Culture medium, oral mucosa epithelial cell model as well as preparation method and application of oral mucosa epithelial cell model

Abstract

The invention provides a culture medium, an oral mucosa epithelial cell model and a preparation method and application of the oral mucosa epithelial cell model, and belongs to the technical field of biology. The culture medium is successfully constructed, the in-vitro oral mucosa epithelium three-dimensional model highly similar to the human oral mucosa epithelium tissue in structure and function is successfully constructed through the culture medium, large-area application and popularization in the global range can be achieved, experimental materials are easy to obtain, cost is low, the period is short, and the method is suitable for large-scale popularization and application. Various research requirements related to the oral mucosa epithelium function can be met, great significance is achieved for promoting oral medicine research and clinical treatment, and good application prospects are achieved in preparation of medicine screening and anti-tumor medicine models.

Description

Technical Field

[0001] The invention belongs to the field of material processing, and specifically relates to a culture medium, an oral mucosal epithelial cell model, and a preparation method and use thereof. Background Art

[0002] As the first barrier of human defense, the integrity of the structure and function of the oral mucosal epithelium is crucial to maintaining oral health. In studies related to the regulation of oral mucosal epithelial function, clinical biopsy tissues or some animal model tissues are usually used as research objects, which have disadvantages such as weak operability and significant individual differences, and involve risks in medical ethics. Therefore, a widely applicable research model and strategy are urgently needed. The construction of an in vitro three-dimensional model of oral mucosal epithelium can provide support for the study of mucosal epithelial function, the exploration of disease mechanisms, and the development of new therapies. Currently, common oral mucosal epithelial research models include two-dimensional cell culture and cell-based three-dimensional culture. The biological characteristics and cell behaviors of two-dimensional models are quite different from those in vivo, and they cannot simulate complex physiological and pathological microenvironments. Although the existing three-dimensional models have made some improvements in simulating tissue microenvironments, their construction methods are complex and costly, and they are limited by experimental techniques and materials, making them difficult to promote and use.

[0003] Traditional human oral keratinocyte (HOK) culture medium is usually based on the KSFM serum-free culture medium scheme, supplemented with 1% penicillin / streptomycin (P / S). This basic culture medium scheme has the following problems when constructing a three-dimensional model: (1) The cell state is unstable, and the cells may fall off due to vibration, resulting in poor cell adhesion; (2) It cannot completely simulate the physiological environment of HOK cells in the body, especially when studying cell differentiation and three-dimensional tissue formation, and cannot form a good three-dimensional structure. Summary of the invention

[0004] In order to solve the problems in the prior art, the purpose of the present invention is to use the most widely used oral keratinocyte cell line to construct an in vitro three-dimensional model with high repeatability, easy operation, and universal applicability, so as to provide technical support for the research on oral mucosal epithelial function and treatment plan research. The present invention provides a culture medium, an oral mucosal epithelial cell model, and a preparation method and use thereof.

[0005] The invention provides a culture medium, which consists of a basal culture medium, non-essential amino acids, glucocorticoids, a cell metabolism supplement, a nucleic acid base supplement, a calcium ion supplement, vitamin C and serum; wherein the concentration of the non-essential amino acids is 2-6 mM, the concentration of the glucocorticoids is 1-2 mM, the concentration of the cell metabolism supplement is 0.05-0.15 mM, the concentration of the nucleic acid base supplement is 0.1-0.3 mM, the calcium ion concentration in the calcium ion supplement is 0-2 mM, the concentration of the vitamin C is 0-50 ug / ml, and the volume of the serum is 0-2% of the total volume of the culture medium.

[0006] Furthermore, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0~1mM, the concentration of the vitamin C is 0~50 ug / ml, and the volume of the serum is 0~2% of the total volume of the culture medium.

[0007] Further, The basal culture medium is a DMEM / F12 (1:1) culture medium in which DMEM culture medium and F12 culture medium are mixed in a ratio of 1:1; The non-essential amino acid is L-glutamine; The glucocorticoid is hydrocortisone; The cell metabolism supplement is O-phosphoethanolamine; The nucleic acid base supplement is adenine; The calcium ion supplement is calcium chloride; The serum is fetal bovine serum.

[0008] Further, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0.25 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 0% of the total volume of the culture medium; Or, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 1.0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 0% of the total volume of the culture medium; Or, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium; Or, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0.25 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium; Or, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 1.0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium.

