cell culture three-antibiotics to obtain serum-free DMEM / F12 culture solution. The recombinant human insulin-like growth factor-1 is purchased from Yixing Biotech Co., Ltd. (Shanghai), the DMEM / F12 culture solution is purchased from Thermo Fisher Scientific, and the cell culture three-antibiotics are penicillin-streptomycin-amphotericin B solution (100X) purchased from Shanghai Biyun Tian Biological Technology Co., Ltd. The volume ratio of cell culture three-antibiotics to DMEM / F12 culture solution is 1:100.

[0076] Preparation of puromycin screening solution, comprising,

[0077] The puromycin is diluted with DMEM / F12 culture solution to a final concentration of 2 μg / mL.

[0078] Dyeing aid, comprising, polybrene.

[0079] A method for preparing an immortalized human hair papilla cell line, comprising,

[0080] S1, inoculate human hair papilla cells into a six-well plate, add complete culture solution containing FBS, and culture at 37°C in a carbon dioxide concentration of 5% for 24 h. When the cell fusion rate reaches 40%, discard the culture solution and wash the cells with phosphate buffered saline (PBS). The human hair papilla cells are purchased from Guangdong Boxi Biological Technology Co., Ltd. The inoculation cell density of human hair papilla cells is 2×10 4 cm 2The volume ratio of serum-free DMEM / F12 culture solution and the second FBS-containing complete culture solution is 1:2. 5

[0081] S2, after adding serum-free DMEM / F12 culture solution, SV40 large T antigen-Lv105 Lentifect TM After 5h of infection with the lentivirus particles (SV40 LT overexpression lentivirus) and the staining aid, the first FBS-containing complete culture solution was added for 19h, and then the culture solution was discarded. The second FBS-containing complete culture solution was added for 48h, and then the culture solution was discarded. The puromycin screening solution was added for 48h for the first screening, and then the culture solution was discarded. The puromycin screening solution was added again for 48h for the second screening. After the second screening, the surviving cells were collected to obtain the successfully infected immortalized human hair papilla cells. The SV40 LT overexpression lentivirus was purchased from Guangzhou Yijin Biotechnology Co., Ltd. The volume ratio of the FBS-containing complete culture solution to the staining aid was 1mL:4μg. The volume ratio of the serum-free DMEM / F12 culture solution to the first FBS-containing complete culture solution was 1:1. The volume ratio of the serum-free DMEM / F12 culture solution to the second FBS-containing complete culture solution was 1:2. The volume ratio of the serum-free DMEM / F12 culture solution to the puromycin screening solution was 1:2.

[0082] S3, after adding trypsin-EDTA for digestion, the FBS-containing complete culture solution was added for resuspension to obtain the digested human hair papilla cells. The volume ratio of trypsin-EDTA to the FBS-containing complete culture solution was 1:2.

[0083] S4, the digested immortalized human hair papilla cells were diluted to a cell density of 1 cell per well by the limited dilution method, and a cell strain with a good growth state was selected for cell expansion. The growth state of the monoclonal cells was observed every day by using a high-content imaging system and a fluorescence microscope. When the confluence of the monoclonal cells reached 90%, the monoclonal cells were digested by trypsin-EDTA for subculture and continued to be cultured. The puromycin screening solution was added for maintenance screening. When the confluence of the monoclonal cells in the 24-well plate reached 90%, the monoclonal cells were digested by trypsin-EDTA for subculture and continued to be cultured. The puromycin screening solution was added for maintenance screening. The cells were cultured until the confluence reached 90% to obtain the immortalized human hair papilla cell line. The volume ratio of the puromycin screening solution to the puromycin screening solution in step S2 was 1:1.

[0084] Example 2

[0085] The FBS-containing complete culture solution was prepared as in Example 1.

[0086] ​Preparation of serum-free DMEM / F12 medium, same as Example 1.

[0087] Preparation of puromycin selection medium, same as Example 1.

[0088] Dyeing aid, same as Example 1.

[0089] A method for preparing an immortalized human hair papilla cell line, compared with Example 1, except that in step S4, the diluted and digested human immortalized human hair papilla cells are replaced by 3 cells per hole, and the other conditions are the same as Example 1.

[0090] Example 3:

[0091] Preparation of complete medium containing FBS, same as Example 1.

[0092] Preparation of serum-free DMEM / F12 medium, same as Example 1.

