Application and method for inducing phenotype transformation from oral mucosa epithelium to dry lip epithelium by linoleic acid

Abstract

The invention provides an application and a method for inducing phenotype transformation from oral mucosa epithelium to dry lip epithelium by linoleic acid, linoleic acid with proper concentration acts on oral mucosa epithelial cells to regulate and control cell differentiation procedures, so that expression of specific markers of the oral mucosa epithelial cells is reduced, expression of specific markers of dry lip epithelial cells is increased, and expression of the specific markers of the oral mucosa epithelial cells is increased. The directional transformation of the cell phenotype is realized. The linoleic acid with the proper concentration is 10-20 [mu] M. The linoleic acid has the effects of regulating keratinization index expression of mucous membrane epithelial cells and enhancing the mucous membrane tissue barrier function for the first time, and the function is not recognized in previous research and clinical application of the linoleic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of facial tissue repair, in particular to application and method of linoleic acid inducing oral mucosa epithelium to transform into dry lip epithelium phenotype. BACKGROUND

[0002] Human lips are divided into dry lips and wet lips, wherein the dry lips, also known as red lips, are a unique oral mucosa with physiological characteristics of adapting to air exposure. The epithelial structure thereof is thinner than the skin epidermis, and can transmit the deep dermal capillary network, thereby showing a red color; at the same time, the barrier function of the dry lips is stronger than that of the wet lips and other oral mucosa, which can effectively reduce the transdermal water loss and resist the problems of dryness, desquamation, cracking and the like which are prone to occur after air exposure.

[0003] Due to the special structure and functional characteristics of the dry lips, it is difficult to find a substitute tissue with complete matching function and structure after the defect of the dry lips, which leads to the dry lip repair becoming one of the key difficulties in the field of facial repair, and this technical problem has not been effectively solved for a long time.

[0004] In the existing clinical practice, the repair method for the dry lip defect mainly relies on surgical treatment, and the specific operation is different according to the defect area: for small area dry lip defect, local direct apposition suture or autologous dry lip free flap transplantation can be used for repair; for larger area dry lip defect, full layer tissue filling repair is usually performed by skin flap transplantation (including local skin flap transplantation and free skin flap transplantation) or mucosal flap transplantation (such as perioral mucosal free muscle flap, lip mucosal flap, buccal mucosal muscle flap and the like). However, the above traditional surgical methods have obvious limitations: the process of obtaining the graft will cause additional damage to the donor site, and the postoperative survival of the graft and the long-term maintenance effect are uncertain, which directly affects the final effect of surgical repair.

[0005] In addition to the above traditional repair methods, tissue engineering lip repair technology has gradually become a research hotspot. This technology constructs a dry lip epithelium reconstruction model (LVERM) by co-culturing skin epidermal keratinocytes and oral mucosa epithelial cells, so as to realize the precise repair of dry lip defect. However, at present, such tissue engineering technology is still in the preclinical research stage, and there are potential risks of ethics and biological safety in the in vitro tissue engineering technology, and there is still a long distance from the actual clinical application.

[0006] Among the existing various dry lip repair methods, full-thickness mucosa transplantation or lip mucosa eversion surgery has obvious advantages over flap repair in terms of color, texture, and vascularization, etc. Among them, lip mucosa eversion and oral mucosa transplantation are the most commonly used and most acceptable dry lip repair methods for patients. However, these methods have a core defect: mucosa does not have the barrier function unique to dry lips, and long-term exposure to air after transplantation can cause dryness, peeling, cracking and other complications, which seriously affect the repair effect and the quality of life of patients.

[0007] Currently, there is no systematic research results and targeted treatment method for the dryness, cracking and other complications caused by air exposure after mucosa transplantation. In clinical practice, symptomatic treatment is usually used to relieve symptoms, including applying Vaseline to prevent cracking, rinsing with physiological saline to relieve pain after cracking, and applying antibiotic ointment to treat concurrent infections. However, the above treatment methods can only temporarily relieve symptoms and cannot fundamentally solve the core problem of insufficient barrier function of mucosa. In addition, they need to be used frequently, have limited treatment effect, and are difficult to effectively improve the postoperative quality of life of patients.

