HaCaT cell model capable of stably expressing green fluorescent protein as well as construction method and application of HaCaT cell model

Abstract

The invention provides a HaCaT cell model capable of stably expressing green fluorescent protein as well as a construction method and application of the HaCaT cell model, and belongs to the technical field of cell model construction. The construction method of the cell model comprises the following steps: mixing a PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, a Super PiggyBac Transspobase plasmid, a cell culture solution and a transfection reagent, so as to obtain a GFP (Green Fluorescent Protein) transfection reagent; mixing HaCaT cells with the obtained GFP transfection reagent, transfecting, adding puromycin for screening after the transfection is finished, and stopping adding puromycin after the screening is finished, so as to obtain the cells which are the HaCaT cell model for stably expressing the green fluorescent protein. The cell model can be used for revealing the interaction between skin cells, screening drugs for preventing or treating skin diseases and evaluating the efficacy of cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cell model construction, and in particular to a HaCaT cell model stably expressing green fluorescent protein, and a construction method and application thereof. Background Art

[0002] The interaction between skin cells is a key factor in producing various skin irritation reactions. In order to evaluate the efficacy of cosmetics more scientifically and accurately, we introduced an in vitro cell co-culture model. However, cells in the co-culture state are difficult to distinguish with the naked eye under a microscope, which limits our accurate judgment of their growth activity and rate.

[0003] The inventors found that the autofluorescence properties of green fluorescent protein (GFP) can help locate and distinguish co-cultured cells, thereby accurately detecting the growth activity and rate of each cell, which is conducive to selecting the optimal ratio of inoculated cells and building a more optimized in vitro cell co-culture model. In the selection of co-cultured cells, the inventors noticed that keratinocytes account for a high proportion of surface skin cells and participate in multiple cell biological processes. As a representative of self-immortalized keratinocytes, HaCaT cells have the advantages of biological similarity, easy culture and intuitive observation, making them the preferred cells for establishing in vitro cell co-culture models.

[0004] Therefore, establishing a HaCaT cell line that can stably express GFP can provide strong support for in-depth research on the effects of HaCaT cells on other cells and reveal the interaction mechanism between skin cells, and provide a stable and reliable in vitro co-culture cell model for drug screening and cosmetic efficacy evaluation. Summary of the invention

[0005] The purpose of the present invention is to provide a HaCaT cell model that stably expresses green fluorescent protein and its construction method and application, so as to provide a stable and reliable in vitro co-culture cell model for revealing the interaction between skin cells, screening drugs for preventing or treating skin diseases, and evaluating the efficacy of cosmetics.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for constructing a HaCaT cell model that stably expresses green fluorescent protein, comprising the following steps:

[0008] (1) Mixing the PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, the Super PiggyBac Transposase plasmid, the cell culture medium and the transfection reagent to obtain the GFP transfection reagent;

[0009] (2) HaCaT cells are mixed with the GFP transfection reagent obtained in step (1) and then transfected. Puromycin is added for screening after the transfection is completed. After the screening is completed, the addition of puromycin is stopped. The cells obtained by screening are the HaCaT cell model that stably expresses green fluorescent protein.

[0010] Preferably, the cell culture medium in step (1) is DMEM culture medium, and the transfection reagent is Lipo8000 TM Transfection reagents;

[0011] The mass volume ratio of the piggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, the Super piggyBac Transposase plasmid, the cell culture fluid and the transfection reagent when mixed is 0.8-1.2 μg: 1.6-2.4 μg: 100-150 μL: 2-6 μL.

[0012] Preferably, the HaCaT cells in step (2) are HaCaT cells cultured to a confluency of 80-95%;

[0013] The transfection time is 40 to 56 hours;

[0014] The concentration of puromycin used in the screening is 0.8-1.2 μg / mL, and the screening time is 6-8 days.

[0015] Preferably, in step (2), the screened cells are further induced by doxycycline hydrochloride, and the concentration of doxycycline hydrochloride used during induction is 0.8-1.2 μg / mL.

[0016] Preferably, in step (2), the screened cells are further subjected to monoclonal culture.