[0093] Preparation of puromycin selection medium, same as Example 1.

[0094] Preparation of quaternary ammonium salt surfactant, including,

[0095] Mix cocodimethyl tertiary amine and anhydrous ethanol, add concentrated hydrochloric acid and stir for 25 min, slowly drop epoxy chloropropane, react at 60℃ for 2.5h, after the reaction is completed, add oil dimethyl tertiary amine, react at 90℃ for 24h, after the reaction is completed, distill under reduced pressure, wash with anhydrous ether 3 times, and recrystallize with recrystallization agent 3 times to obtain quaternary ammonium salt surfactant. The amount ratio of cocodimethyl tertiary amine and anhydrous ethanol is 1g:10mL; the mass concentration of concentrated hydrochloric acid is 37%, the volume ratio of anhydrous ethanol and concentrated hydrochloric acid is 1:0.05; the mass ratio of cocodimethyl tertiary amine and epoxy chloropropane is 1:1.5; the mass ratio of cocodimethyl tertiary amine and oil dimethyl tertiary amine is 1:3; the volume ratio of anhydrous ethanol and anhydrous ether is 1:1; the recrystallization agent includes acetone and ethyl acetate, the volume ratio of acetone and ethyl acetate is 1:3; the volume ratio of anhydrous ethanol and recrystallization agent is 1:1.

[0096] Dyeing aid, compared with Example 1, except that 30% of the mass of polybrene is added as quaternary ammonium salt surfactant, and the other conditions are the same as Example 1.

[0097] A method for preparing an immortalized human hair papilla cell line, compared with Example 1, except that in step S2, the dyeing aid is replaced by the dyeing aid prepared in this embodiment, and the other conditions are the same as Example 1.

[0098] Example 4:

[0099] Preparation of complete medium containing FBS, same as Example 1.

[0100] Preparation of serum-free DMEM / F12 medium, same as Example 1.

[0101] Preparation of puromycin selection medium, same as Example 1.

[0102] Preparation of quaternary ammonium surfactant, same as Example 3.

[0103] Dyestuff aid, same as Example 1 except that 10% of the mass of the polybrene is replaced by quaternary ammonium surfactant.

[0104] Method for preparing an immortalized human hair papilla cell line, same as Example 1 except that in step S2 the dyestuff aid is replaced by the dyestuff aid prepared in this Example.

[0105] Example 5:

[0106] Preparation of complete medium with FBS, same as Example 1.

[0107] Preparation of serum-free DMEM / F12 medium, same as Example 1.

[0108] Preparation of puromycin selection medium, same as Example 1.

[0109] Preparation of quaternary ammonium surfactant, same as Example 3.

[0110] Dyestuff aid, same as Example 1 except that 30% of the mass of the polybrene is replaced by quaternary ammonium surfactant and 5% of the mass of the polybrene is replaced by lauryl sodium sulfosuccinate.

[0111] Method for preparing an immortalized human hair papilla cell line, same as Example 1 except that in step S2 the dyestuff aid is replaced by the dyestuff aid prepared in this Example.

[0112] Example 6:

[0113] Preparation of complete medium with FBS, same as Example 1.

[0114] Preparation of serum-free DMEM / F12 medium, same as Example 1.

[0115] Preparation of puromycin selection medium, same as Example 1.

[0116] Preparation of quaternary ammonium surfactant, same as Example 3.

[0117] Dyeing aid, compared with Example 1, except that 30% of the mass of the polybrene is additionally added as the quaternary ammonium salt surfactant prepared in this example, and 0.5% of the mass of the polybrene is additionally added as the disodium lauryl sulfosuccinate, and the other conditions are the same as in Example 1.

[0118] A method for preparing an immortalized human hair papilla cell line, compared with Example 1, except that in step S2, the dyeing aid is replaced by the dyeing aid prepared in this example, and the other conditions are the same as in Example 1.

[0119] Comparative Example 1:

[0120] Preparation of complete medium containing FBS, the same as in Example 1.

[0121] Preparation of serum-free DMEM / F12 medium, the same as in Example 1.

[0122] Preparation of puromycin selection medium, the same as in Example 1.

[0123] Preparation of quaternary ammonium salt surfactant, compared with Example 3, except that no oleyl dimethyl tertiary amine is used, and the other conditions are the same as in Example 3.