[0009] The present invention also provides a method for preparing the culture medium, which comprises the following steps: weighing raw materials of the culture medium in proportion, mixing, and obtaining the culture medium.

[0010] The present invention also provides use of the culture medium in preparing an oral mucosal epithelial cell model.

[0011] The present invention also provides an oral mucosal epithelial cell model, which is a model obtained by culturing oral mucosal epithelial cells using the above culture medium.

[0012] Furthermore, the oral mucosal epithelial cells are human oral keratinocytes HOK, human immortalized keratinocytes Hacat, human normal squamous epithelial cells NOK, abnormally proliferating oral mucosal keratinocytes DOK or tumor cells Cal27.

[0013] The present invention also provides a method for constructing the above-mentioned oral mucosal epithelial cell model, which comprises the following steps: using an air-liquid interface culture method, on day 0, cells are inoculated into a culture chamber, and the above-mentioned culture medium is added to the upper and lower layers; on day 2, an air interface is constructed and the upper culture medium is removed; on days 12 to 16, the lower culture medium is replaced for culture; after the culture is completed, the three-dimensional tissue is collected and processed to obtain the oral mucosal epithelial cell model.

[0014] The present invention also provides the use of the oral mucosal epithelial cell model in drug screening and anti-tumor drug efficacy.

[0015] The present invention has achieved the following beneficial effects: The present invention provides a culture medium, by which an in vitro three-dimensional oral mucosal epithelium model with a structure and function highly similar to human oral mucosal epithelial tissue is successfully constructed, which can be widely promoted and used worldwide, and the experimental materials are easy to obtain, the cost is low, and the cycle is short. It can meet various research needs related to the function of oral mucosal epithelium, and is of great significance to promoting the progress of oral medicine research and clinical treatment, and has good application prospects in the preparation of drug screening and anti-tumor drug models.

[0016] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0017] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram for the construction of the 3D cell model.

[0019] Figure 2 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of HOK cells.

[0020] Figure 3 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of Hacat cells.

[0021] Figure 4 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of NOK cells.

[0022] Figure 5 To investigate the effects of different culture medium compositions on the construction of three-dimensional culture models of DOK cells.

[0023] Figure 6 To investigate the effects of different culture medium compositions on the three-dimensional culture model of tumor cells Cal27.

[0024] Figure 7 The three-dimensional model constructed for tumor cells Cal27 was used to verify the effect of anti-tumor drugs: (A) Representative three-dimensional model staining; (B) Thickness statistics of the three-dimensional model after treatment with different drugs. DETAILED DESCRIPTION

[0025] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0026] The invention purchases the most commonly used cell lines in oral medicine research, including: human oral keratinocytes HOK, human immortalized keratinocytes Hacat, human normal squamous epithelial cells NOK, abnormally proliferated oral mucosal keratinocytes DOK and tumor cells Cal27.

[0027] DMEM / F12 (1:1) medium refers to DMEM / F12 medium in which DMEM medium and F12 medium are mixed in a ratio of 1:1, F12 (Sigma-aldrich, N6658, 500ml), DMEM (Gbico, C11995500BT, 500ml).

[0028] Example 1. Preparation of cell culture medium 500 ml of DMEM / F12 (1:1) medium was taken as the basal medium, and L-glutamine with a final concentration of 4 mM, hydrocortisone with a final concentration of 1.48 μM, O-phosphoethanolamine with a final concentration of 0.1 mM, and adenine with a final concentration of 0.18 mM were added thereto, and the calcium chloride, L-ascorbic acid and serum contents were adjusted as shown in Table 1 to obtain the cell culture medium of the present invention.

[0029] Table 1 Formulas of different cell culture media Example 2: Establishment of an in vitro three-dimensional HOK cell culture model Cell culture conditions: 37 ℃, 5% CO2.

[0030] according to Figure 1 The cell model was constructed according to the schematic diagram. The specific operation is as follows: a 24-well plate cell culture chamber is used. On Day 0, HOK cells are plated in the chamber and medium 1 is added to the upper and lower layers to maintain the upper and lower liquid interfaces; on Day 2, an air interface is constructed and the upper layer of medium in the chamber is removed; the lower layer of medium is replaced every day from Day 12 to Day 16. Finally, the multi-layer three-dimensional tissue cultured in the chamber is collected, fixed, embedded, and sliced ​​to obtain a three-dimensional culture model of HOK cells.