[0008] From the histological basis, the human dry lip epithelium is in a transitional state between the skin epidermis and the oral mucosa epithelium in terms of structure and molecular expression, and all three are composed of stratified squamous epithelium, which has histological conditions for mutual transformation. Based on this characteristic, promoting the transdifferentiation of mucosal epithelial cells to epidermal keratinocytes, up-regulating the expression of epidermal barrier-related proteins in mucosal epithelium, and promoting the transformation of mucosal epithelium to dry lip epithelium are the fundamental ways to fundamentally solve the dryness-related complications caused by air exposure of mucosa.

[0009] However, there is no effective technical solution in the prior art that can achieve the above transformation, and the existing surgical methods for dry lip repair still have their own limitations: local direct apposition suture is only suitable for small defects and cannot meet the repair needs of large area defects; autologous dry lip flap transplantation is limited by the scarcity of autologous dry lip tissue source and is also difficult to adapt to large defect filling; the graft of flap transplantation has a large gap in color and texture from the original dry lip, which easily leads to functional and aesthetic problems; although mucosal flap transplantation has more advantages in color and texture simulation, the complications caused by the lack of barrier function have not been fundamentally solved. Therefore, there is an urgent need for a technical solution that can meet the needs of dry lip defect repair effect and complication eradication to fill the gap in the prior art. SUMMARY

[0010] In view of the problems in the prior art, the purpose of the present application is to provide an application and method of linoleic acid-induced oral mucosa epithelium to dry lip epithelium phenotype transformation.

[0011] The linoleic acid concentration provided by the application can effectively induce oral mucosa epithelial cells to transform into a dry lip epithelial cell phenotype, and specifically, the linoleic acid regulates the cell differentiation program, down-regulates the expression of oral mucosa epithelial cell specific markers, up-regulates the expression of skin epidermal cell specific markers, and realizes the directional transformation of the mucosa epithelial cells into the dry lip epithelial cell phenotype.

[0012] Preferably, the suitable concentration of linoleic acid is 10-20 μM.

[0013] Preferably, the oral mucosa epithelial cells are derived from mouse and human lip mucosa tissues.

[0014] Preferably, the oral mucosa epithelial cell specific markers are cytokeratin 13 and SPRR3, the dry lip epithelial cell specific markers are cytokeratin 10, keratin 16, Involucrin and Loricrin, and after the linoleic acid acts, the epidermal differentiation marker cytokeratin 10, the early terminal differentiation marker Involucrin and the late terminal differentiation marker Loricrin are all up-regulated.

[0015] Preferably, after the linoleic acid acts on the oral mucosa epithelial cells, the expression of the oral mucosa epithelial differentiation complex member SPRR3 does not change significantly.

[0016] The method for inducing oral mucosa epithelial cells to transform into a dry lip epithelial cell phenotype provided by the application comprises the following steps: Step S1: isolating and obtaining oral mucosa epithelial cells, inoculating in a cell culture system for conventional culture; Step S2: when the cell fusion degree reaches 60-80%, adding linoleic acid into the culture system, so that the final concentration of the linoleic acid in the system is 10-20 μM; Step S3: continuing to culture for 24-48 h, and confirming the phenotype transformation effect by detecting the expression of cell markers.

[0017] Preferably, the cell culture system in step S1 is an HD-DMEM culture medium containing 10% fetal bovine serum and 1% double antibody, and the culture condition is 37℃, 5% CO2.