[0124] Dyeing aid, compared with Example 1, except that 30% of the mass of the polybrene is additionally added as the quaternary ammonium salt surfactant prepared in this example, and 0.5% of the mass of the polybrene is additionally added as the disodium lauryl sulfosuccinate, and the other conditions are the same as in Example 1.

[0125] A method for preparing an immortalized human hair papilla cell line, compared with Example 1, except that in step S2, the dyeing aid is replaced by the dyeing aid prepared in this example, and the other conditions are the same as in Example 1.

[0126] Comparative Example 2:

[0127] Preparation of complete medium containing FBS, the same as in Example 1.

[0128] Preparation of serum-free DMEM / F12 medium, the same as in Example 1.

[0129] Preparation of puromycin selection medium, the same as in Example 1.

[0130] Dyeing aid, compared with Example 1, except that 5% of the mass of the polybrene is additionally added as the disodium lauryl sulfosuccinate, and the other conditions are the same as in Example 1.

[0131] A method for preparing an immortalized human hair papilla cell line, compared with Example 1, except that in step S2, the dyeing aid is replaced by the dyeing aid prepared in this example, and the other conditions are the same as in Example 1.

[0132] Experimental Example:

[0133] 1. Cell morphology of the immortalized human hair papilla cell line

[0134] The immortalized human hair papilla cell line prepared in Example 1 was observed using an electron microscope. Figure 1 The cell morphology of the immortalized human hair papilla cell line is shown in the figure, with a scale of 150 μm. This indicates that the immortalized human hair papilla cell line is successfully prepared.

[0135] The immortalized human hair papilla cells prepared in Example 1 and Example 2 were observed using a high-content cell imaging system, respectively. Figure 2 The well containing only one immortalized human hair papilla cell monoclonal selected is shown in the figure, Figure 3 The well containing three immortalized human hair papilla cell monoclonals discarded is shown in the figure. This indicates that the immortalized human hair papilla cell monoclonal group is successfully screened.

[0136] 2. qPCR experiment of immortalized human hair papilla cell monoclonal

[0137] Referring to the preparation method of Example 1, 11 immortalized human hair papilla cell monoclonals were obtained, which were numbered A10, B6, B11, C7, D4, H7, A1, D1, C6, C11 and H11 in sequence, and the relative expression levels of the 11 immortalized human hair papilla cell monoclonals were detected. SV40T The specific steps are as follows: (1) The immortalized human hair papilla cells were digested with trypsin-EDTA and divided into two groups, which were denoted as group A and group B. 1x10 6 cells were taken into group A, and the remaining cells were taken into group B; (2) 1 mL of serum-free cell freezing solution was added to resuspend the cells in group A to obtain frozen cells. The serum-free cell freezing solution was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. The amount of serum-free cell freezing solution used was 1 mL; (3) The RNA of the cells in group B was extracted using an RNA rapid extraction kit. After the RNA was extracted, 1 μg of RNA was subjected to reverse transcription using a one-step reverse transcription kit to obtain cDNA, and then a qPCR experiment was performed using a dye method fluorescent quantitative PCR premix to detect the relative expression level of SV40T In the qPCR experiment, a blank control group was set, in which no cDNA template was added; each group in the qPCR experiment was subjected to four technical repeats. The RNA rapid extraction kit, the one-step reverse transcription kit and the dye method fluorescent quantitative PCR premix were all purchased from Shanghai Adamas Reagent Co., Ltd. In the qPCR experiment, the forward primer sequence for detecting the target gene SV40T was 5'-AATTTGCCCTTGGACAGGCT-3', the nucleotide sequence of which is shown as SEQ ID No. 1, SV40T the reverse primer sequence of which is 5'-GCCTGAAATGAGCCTTGGGA-3', the nucleotide sequence of which is shown as SEQ ID No. 2; the forward primer sequence for detecting the housekeeping gene GADPHThe 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.

[0138] Figure 4 The results represent the qPCR identification 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 monoclonal cells, respectively. BC represents 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... SV40T It exhibits the highest relative expression level and can be used for subsequent large-scale culture and cell library construction.

[0139] Following the qPCR experimental method described above, the immortalized human dermal papilla cell lines prepared in Examples 3-6 and Comparative Examples 1-2 were measured. SV40T Relative expression levels. The immortalized human dermal papilla cell line 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. Table 1 is... SV40T Relative expression level.