[0031] Example 3: Establishment of an in vitro three-dimensional culture model of Hacat cells Referring to the method of Example 2, an in vitro three-dimensional culture model of Hacat cells was established, the only difference being that the culture medium 1 was replaced by the culture medium 2, and the HOK cells were replaced by Hacat cells, to obtain a three-dimensional culture model of Hacat cells.

[0032] Example 4: Establishment of an in vitro three-dimensional NOK cell culture model Referring to the method of Example 2, an in vitro three-dimensional culture model of Hacat cells was established, the only difference being that the culture medium 1 was replaced by the culture medium 3 and the HOK cells were replaced by the NOK cells, thereby obtaining a three-dimensional culture model of NOK cells.

[0033] Example 5: Establishment of an in vitro three-dimensional DOK cell culture model Referring to the method of Example 2, an in vitro three-dimensional culture model of DOK cells was established, the only difference being that the culture medium 1 was replaced by the culture medium 4, and the HOK cells were replaced by the DOK cells, to obtain the three-dimensional culture model of DOK cells.

[0034] Example 6: Establishment of an in vitro three-dimensional culture model of Cal27 cells Referring to the method of Example 2, an in vitro three-dimensional culture model of Hacat cells was established, the only difference being that culture medium 1 was replaced by culture medium 5, and HOK cells were replaced by Cal27 cells, thereby obtaining a three-dimensional culture model of Cal27 cells.

[0035] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0036] Experimental Example 1: Screening of conditions for constructing a three-dimensional cell culture model in vitro 1. Experimental methods 1.1. Hematoxylin-eosin staining (1) Place the paraffin sections in a 65°C constant temperature oven and bake for 1.5 h. (2) Paraffin sections were dewaxed and hydrated in sequence (dewaxing in xylene I solution for 20 min, dewaxing in xylene II solution for 15 min, and hydrating in 100% I, 100% II, 95%, 80%, and 75% graded alcohol for 5 min each); (3) Rinse with distilled water 3 times, 5 min each time; (4) Mayer hematoxylin staining for 1 min, 1% hydrochloric acid alcohol staining for 3-5 s, and tap water anti-blueing for 15 min; (5) Counterstain with 0.5% eosin for 1–2 min; (6) Dehydration in 80%, 90%, and 100% alcohol for 5 min each; (7) Transparent in xylene I or II solution, and seal with neutral gum; (8) Observation under a microscope.

[0037] 1.2 Immunofluorescence staining Cells were cultured on coverslips placed in 24-well plates. Subsequently, plasmid transfection was performed using Lipofectamine 2000 (Invitrogen). Eight hours after transfection, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with PBS containing 0.25% Triton X-100. Next, cells were blocked with 10% goat serum and then incubated with primary antibodies against HA tags overnight at 4°C. Afterwards, cells were washed with PBS and incubated with ALEXAFLUOR 488 secondary antibodies. Finally, slides were mounted using ProLong® Gold Antifade reagent (Invitrogen) containing DAPI, and confocal imaging was performed using a S3000 confocal microscope (HOOKEInstruments) to capture images.

[0038] 2. Experimental results (1) Screening of HOK cell culture conditions Refer to the method in Example 1, the only difference is that the HOK cell three-dimensional culture model is established according to the culture medium composition shown in Table 2.

[0039] Table 2. Effects of different culture medium compositions on the three-dimensional culture model of HOK cells Note: “-” means the substance is not added.

[0040] Through Table 2 and Figure 2 The three-dimensional model of HOK cells was constructed with different culture medium compositions, and it can be seen that: Ca 2+ Ion concentration affects the layered structure of the three-dimensional model. 2+ At a concentration of 0.25mM, a Vc concentration of 50ug / ml, and without serum, the oral mucosal epithelial model constructed by HOK cells showed significant stratification characteristics and could obtain a better three-dimensional structural model. Compared with the three-dimensional model constructed by traditional HOK culture medium, it has a better stratification effect and can better reflect the state of real oral mucosal tissue.

[0041] (2) Screening of Hacat cell culture conditions Refer to the method in Example 1, the only difference is that the three-dimensional culture model of Hacat cells is established according to the culture medium composition shown in Table 3.