[0018] Compared with the prior art, the application has the following beneficial effects: 1. The application first discovers and confirms that linoleic acid has the functions of regulating the expression of keratinization indicators of mucosa epithelial cells and enhancing the barrier function of mucosa tissues, which is not recognized in the previous research and clinical application of linoleic acid. 2. The application provides a new treatment strategy based on linoleic acid for the complications such as dryness and cracking of the dry lip (red lip) caused by the defect of mucosa barrier function after the repair of the oral mucosa. 3. The present application first proves that linoleic acid regulates the specific pathway of keratinization indicators of epithelial cells, i.e. LA / ALOX15 / 13S-HODE / PPARs, and further determines the direct and indirect regulation pathways between PPARs and keratinization indicators. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of the provided drawings. Other features, objects and advantages of the present application will become more apparent through reading the following detailed description of the non-limiting embodiments with reference to the accompanying drawings: Figure 1 The gray scale graph and quantitative statistical column graph for detecting the changes of barrier-related indicators of oral mucosa epithelial cells after different dosing times of linoleic acid in the WB experiment in the present application; Figure 2 The quantitative statistical column graph for detecting the transcription level of barrier-related indicators of oral mucosa epithelial cells after 24h of treatment with different concentrations of linoleic acid in the RT-qPCR experiment in the present application; Figure 3 The actual photo and statistical analysis graph of the scratch experiment under the visible light microscope for detecting the effect of linoleic acid on the transverse migration ability of mouse oral mucosa epithelial cells in the present application; Figure 4 The statistical analysis column graph for the activity of oral mucosa epithelial cells after 24h of treatment with linoleic acid and solvent in the present application; Figure 5 The gray scale graph and quantitative statistical column graph for detecting the changes of keratinization indicators and ALOX15 of mouse oral mucosa epithelial cells after 24h of dosing with effective concentration of linoleic acid and dosing with effective concentration of linoleic acid combined with ALOX15 inhibitor in the WB experiment in the present application; Figure 6 The gray scale graph and quantitative statistical column graph for detecting the expression changes of keratinization indicators of human epidermal keratinocytes after 24h of dosing with effective concentration of linoleic acid in the WB experiment in the present application; Figure 7 The gray scale graph and quantitative statistical column graph for detecting the expression changes of keratinization indicators and ALOX15 of mouse oral mucosa epithelial cells after 24h of treatment with different concentrations of 13S-HODE in the WB experiment in the present application; Figure 8 The gray scale graph and quantitative statistical column graph for detecting the protein expression of PPARα and PPARγ by 10uM linoleic acid in the WB experiment in the present application, and the quantitative statistical column graph for detecting the transcription level by RT-qPCR experiment. Figure 9 The gray scale chart and quantitative statistical column chart of the expression of PPARa and PPARy protein in the WB experiment in the application for detecting the effect of 13S-HODE with different concentrations on the expression of PPARa and PPARy protein, and the quantitative statistical column chart of the transcription level in the RT-qPCR experiment; Figure 10 CK10 immunohistochemical staining of the human lip mucosa full-thickness tissue cultured in the Transwell chamber in the application; Figure 11 The mechanism diagram of the regulation of the keratinization-related indicators of the oral mucosa epithelial cells mediated by PPARs in the application; Figure 12 The result chart of the Chip-seq analysis of the direct regulation of PPARy on the target gene and the result chart of the luciferase reporter gene experiment verifying the transcriptional regulation of the KRT10 gene in the application. DETAILED DESCRIPTION

[0020] The application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the application, but do not limit the application in any form. It should be noted that, for those skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made. These all belong to the protection scope of the application.

[0021] Linoleic acid (LA) is a key component of the skin barrier function, not only directly constituting the stratum corneum, but also regulating the proliferation, differentiation and keratinization of keratinocytes through multiple mechanisms, thereby enhancing the skin barrier function.

[0022] The application confirms that appropriate concentration of linoleic acid can induce the transformation of the oral mucosa epithelial cell phenotype to the dry lip epithelial cell phenotype by isolating mouse and human lip mucosa epithelial cells. At the tissue level, the directional transformation of the mucosa epithelial cell phenotype to the dry lip epithelial cell phenotype is successfully realized by the linoleic acid administration with the help of the Transwell chamber culture simulating the physiological environment after the air exposure of the mucosa, which further verifies the core hypothesis of the application.

[0023] The research results innovatively confirm that the oral mucosa epithelial cells such as the lip mucosa can be precisely regulated in the differentiation program and transformed into the epithelial cells with the characteristics of the dry lip under the induction of the active lipid (linoleic acid). This discovery not only expands the role boundary of linoleic acid in the regulation of the function of the stratified squamous epithelium, but also clearly defines its new function as a bioactive lipid.