[0140] Table 1 SV40T relative expression level

[0141]

[0142] As shown in Table 1, the immortalized human dermal papilla cell lines prepared in Examples 3-4 of this invention... SV40T The relative expression level was significantly higher than that in Example 1 because, in the preparation of the immortalized human dermal papilla cell line, Examples 3-4 introduced quaternary ammonium salt surfactants into the dyeing agent, while Example 1 did not introduce quaternary ammonium salt surfactants into the dyeing agent; the immortalized human dermal papilla cell line prepared in Example 3... SV40T The relative expression level was significantly higher than in Example 4 due to the different amount of quaternary ammonium salt surfactant used. This indicates that the introduction of quaternary ammonium salt surfactant into the staining agent in this invention helps to improve the infection effect of SV40 LT overexpression lentivirus and significantly enhances the prepared immortalized human hair papilla cell line. SV40T Relative expression level.

[0143] The immortalized human hair papilla cell line prepared in Example 3 of this invention SV40TThe relative expression level was significantly higher than that of Comparative Example 1 because, in the preparation of the quaternary ammonium salt surfactant, Example 3 used cocoyl dimethyl tertiary amine to react with epichlorohydrin first, and then with oleyl dimethyl tertiary amine to obtain the quaternary ammonium salt surfactant; while Comparative Example 1 only used cocoyl dimethyl tertiary amine to react with epichlorohydrin first to obtain the quaternary ammonium salt surfactant. This indicates that the present invention, which uses cocoyl dimethyl tertiary amine, epichlorohydrin, and oleyl dimethyl tertiary amine to obtain the quaternary ammonium salt surfactant, can effectively improve the infection effect of SV40 LT overexpression lentivirus and significantly enhance the efficacy of the prepared immortalized human dermal papilla cell line. SV40T Relative expression level.

[0144] The immortalized human dermal papilla cell lines prepared in Examples 5-6 of this invention SV40T The relative expression level was significantly higher than in Example 3 because, in the preparation of the immortalized human dermal papilla cell line, Examples 5-6 further introduced disodium lauryl sulfosuccinate into the dyeing agent; the immortalized human dermal papilla cell line prepared in Example 5... SV40T The relative expression level was significantly higher than in Example 6 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 was significantly higher than that of Comparative Example 2 because, in the preparation of the immortalized human dermal papilla cell line, Example 5 synergistically introduced a quaternary ammonium salt surfactant and disodium lauryl sulfosuccinate into the dyeing agent, while Comparative Example 2 only introduced disodium lauryl sulfosuccinate alone. This indicates that the synergistic introduction of a quaternary ammonium salt surfactant and disodium lauryl sulfosuccinate into the dyeing agent in this invention helps to further improve the infection effect of SV40 LT overexpression lentivirus, and further significantly enhances the expression level of the prepared immortalized human dermal papilla cell line. SV40T Relative expression level.

[0145] 3. Cell proliferation and activity detection experiments of immortalized human dermal papilla cell lines

[0146] Using the CCK-8 reagent kit, cell proliferation and viability assays were performed on the immortalized human dermal papilla cell line and human dermal papilla cells prepared in Example 1. The human dermal papilla cells were purchased from Guangdong Boxi Biotechnology Co., Ltd., and the CCK-8 reagent was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The specific steps are as follows: Cells were passaged to P10, and then... 5 Add DMEM / F12 culture medium to the cells at a dosage of cells / mL to prepare a cell suspension; take 100µL of cell suspension, and the number of cells per well is 2×10⁶. 4After 24 h of culture at 37℃, 10 μL of CCK-8 reagent was added and mixed, and then incubated at 37℃ for 1 h; after the incubation, the absorbance at 450 nm was measured by using an enzyme-labeled instrument, and the number of cells in each well after incubation was calculated according to the instruction of the CCK-8 kit. The difference in the proliferation ability of the two kinds of cells was compared, and the cell proliferation rate was calculated, the cell proliferation rate (%) = the number of cells in each well after incubation / the number of cells in each well before incubation x 100%, wherein the number of cells in each well before incubation was 2 x 10 4

[0147] Figure 5 The cell proliferation rate of the immortalized human hair papilla cell line prepared in Example 1 was higher than that of the human hair papilla cells, which indicated that the immortalized human hair papilla cells prepared in the application had a faster proliferation rate than the hair papilla cells. Figure 5

[0148] The cell proliferation rate of the immortalized human hair papilla cell line prepared in Example 1 was higher than that of the human hair papilla cells, which indicated that the immortalized human hair papilla cells prepared in the application had a faster proliferation rate than the hair papilla cells.