[0042] Table 3. Effects of different culture medium compositions on the three-dimensional culture model of Hacat cells Note: “-” means the substance is not added.

[0043] Through Table 3 and Figure 3 The three-dimensional model of Hacat cells was constructed with different culture medium compositions, and it can be seen that: Ca 2+ Ion and serum concentrations affect the layered structure of the three-dimensional model. 2+ When the concentration was 1mM and the Vc concentration was 50ug / ml, in the absence of serum, the oral mucosal epithelial model constructed by Hacat cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0044] (3) Screening of NOK cell culture conditions The method in Reference Example 1 was used, except that the three-dimensional culture model of NOK cells was established according to the culture medium composition shown in Table 4.

[0045] Table 4. Effects of different culture medium compositions on NOK cell three-dimensional culture model Note: “-” means the substance is not added.

[0046] Through Table 4 and Figure 4 The three-dimensional model of NOK cells was constructed with different culture medium compositions. It can be seen that serum concentration affects the hierarchical structure of the three-dimensional model. 2+ When the concentration was 0mM, the Vc concentration was 50ug / ml, and the serum was 1%, the oral mucosal epithelial model constructed by NOK cells showed significant stratification characteristics and could obtain a better three-dimensional structure model.

[0047] (4) Screening of DOK cell culture conditions Refer to the method in Example 1, the only difference is that the DOK cell three-dimensional culture model is established according to the culture medium composition shown in Table 5.

[0048] Table 5. Effects of different culture medium compositions on the three-dimensional culture model of DOK cells Through Table 5 and Figure 5 The three-dimensional model of DOK cells was constructed with different culture medium compositions. It can be seen that serum concentration affects the hierarchical structure of the three-dimensional model. 2+ When the concentration was 0.25mM, the Vc concentration was 50ug / ml, and the content of 1% serum, the oral mucosal epithelial model constructed by DOK cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0049] (5) Screening of Cal27 cell culture conditions The method in Reference Example 1 was used, except that the Cal27 cell three-dimensional culture model was established according to the culture medium composition shown in Table 6.

[0050] Table 6. Effects of different culture medium compositions on the three-dimensional culture model of Cal27 cells Note: “-” means the substance is not added.

[0051] Through Table 6 and Figure 6 The three-dimensional model of Cal27 cells was constructed with different culture medium compositions. It can be seen that serum concentration affects the hierarchical structure of the three-dimensional model. 2+ When the concentration was 1 mM, the Vc concentration was 50 ug / ml, and the content of 1% serum, the oral mucosal epithelial model constructed by Cal27 cells showed significant stratification characteristics and could obtain a better three-dimensional structural model.

[0052] Experimental Example 2: Application of the in vitro Cal27 cell three-dimensional culture model in the study of the efficacy of anti-tumor drugs Based on the three-dimensional culture model constructed for Cal27 cells, the present invention studied the combined effects of two anti-tumor drugs. The advantage of this model is that the culture time is significantly longer than the two-dimensional cell level, so long-term drug treatment studies can be performed in vitro. The present invention evaluated the proliferation of cells under the conditions of drug 1 (Drug1), drug 2 (Drug2), and combination therapy (Comb), where CON is the control study group. Within a 14-day period, the results showed that the combination of the two drugs had a better ability to resist Cal27 cell proliferation ( Figure 7 A), by statistically analyzing the thickness of the three-dimensional model under different conditions, it was found that the combination of drugs significantly reduced the three-dimensional structure formed by Cal27 ( Figure 7 B). The above studies show that this model can be widely used in drug screening, anti-tumor drug efficacy research, etc.

[0053] In summary, the present invention found through screening that HOK cells use the screened culture medium formula (Ca 2+ The concentration was 0.25 mM, the Vc concentration was 50 ug / ml, and there was no serum). Hacat cells were cultured using the selected medium formula (Ca 2+ NOK cells were cultured using a culture medium formulated with a Ca concentration of 1.0 mM and a Vc concentration of 50 ug / ml in the absence of serum. 2+ DOK cells were cultured in a medium with a Ca concentration of 0 mM, a Vc concentration of 50 ug / ml, and 1% serum. 2+The Cal27 cells were cultured in a culture medium with a concentration of 0.25 mM, a Vc concentration of 50 ug / ml, and 1% serum. 2+ When the concentration was 1.0 mM, the Vc concentration was 50 ug / ml, and the serum was 1%, it could show significant stratification characteristics in the oral mucosal epithelial model and obtain a better three-dimensional structure model.