[0017] The invention provides a HaCaT cell model stably expressing green fluorescent protein which is constructed according to the method.

[0018] The present invention provides an application of the cell model in preparing and screening products for preventing or treating skin diseases.

[0019] Preferably, the product comprises an antibody, a vaccine or a drug.

[0020] The present invention provides an application of the cell model in evaluating the efficacy of cosmetics, wherein the efficacy of cosmetics includes promoting or inhibiting the transmission of melanin.

[0021] The present invention provides an application of the cell model for non-diagnostic and therapeutic purposes in studying the interaction mechanism between HaCaT cells and other cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention successfully constructs a HaCaT cell model capable of stably expressing green fluorescent protein, which can be used to reveal interactions between skin cells, screen drugs for preventing or treating skin diseases, and evaluate the efficacy of cosmetics. The present invention can simplify the process of constructing a co-culture model with the HaCaT cell line as the core, provide an intuitive research tool for constructing a co-culture model with the HaCaT cell line as the core, and provide a new model for studying the mechanism of cell-cell interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 This is a structural diagram of the PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid in Example 1;

[0026] Figure 2 is a structural diagram of the SuperPiggyBac Transposase plasmid in Example 1;

[0027] Figure 3 The morphology of the HaCaT cells stably expressing GFP and the HaCaT cell model stably expressing GFP obtained by monoclonal culture observed under bright field and GFP excitation light source in Example 1; wherein A is the state of the HaCaT cells stably expressing GFP under bright field, B is the morphology of the HaCaT cells stably expressing GFP under GFP excitation light source, C is the state of the HaCaT cell model stably expressing GFP obtained by monoclonal culture under bright field, and D is the morphology of the HaCaT cell model stably expressing GFP obtained by monoclonal culture under GFP excitation light source;

[0028] Figure 4 The calculation results of the relative growth rate of B16 cells in the co-cultured cells of each treatment group in Example 2;

[0029] Figure 5 The calculation results of the relative growth rate of HaCaT cells in the co-cultured cells in each treatment group in Example 2;

[0030] Figure 6The results of observation of the HaCaT cell and B16 cell co-culture model constructed in Example 2 under bright field (A) and GFP excitation light source (B). DETAILED DESCRIPTION

[0031] The present invention provides a method for constructing a HaCaT cell model that stably expresses green fluorescent protein, comprising the following steps:

[0032] (1) Mixing the PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, the Super PiggyBac Transposase plasmid, the cell culture medium and the transfection reagent to obtain the GFP transfection reagent;

[0033] (2) HaCaT cells are mixed with the GFP transfection reagent obtained in step (1) and then transfected. Puromycin is added for screening after the transfection is completed. After the screening is completed, the addition of puromycin is stopped. The cells obtained by screening are the HaCaT cell model that stably expresses green fluorescent protein.

[0034] In the present invention, the cell culture medium in step (1) is DMEM culture medium, and the transfection reagent is Lipo8000 TM Transfection reagents;

[0035] The mass-to-volume ratio of the PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, Super PiggyBac Transposase plasmid, cell culture fluid and transfection reagent when mixed is 0.8-1.2 μg: 1.6-2.4 μg: 100-150 μL: 2-6 μL, preferably 0.9-1.1 μg: 1.8-2.2 μg: 110-140 μL: 3-5 μL, and further preferably 1 μg: 2 μg: 125 μL: 4 μL.

[0036] In the present invention, the HaCaT cells in step (2) are HaCaT cells cultured to a confluence of 80 to 95%, preferably HaCaT cells cultured to a confluence of 80 to 90%, and more preferably HaCaT cells cultured to a confluence of 90%;

[0037] The transfection time is 40 to 56 hours, preferably 42 to 54 hours, more preferably 44 to 52 hours, and more preferably 48 hours;

[0038] The concentration of puromycin used in the screening is 0.8 to 1.2 μg / mL, preferably 0.9 to 1.1 μg / mL, and more preferably 1 μg / mL. The screening time is 6 to 8 days, and preferably 7 days.

[0039] In the present invention, in step (2), the screened cells are further induced by doxycycline hydrochloride, and the concentration of doxycycline hydrochloride used during induction is 0.8 to 1.2 μg / mL, preferably 0.9 to 1.1 μg / mL, and more preferably 1 μg / mL.