[0024] Inducing oral mucosal epithelial cells to transform into dry lip epithelial cell phenotypes using appropriate concentrations of linoleic acid can significantly enhance the oral mucosal barrier function, potentially solving the clinical problem of persistent dryness and cracking after mucosal transplantation or eversion from the root, greatly improving the effect of mucosal repair of dry lips, and making up for the current shortcoming of insufficient effective tissue source in dry lip repair.

[0025] In clinical applications, it is expected that direct topical application of linoleic acid after mucosal flap transplantation or lip mucosal eversion surgery can further induce the transformation of oral mucosal epithelium into dry lip epithelium on the basis of simple moisturizing and lubrication. With long-term use, it is even expected to achieve the ideal effect of not needing continuous topical administration.

[0026] For effectiveness studies, such as Figure 1 As shown, Western blotting (WB) was performed to detect changes in oral mucosal epithelial cell barrier-related indicators at 6h, 9h, 12h, and 24h after linoleic acid (LA) administration. (a) is a WB grayscale image, and (b)-(e) are bar charts (mean ± standard deviation) of the WB grayscale images. The control group and the internal reference protein (β-actin) were used as controls, with n=3. For ease of representation, the post-hoc SNK statistical analysis results are labeled above the LA treatment groups showing statistical differences from the control group. Significant differences are expressed as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0027] like Figure 2 As shown, the transcriptional levels of oral mucosal epithelial cell barrier-related indicators were detected using RT-qPCR after 24 hours of treatment with different concentrations of linoleic acid. The results were presented as follows: -△△Ct Statistical analysis was performed, with a control group and an internal reference protein (β-actin) as controls, n=3. Significant differences were expressed as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0028] Figure 1WB results showed that 10 mM linoleic acid treatment for 24 hours can effectively up-regulate the protein expression levels of CK10 (P<0.0001), Involucrin (P<0.0001), and Loricrin (P<0.01) in mouse oral mucosa epithelial cells. The above indicators are highly expressed in dry lips (also known as red lips) under physiological conditions, while they are lowly expressed or not expressed in mucosa. Therefore, we believe that 10 mM linoleic acid treatment for 24 hours can significantly induce the phenotype transformation of mouse mucosa epithelial cells to dry lip epithelial cells. The stable expression of the barrier-related indicator Sprr3 in the mouse oral mucosa epithelial cells after 10 mM linoleic acid induction for 24 hours suggests that the mouse oral mucosa epithelial cells still retain some mucosa characteristics on the basis of linoleic acid-induced transdifferentiation, which is highly consistent with the characteristics of dry lip epithelium in the mucosa epithelium-skin epidermis transition state.

[0029] The RT-qPCR results of CK10 and Involucrin ( Figure 2 ) are basically consistent with the WB results, confirming the stability of the results at the transcriptional and protein translation levels.

[0030] For safety studies, as shown in Figure 3 , the scratch experiment photos at 0h, 12h, 24h, 36h, and 48h under a visible light microscope were used to explore the effect of effective concentration linoleic acid on the transverse migration ability of mouse mucosa epithelial cells. The statistical analysis chart of the scratch healing rate in different groups at 12h after scratch modeling was also included. The significant differences in the results are represented as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0031] As shown in Figure 4 , the statistical analysis column chart of the oral mucosa epithelial cell activity in different groups after 24 hours of linoleic acid and 0.1% DMSO treatment is shown. The significant differences in the results are represented as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] As shown in Figure 6 , the WB experiment detected the changes in keratinization indicators of human epidermal keratinocytes after 24 hours of effective concentration linoleic acid administration. (a) is the WB grayscale image, and (b)-(g) are the WB grayscale image quantitative statistical analysis column chart (mean ± standard deviation), with the control group and the internal reference protein (β-actin) as the control, n=3. The significant differences in the results are represented as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0033] The CCK-8 experiment results suggest that Figure 4), the effective concentration of linoleic acid slightly inhibited the activity of mouse oral mucosa epithelial cells. The 0.1% DMSO group and the two different concentrations of linoleic acid treatment groups all slightly decreased the cell activity (P<0.05), and the decrease was consistent, so we speculate that the slight cytotoxicity is mainly from the solvent 0.1% DMSO, not linoleic acid itself. This further suggests that the mucosal epithelial cells may be more sensitive to lipid-soluble drugs, because 0.1% DMSO does not cause significant toxicity to most cells.