[0149] Table 2 Cell proliferation rate (%)

[0150]

[0151] As shown in Table 2, the cell proliferation rate of the immortalized human hair papilla cell line prepared in Examples 3-4 was significantly higher than that of Example 1, because in the preparation of the immortalized human hair papilla cell line, Examples 3-4 introduced quaternary ammonium salt surfactants into the auxiliary dyeing agent, while Example 1 did not introduce quaternary ammonium salt surfactants into the auxiliary dyeing agent; the cell proliferation rate of the immortalized human hair papilla cell line prepared in Example 3 was significantly higher than that of Example 4, because the amount of quaternary ammonium salt surfactants used was different. This indicates that the introduction of quaternary ammonium salt surfactants into the auxiliary dyeing agent significantly improves the cell proliferation rate of the prepared immortalized human hair papilla cell line.

[0152] ​​The cell proliferation rate of the immortalized human hair papilla cell line prepared in Example 3 is significantly higher than that of Comparative Example 1, because in the preparation of the quaternary ammonium salt surfactant, Example 3 uses cocodimethyl tertiary amine to react with epichlorohydrin first, and then with oleyl dimethyl tertiary amine to obtain the quaternary ammonium salt surfactant; while Comparative Example 1 only uses cocodimethyl tertiary amine to react with epichlorohydrin to obtain the quaternary ammonium salt surfactant. This shows that the quaternary ammonium salt surfactant obtained by reacting cocodimethyl tertiary amine, epichlorohydrin and oleyl dimethyl tertiary amine can significantly improve the cell proliferation rate of the prepared immortalized human hair papilla cell line.

[0153] The cell proliferation rate of the immortalized human hair papilla cell line prepared in Examples 5-6 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 aid; 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 introduces the quaternary ammonium salt surfactant and disodium lauryl sulfosuccinate into the dyeing aid, while Comparative Example 2 only introduces disodium lauryl sulfosuccinate alone. This shows that the simultaneous introduction of the quaternary ammonium salt surfactant and disodium lauryl sulfosuccinate into the dyeing aid can further significantly improve the cell proliferation rate of the prepared immortalized human hair papilla cell line.

[0154] 4. Cell activity and cell counting experiments of the immortalized human hair papilla cell line

[0155] Calcein / PI cell activity and cytotoxicity detection kit was used to perform cell activity and cell counting experiments on the immortalized human hair papilla cell line prepared in Example 1 and human hair papilla cells, each group of experiments was repeated 3 times, the human hair papilla cells were purchased from Guangdong Boxi Biological Technology Co., Ltd., and the Calcein / PI cell activity and cytotoxicity detection kit was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd., the specific steps are as follows: the cells were subcultured to P10 generation 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 incubated at 37°C for 30 min in the dark; the stained cells were photographed using a high-content cell imaging system, and cell counting was performed using the analysis function of the system.

[0156] Figure 6 The cell activity results of the human hair papilla cells are as follows, Figure 7 The cell activity results of the immortalized human hair papilla cell line are as follows. Figure 6 and Figure 7In comparison, the green fluorescence intensity of the immortalized human hair papilla cell line prepared in Example 1 is stronger, which indicates that the immortalized human hair papilla cell line prepared in Example 1 exhibits higher cell viability and the cell proliferation speed is obviously greater than that of the limited passage human hair papilla cells. Table 3 is the cell counting results of the immortalized human hair papilla cell line and the human hair papilla cells.

[0157] Table 3 Cell counting results of the immortalized human hair papilla cell line and the human hair papilla cells

[0158]

[0159] As can be seen from Table 3, the number of living cells of the immortalized human hair papilla cell line prepared in Example 1 is obviously more than that of the human hair papilla cells. This indicates that after the human hair papilla cells are immortalized, the cell viability is improved and the cell proliferation speed is also accelerated to a certain extent.