[0040] In the present invention, the cells screened in step (2) are further subjected to monoclonal culture.

[0041] The invention provides a HaCaT cell model stably expressing green fluorescent protein which is constructed according to the method.

[0042] The present invention provides an application of the cell model in preparing and screening products for preventing or treating skin diseases.

[0043] In the present invention, the product includes an antibody, a vaccine or a drug.

[0044] The present invention provides an application of the cell model in evaluating the efficacy of cosmetics, wherein the efficacy of cosmetics includes promoting or inhibiting the transmission of melanin.

[0045] The present invention provides an application of the cell model for non-diagnostic and therapeutic purposes in studying the interaction mechanism between HaCaT cells and other cells.

[0046] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] Example 1

[0048] A method for constructing a cell model stably expressing green fluorescent protein (GFP), comprising the following steps:

[0049] (1) 1 μg of PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid (structure diagram as shown in the figure) was added. Figure 1 As shown), 2 μg SuperPiggyBac Transposase plasmid (structure as shown Figure 2 As shown), 125 μL DMEM culture medium and 4 μL Lipo8000 TM The transfection reagent (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was gently pipetted and mixed to obtain the GFP transfection reagent (the transfection reagent was used within 6 hours after preparation);

[0050] (2) HaCaT cells (purchased from Wuhan Pronocell Life Science Co., Ltd.) were added to a 6-well plate containing DMEM culture medium, and the cells were cultured and grown to about 90% confluency before transfection. Before transfection, the culture medium in the 6-well plate was replaced with complete culture medium (complete culture medium is DMEM culture medium containing 10% fetal bovine serum and 0.1% penicillin-streptomycin dual antibody solution), and the amount of complete culture medium per well was 2 mL; during transfection, the GFP transfection reagent prepared in step (1) was evenly added dropwise to the 6-well plate at an amount of 125 μL per well, and the transfection was performed for 48 hours;

[0051] (3) After the transfection in step (2), the culture medium in the 6-well plate was replaced with a culture medium containing 1 μg / mL puromycin for screening. The culture medium containing 1 μg / mL puromycin was replaced every 24 hours for a total of one week. When the cells in the 6-well plate showed no obvious death under a microscope, the culture medium was replaced with a culture medium containing 0.5 μg / mL puromycin, and 1 μg / mL doxycycline hydrochloride was added for induction. After 24 hours, the cells were placed under a fluorescence microscope for observation. If the green fluorescence corresponded to the bright field cells one by one, HaCaT cells stably expressing GFP were obtained.

[0052] (4) After the HaCaT cells stably expressing GFP obtained in step (3) have expanded from single cells to small cell clusters, add 1 mL of 0.25% Trypsin-EDTA preheated in a 37°C water bath, and incubate in a carbon dioxide incubator for 5 min. At this time, the cells are still attached to the wall normally, and the 0.25% Trypsin-EDTA is sucked away with an electric aspirator. The cell clusters of single cells are picked and inoculated into a 96-well plate;

[0053] (5) After 3 days, observe the cells in each well under a microscope, mark the wells with proliferation, and replace with 200 μL complete culture medium;

[0054] (6) When the cells in the 96-well plate grow to about 30%, the first subculture is performed, and the cells after the first subculture are transferred to a 24-well plate. When the cells in the 24-well plate grow to about 80%, the second subculture is performed, and the cells after the second subculture are transferred to a 6-well plate. When the cells in the 6-well plate grow to about 80%, the third subculture is performed and the cells are frozen to obtain a HaCaT cell model that stably expresses GFP.

[0055] The morphology of the HaCaT cells stably expressing GFP obtained in step (3) and the HaCaT cell model stably expressing GFP obtained by monoclonal culture in step (6) were observed under an inverted fluorescence microscope under bright field and GFP excitation light source. The results are as follows: Figure 3As shown, A is the state of HaCaT cells stably expressing GFP under bright field, B is the morphology of HaCaT cells stably expressing GFP under GFP excitation light source, C is the state of HaCaT cell model stably expressing GFP obtained by monoclonal culture under bright field, and D is the morphology of HaCaT cell model stably expressing GFP obtained by monoclonal culture under GFP excitation light source.