[0054] In summary, the present invention provides a culture medium, an oral mucosal epithelial cell model, and a preparation method and use thereof. The present invention successfully constructs a culture medium, and uses the culture medium to successfully construct an in vitro oral mucosal epithelial three-dimensional model that is highly similar to the structure and function of human oral mucosal epithelial tissue, which can be widely promoted and used worldwide, and the experimental materials are easy to obtain, low cost, and short cycle. It can meet various research needs related to the function of oral mucosal epithelial cells, and is of great significance to the advancement of oral medical research and clinical treatment, and has good application prospects in the preparation of drug screening and anti-tumor drug models.

Claims

1. A culture medium, characterized in that It consists of a basal culture medium, non-essential amino acids, glucocorticoids, cell metabolism supplements, nucleic acid base supplements, calcium ion supplements, vitamin C and serum; wherein the concentration of the non-essential amino acids is 2-6 mM, the concentration of the glucocorticoids is 1-2 mM, the concentration of the cell metabolism supplements is 0.05-0.15 mM, the concentration of the nucleic acid base supplements is 0.1-0.3 mM, the calcium ion concentration in the calcium ion supplement is 0-2 mM, the concentration of the vitamin C is 0-50 ug / ml, and the volume of the serum is 0-2% of the total volume of the culture medium.

2. The culture medium according to claim 1, characterized in that The concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0~1 mM, the concentration of the vitamin C is 0~50ug / ml, and the volume of the serum is 0~2% of the total volume of the culture medium.

3. The culture medium according to claim 1, characterized in that The basal culture medium is a DMEM / F12 (1:1) culture medium in which DMEM culture medium and F12 culture medium are mixed in a ratio of 1:1; The non-essential amino acid is L-glutamine; The glucocorticoid is hydrocortisone; The cell metabolism supplement is O-phosphoethanolamine; The nucleic acid base supplement is adenine; The calcium ion supplement is calcium chloride; The serum is fetal bovine serum.

4. The culture medium according to any one of claims 1 to 3, characterized in that The concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0.25 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 0% of the total volume of the culture medium; Or, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 1.0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 0% of the total volume of the culture medium; Or, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium; Or, the concentration of the non-essential amino acid is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 0.25 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium; Or, the concentration of the non-essential amino acids is 4 mM, the concentration of the glucocorticoid is 1.48 mM, the concentration of the cell metabolism supplement is 0.1 mM, the concentration of the nucleic acid base supplement is 0.18 mM, the calcium ion concentration in the calcium ion supplement is 1.0 mM, the concentration of the vitamin C is 50 ug / ml, and the volume of the serum is 1% of the total volume of the culture medium.

5. A method for preparing the culture medium according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: weighing raw materials of the culture medium in proportion, mixing, and obtaining the culture medium.

6. Use of the culture medium according to any one of claims 1 to 4 in preparing an oral mucosal epithelial cell model.

7. An oral mucosal epithelial cell model, characterized in that: The model is obtained by culturing oral mucosal epithelial cells using the culture medium described in any one of claims 1 to 4.

8. The oral mucosal epithelial cell model according to claim 7, characterized in that: The oral mucosal epithelial cells are human oral keratinocytes HOK, human immortalized keratinocytes Hacat, human normal squamous epithelial cells NOK, abnormally proliferated oral mucosal keratinocytes DOK or tumor cells Cal27.

9. A method for constructing the oral mucosal epithelial cell model according to claim 7 or 8, characterized in that: The method comprises the following steps: using an air-liquid interface culture method, on day 0, cells are inoculated into a culture chamber, and the culture medium described in any one of claims 1 to 4 is added to the upper and lower layers; on day 2, an air interface is constructed and the upper culture medium is removed; on days 12 to 16, the lower culture medium is replaced for culture; after the culture is completed, the three-dimensional tissue is collected and processed to obtain an oral mucosal epithelial cell model.

10. Use of the oral mucosal epithelial cell model according to claim 7 or 8 in drug screening and anti-tumor drug efficacy.

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