[0160] 5. Cell senescence β-galactosidase staining of the immortalized human hair papilla cell line

[0161] The β-galactosidase staining of the immortalized human hair papilla cell line prepared in Example 1 and the human hair papilla cells was performed using a cell senescence β-galactosidase staining kit. The human hair papilla cells were purchased from Guangdong Boxi Biological Technology Co., Ltd. and the cell senescence β-galactosidase staining kit was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd. The specific steps are as follows: the cells were subcultured to P10 generation, DMEM / F12 culture solution was added to the cells at a dosage of 1 x 10 5 cells / mL to prepare a cell suspension; 500 μL of the cell suspension was added to a 24-well plate so that the number of cells per well was 5 x 10 4 cells / mL, and the cells were cultured at 37°C for 48 h. The culture solution was discarded, the cells were washed with PBS, 250 μL of a 4% polyformaldehyde solution (purchased from Shanghai Biyun Tian Biological Technology Co., Ltd.) was added to each well, the solution was aspirated, and the cells were washed with PBS for 3 times, each time for 3 min. After the washing was completed, 250 μL of a β-galactosidase staining working solution was added to each well, which was mixed and incubated at 37°C overnight. After the incubation was completed, the cells were observed and photographed under a microscope to evaluate the senescence of the cells.

[0162] Figure 8 The senescence of the human hair papilla cells is shown in Figure 6, and the senescence of the immortalized human hair papilla cell line prepared in Example 1 is shown in Figure 7. Figure 9 The senescence of the human hair papilla cells is shown in Figure 6, and the senescence of the immortalized human hair papilla cell line prepared in Example 1 is shown in Figure 7. Figure 8 and Figure 9 As can be seen from Figures 6 and 7, the human hair papilla cells exhibit obvious high β-galactosidase positive expression, and the positive expression degree is obviously higher than that of the immortalized human hair papilla cell line prepared in Example 1. This indicates that the immortalized human hair papilla cells prepared in Example 1 are less likely to exhibit senescence characteristics.

[0163] 6. 3D cell culture experiment of immortalized human hair papilla cell line

[0164] The human hair papilla cells were subcultured to P8 generation, and the human hair papilla cells were purchased from Guangdong Boxi Biological Technology Co., Ltd. The immortalized human hair papilla cell line prepared in Example 1 was subcultured to P10 generation, and then P8 generation human hair papilla cells and P10 generation immortalized human hair papilla cell line were used for 3D cell culture, respectively. The specific steps are as follows: DMEM / F12 culture solution was added to the cells at a dosage of 1x10 5 cells / mL to prepare a cell suspension; 100 μL of the cell suspension was added to a 96-well plate, so that the number of cells per well was 1x10 4 cells, and the cells were cultured at 37°C on a shaking table at a speed of 80 r / min for 36 h. High-content cell imaging system was used to take pictures and observe 3D cell spheres.

[0165] Figure 10 The 3D cell spheres of the human hair papilla cells were Figure 11 The 3D cell spheres of the immortalized human hair papilla cell line were Figure 10 and Figure 11 It can be seen that the immortalized human hair papilla cell line prepared in Example 1 can form 3D cell spheres in the ultra-low adsorption culture plate, like the P8 generation human hair papilla cells. This shows that the immortalized human hair papilla cell line prepared in Example 1 has not lost the ability to induce hair follicle formation.

[0166] 7. Immunofluorescence identification of immortalized human hair papilla cell line

[0167] The human hair papilla cells were subcultured to P12 generation, and the human hair papilla cells were purchased from Guangdong Boxi Biological Technology Co., Ltd. The immortalized human hair papilla cell line prepared in Example 1 was subcultured to P8 generation, and then P12 generation human hair papilla cells and P8 generation immortalized human hair papilla cell line were used for immunofluorescence identification, respectively. The specific steps are as follows: DMEM / F12 culture solution was added to the cells at a dosage of 1x10 5 cells / mL to prepare a cell suspension; 500 μL of the cell suspension was added to a 24-well plate, so that the number of cells per well was 5x10 4 / hole; mix, put into 37℃ incubator, culture for 24h. Discard the culture medium, wash once with PBS, add 500μL of anhydrous methanol to 4℃, fix for 5min; use TBST buffer, TBST buffer is purchased from Yixing Biotech Co., Ltd. (Shanghai), rinse 3 times, add 500μL of 5% goat serum solution to each hole, the goat serum solution is purchased from Shanghai Blueyuan Biotech Co., Ltd., block for 1h; add 300μL of primary antibody LEPR polyclonal antibody to each hole, the primary antibody LEPR polyclonal antibody is purchased from Wuhan Sanying Biotechnology Co., Ltd., incubate overnight at 4℃; discard the primary antibody, rinse 3 times with TBST buffer, add 300μL of Cy3-labeled goat anti-rabbit IgG (H+L) secondary antibody to each hole, the Cy3-labeled goat anti-rabbit IgG (H+L) secondary antibody is purchased from Shanghai Blueyuan Biotech Co., Ltd., incubate for 2h at room temperature; discard the secondary antibody, rinse 3 times with TBST buffer, add 300μL of 1.43μmol / L 4', 6-diamidino-2-phenylindole (DAPI) to each hole, the DAPI is purchased from Aibishun (Shanghai) Biotechnology Co., Ltd., incubate for 10min at room temperature in the dark to stain the cell nucleus; discard the DAPI, rinse 3 times with TBST buffer, and then take pictures using a fluorescence microscope.