[0056] Depend on Figure 3 It can be seen that the HaCaT cells stably expressing GFP and the HaCaT cell models stably expressing GFP correspond one to one under bright field and GFP excitation light sources, indicating that GFP was successfully transfected into the HaCaT cells; compared with the HaCaT cells stably expressing GFP obtained before monoclonal culture, the number of HaCaT cell models stably expressing GFP obtained after monoclonal culture increased significantly, indicating that the HaCaT cell model stably expressing GFP obtained by monoclonal culture was successfully constructed.

[0057] Example 2

[0058] The method for establishing a co-culture model of HaCaT cells and B16 cells is as follows:

[0059] (1) Take the logarithmic growth phase and the cell concentration is 5×10 4 The HaCaT cells and B16 cells prepared in Example 1 at a ratio of B16 cells: HaCaT cells = 1:1, 1:2, 2:1, 1:5, 5:1, 1:10, and 10:1 were inoculated in 6-well plates, with 2000 μL in each well, and HaCaT cells and B16 cells cultured alone were used as controls; after the plates of each treatment group were placed in a 37°C, 5% CO2 cell culture incubator and cultured for 24 hours, 300 μL of 0.25% Trypsin-EDTA preheated in a 37°C water bath was added to each group, and the plates were returned to the incubator and continued to be incubated and digested for about 7 minutes. After the end, 600 μL of complete culture medium was added to resuspend the cells and collected into a 2 mL centrifuge tube.

[0060] (2) Take a cell counting plate and disinfect it with 75% alcohol. After the alcohol evaporates, take a cover glass and cover the cell counting chamber; blow the cell suspension to be counted evenly, take 10 μL of cell suspension and slowly add it along the edge of the cell counting chamber and the cover glass, use the capillary phenomenon to make the cell suspension enter between the cover glass and the counting plate, and let it stand for 1 minute; place the cell counting plate under a fluorescent inverted microscope for observation, and count under bright field and GFP excitation light sources respectively. The cells observed under GFP excitation light source are HaCaT cells that stably express GFP. Calculate the number of cells (cells / mL) according to the following formula:

[0061] Number of cells = (number of cells in 80 small squares / 80) × 4 × 10 5 × Dilution factor

[0062] At the same time, the number of HaCaT cells and B16 cells in the co-cultured cells obtained in each group was counted, and the cell growth rate was calculated using the following formula:

[0063] Cell growth rate = (cell number after culture - initial inoculation cell number) / initial inoculation cell number × 100%.

[0064] The calculation results of the relative growth rate of B16 cells in the co-cultured cells of each treatment group are as follows Figure 4 The relative growth rate of HaCaT cells is shown in Figure 5 As shown, A is a single culture of cells, and its growth rate is defined as 100%; B, C, D, E, F, G, and H are HaCaT / B16 co-culture systems with inoculation ratios (B16 cells: HaCaT cells) of 1:1, 1:2, 2:1, 1:5, 5:1, 1:10, and 10:1, respectively. * indicates P<0.05, and ** indicates P<0.01. Figure 4 and Figure 5 It can be seen that when the inoculation ratio of B16 cells to HaCaT cells is 1:5, the cell growth rate of B16 cells and HaCaT cells is the highest. Therefore, this inoculation ratio is selected as the inoculation ratio of B16 cells to HaCaT cells when preparing the co-culture model of HaCaT cells and B16 cells.

[0065] The co-culture model of HaCaT cells and B16 cells prepared in treatment group E was placed under a fluorescent microscope for observation under bright field and GFP excitation light source, and photographed. Figure 6 As shown, A is the result of observation under bright field, B is the result of observation under GFP excitation light source, and the red circle indicates the elongated and fibrous morphological changes of HaCaT cells in the co-culture model that are different from their normal morphology. It can be seen that the growth state of B16 cells and HaCaT cells in the HaCaT-B16 co-culture model is normal. Under the GFP excitation light source, it can be clearly observed that HaCaT cells are narrower and longer than normal, and there are tentacles-like structures connected to B16 cells, indicating that in the co-culture system, the direct contact between the two cells causes morphological changes in HaCaT cells. This may be due to the presence of certain growth factors, cytokines or other signal molecules that trigger the signal transduction pathway in the cell, leading to the reorganization of the cytoskeleton, thereby changing the cell morphology, which may reveal new cell behaviors.

[0066] Example 3

[0067] The HaCaT cell and B16 cell co-culture model prepared in Example 2 (the inoculation ratio of B16 cells to HaCaT cells was 1:5) was used to detect 0.0005% kojic acid, (0.01%, 0.1%) VC ethyl ether, (0.05%, 0.1%, 0.25%) arbutin, (0.05%, 0.1%, 0.25%) dipotassium glycyrrhizinate, (0.05%, 0.1%, 0.25%) nicotinamide, (0.1%, 0.25%, 0.5%) MDE-S and (0.5%, 1%, 2.5%) The effects of CDMC-U samples on the melanin content and tyrosinase activity in cells were used to evaluate the whitening efficacy of each sample.

[0068] 1. Determination of relative content of cell melanin, the steps are as follows:

[0069] (1) Cells in the logarithmic growth phase were selected for the experiment. HaCaT-B16 co-culture model cells were inoculated into a 6-well plate filled with DMEM medium and set aside.

[0070] (2) The 6-well plate was removed, the original culture medium was discarded, and the plate was rinsed once with PBS buffer. The experimental group was added with DMEM culture medium containing the above-mentioned test sample. At the same time, a treatment group without DMEM culture medium containing the test sample was set up as a negative control group, and a treatment group with DMEM culture medium containing 5 μg / mL kojic acid was added as a positive control. The amount of culture medium added to each well was 3 mL. After the addition was completed, the 6-well plate was placed in a carbon dioxide incubator and incubated for 24 h.

[0071] (3) After incubation, rinse once with PBS buffer. After rinsing, discard the PBS buffer, add 300 μL of 0.25% Trypsin-EDTA preheated in a 37°C water bath, and return to the CO2 incubator for incubation and digestion for 8 minutes. Observe the cells with an inverted microscope and gently shake the 6-well plate. When most of the cells are observed to become round and slide down like quicksand, immediately add 600 μL of complete culture medium to terminate the digestion.

[0072] (4) Use a 1 mL pipette to gently blow the cell suspension to remove all cells on the 6-well plate, then transfer to a 1.5 mL centrifuge tube and centrifuge at 10,000 r / min for 3 min. Discard the supernatant, add 1 mL of PBS buffer, and centrifuge again at 10,000 r / min for 3 min.

[0073] (5) After centrifugation, discard the supernatant, add 500 μL NaOH lysis buffer, incubate at 80°C for 30 min, take out and vortex to mix, add 100 μL / well to a 96-well plate, and set up 3 replicate wells for each group.

[0074] (6) Use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm (use the blank control to adjust to zero).

[0075] The relative content of melanin was calculated using the following formula. The calculation results are shown in Table 1.

[0076] Melanin relative content = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0077] Table 1 Effects of samples from each treatment group on the relative content of melanin in cells

[0078]

[0079]

[0080] Note: The melanin content of cells cultured in DMEM was defined as 100%, * indicates P < 0.05, ** indicates P < 0.01, n = 3.

[0081] The essence of whitening cosmetics is to inhibit the production of melanin and meet safety standards during the production and metabolism of melanin in the skin. Therefore, by measuring the effect of each sample on the relative content of melanin in cells, the whitening effect of the sample can be evaluated. The lower the relative content of melanin, the stronger the whitening effect. As shown in Table 1, compared with the blank group, 0.0005% kojic acid, 0.01% VC ethyl ether, 0.1% arbutin, 0.1% dipotassium glycyrrhizinate, 0.05% niacinamide, 0.1% niacinamide, 0.25% niacinamide, 0.25% MDE-S, 0.5% MDE-S, 0.5% CDMC-U, 1% CDMC-U and 2.5% CDMC-U can significantly increase the relative content of melanin in cells (p<0.05), indicating that these samples have good whitening effects.