[0168] Figure 12 The results of immunofluorescence identification of the immortalized human hair papilla cell line are shown, in which DPCs represent hair papilla cells, SV40T-DPCs represent immortalized hair 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 hair papilla cells and is involved in the hair follicle induction signaling pathway. According to the results of immunofluorescence Figure 12 , it is shown that both immortalized human hair papilla cells and limited passage human hair papilla cells can express the specific protein LEPR of hair papilla cells, and there is no difference in the fluorescence intensity of the two types of cells. This indicates that the immortalization process does not affect the phenotype maintenance of human hair papilla cells, and the immortalized human hair papilla cells can still express the marker protein of hair papilla cells and retain the characteristics of hair follicle induction function.

[0169] 8. Material characterization

[0170] The quaternary ammonium salt surfactant prepared in Example 3 of the application was collected and detected using a Fourier infrared spectrometer.

[0171] Figure 13 The infrared spectrum of the quaternary ammonium salt surfactant is shown, with an absorption peak of -OH at 3410cm -1 , an absorption peak of -CH3 at 2900cm -1 , and an absorption peak of -CH2- at 2800cm -1an absorption peak of -CH2appears near 1720 cm -1 an absorption peak of C=O appears near 1630 cm -1 an absorption peak of C=C appears near 1100 cm -1 an absorption peak of C-O appears near.

[0172] The routine operations in the operation steps of the present application are well known to those skilled in the art and will not be described here.

[0173] The above description is only specific embodiments of the present application and is not intended to limit the present application. Any changes and modifications made within the principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for immortalizing a human hair papilla cell line, comprising, The immortalized human hair papilla cell line is obtained by infecting human hair papilla cells with SV40 LT overexpression lentivirus combined with a dyeing aid, treating with a puromycin screening solution, picking single clone cells by limited dilution method and expanding culture; the dyeing aid comprises polybrene, quaternary ammonium salt surfactant and lauryl alcohol sulfosuccinate disodium; the quaternary ammonium salt surfactant is used in an amount of 10-30% of the mass of polybrene; the lauryl alcohol sulfosuccinate disodium is used in an amount of 0.5-5% of the mass of polybrene; in the preparation of the quaternary ammonium salt surfactant, cocodimethyl tertiary amine and anhydrous ethanol are mixed, then cocodimethyl tertiary amine is reacted with epichlorohydrin, and then the oil-based dimethyl tertiary amine is reacted to obtain the quaternary ammonium salt surfactant; in the preparation of the puromycin screening solution, DMEM / F12 culture solution is used to dilute puromycin to a final concentration of 1-10 μg / mL to obtain the puromycin screening solution.

2. The method for preparing an immortalized human hair papilla cell line according to claim 1, characterized in that, The SV40 LT overexpression lentivirus is added in a dose with a multiplicity of infection of 5-20.

3. The method for preparing an immortalized human hair 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 use amount ratio of the cocodimethyl tertiary amine and the anhydrous ethanol is 1g:5-20mL.

5. The method for preparing an immortalized human hair papilla cell line according to claim 1, characterized in that, The mass ratio of the cocodimethyl tertiary amine and the epichlorohydrin is 1:1-3.

6. The method for preparing an immortalized human hair papilla cell line according to claim 1, wherein the step of culturing the cells is performed in the presence of a growth factor. The mass ratio of the cocodimethyl tertiary amine and the oil-based dimethyl tertiary amine is 1:1-5.

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