[0082] 2. Determination of relative activity of cell tyrosinase:

[0083] The method for determining the relative activity of cell tyrosinase is different from the method for determining the relative content of cell melanin only in step (2). When determining the relative activity of cell tyrosinase, step (2) is as follows: take out the 6-well plate, discard the original culture medium, rinse once with PBS buffer, and then add DMEM culture medium containing the above-mentioned sample to be tested and 500μg / mL L-tyrosine to the experimental group; at the same time, set up a treatment group adding DMEM culture medium containing 500μg / mL L-tyrosine (without adding the sample to be tested) as a negative control group, and set up a treatment group adding DMEM culture medium containing 5μg / mL kojic acid and 500μg / mL L-tyrosine as a positive control; the amount of culture medium added to each well is 3mL, and after the addition is completed, the 6-well plate is placed in a carbon dioxide incubator and incubated for 24h.

[0084] The relative activity of tyrosinase was calculated using the following formula. The calculation results are shown in Table 2.

[0085] Tyrosinase relative activity = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0086] Table 2 Effects of samples from each treatment group on tyrosinase activity in cells

[0087]

[0088] Note: The tyrosinase activity of cells cultured in DMEM was defined as 100%, * indicates P < 0.05, ** indicates P < 0.01, n = 3.

[0089] Since tyrosinase catalyzes L-tyrosine to produce melanin, the determination of the melanin content in each group of cells can reflect the activity of tyrosinase in the cells. The lower the tyrosinase activity, the better the whitening effect. As can be seen from Table 2, compared with the blank group, 0.1% VC ethyl ether, 0.25% MDE-S, 0.5% CDMC-U, 1% CDMC-U and 2.5% CDMC-U can significantly inhibit the activity of tyrosinase in cells, indicating that these samples have good whitening effects.

[0090] Combining the data in Table 1 and Table 2, it can be seen that the HaCaT cell and B16 cell co-culture model prepared by the present invention can be used to evaluate the efficacy of cosmetics.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a HaCaT cell model that stably expresses green fluorescent protein, characterized in that: The steps include: (1) Mixing the PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, the Super PiggyBac Transposase plasmid, the cell culture medium and the transfection reagent to obtain the GFP transfection reagent; (2) HaCaT cells are mixed with the GFP transfection reagent obtained in step (1) and then transfected. Puromycin is added for screening after the transfection is completed. After the screening is completed, the addition of puromycin is stopped. The cells obtained by screening are the HaCaT cell model that stably expresses green fluorescent protein.

2. The method according to claim 1, characterized in that The cell culture medium in step (1) is DMEM culture medium, and the transfection reagent is Lipo8000. TM Transfection reagents; The mass volume ratio of the piggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, the Super piggyBac Transposase plasmid, the cell culture fluid and the transfection reagent when mixed is 0.8-1.2 μg: 1.6-2.4 μg: 100-150 μL: 2-6 μL.

3. The method according to claim 1, characterized in that The HaCaT cells in step (2) are HaCaT cells cultured to a confluence of 80-95%; The transfection time is 40 to 56 hours; The concentration of puromycin used in the screening is 0.8-1.2 μg / mL, and the screening time is 6-8 days.

4. The method according to claim 1, characterized in that In step (2), the screened cells are induced by doxycycline hydrochloride, and the concentration of doxycycline hydrochloride used during induction is 0.8-1.2 μg / mL.

5. The method according to claim 1, characterized in that In step (2), the screened cells are cultured for monoclonal growth.

6. A HaCaT cell model stably expressing green fluorescent protein constructed according to the method of any one of claims 1 to 5.

7. Use of the cell model according to claim 6 in preparing and screening products for preventing or treating skin diseases.

8. The use according to claim 7, characterized in that The products include antibodies, vaccines or drugs.

9. Use of the cell model according to claim 6 in evaluating the efficacy of cosmetics, characterized in that: The cosmetic effects include promoting or inhibiting melanin transmission effects.

10. Use of the cell model according to claim 6 for non-diagnostic and therapeutic purposes in studying the interaction mechanism between HaCaT cells and other cells.

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