[0034] The results of the scratch test show that (Fig. 4) Figure 3 ), two concentrations of linoleic acid can promote the transverse migration of oral mucosa epithelial cells, and the scratch healing rate at 12h is significantly higher than that of the control group and the 0.1% DMSO group (P<0.05), indicating that the effective concentration of linoleic acid does not inhibit the normal transverse migration function of epithelial cells, further confirming its cell safety.

[0035] At the same time, 10-20μM linoleic acid has no obvious effect on the barrier index of human epidermal keratinocytes (Fig. 5) Figure 6 ), which means that the concentration of linoleic acid that induces the transformation of oral mucosa epithelium to dry lip epithelium is not enough to change the barrier function of the surrounding epidermis, suggesting that there is a sufficient concentration window for linoleic acid in the future in vivo delivery, further improving its safety of mucosal local application.

[0036] Corresponding to the mechanism of ALOX15 and 13S-HODE in the process of linoleic acid action, as shown in Fig. 6 Figure 5 , the changes of keratinization index of mucosal epithelial cells after 10μM linoleic acid, 10μM linoleic acid+7.75μM ALOX15 inhibitor treatment for 24h. (a) is the WB gray diagram, (b)-(i) are the WB gray diagram quantitative statistical analysis column chart (mean ± standard deviation), with the control group and the internal reference protein (β-actin) as the control, n=3. The significant differences of the results are represented as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0037] As shown in Fig. 7 Figure 7 , the expression of keratinization index of mouse mucosal epithelial cells after 1.5μM-5μM 13S-HODE (product of enzymatic oxidation and metabolism of linoleic acid by ALOX15) treatment for 24h. (a) is the WB gray diagram, (b)-(i) are the WB gray diagram quantitative statistical analysis column chart (mean ± standard deviation), with the control group and the internal reference protein (β-actin) as the control, n=3. The significant differences of the results are represented as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0038] ALOX15 inhibitor can significantly inhibit the regulatory effect of linoleic acid on the barrier index of oral mucosa epithelial cells Figure 5 ), suggesting that ALOX15 is the key node of linoleic acid in the above-mentioned effects. Specifically, 10 μM linoleic acid can promote the expression of CK10 (P<0.01), Involucrin (P<0.01), Loricrin (P<0.0001), Sprr3 (P<0.05), and CK16 (P<0.05), while the addition of 7.75 μM ALOX15 inhibitor significantly reduces the above-mentioned indicators compared with the 10 μM linoleic acid group (P<0.05); 10 μM linoleic acid can significantly inhibit the expression of CK13 (P<0.05) and CK19 (P<0.05), while the addition of 7.75 μM ALOX15 inhibitor significantly increases the expression of CK13 (P<0.01) and CK19 (P<0.05) compared with the linoleic acid group, i.e., ALOX15 inhibitor can partially offset the effect of linoleic acid. The above results show that ALOX15 is an important intermediate factor of linoleic acid in inducing the change of keratinization index of mucosal epithelial cells, and when ALOX15 is abnormal, linoleic acid will lose its regulatory ability on the above-mentioned keratinization index.

[0039] ALOX15 is an enzyme that can promote the enzymatic oxidative metabolism of linoleic acid, and its main stable metabolite is 13S-HODE. We further used purified 13S-HODE to directly act on mucosal epithelial cells to explore whether it is a downstream molecule of linoleic acid-mediated keratinization regulation.

[0040] WB results suggest ( Figure 7 ), 13S-HODE has the same regulatory effect on mucosal epithelial cells as linoleic acid, specifically promoting the expression of CK10 (P<0.05), Involucrin (P<0.05), Loricrin (P<0.05), and CK16 (P<0.01), while inhibiting the expression of CK13 (P<0.001) and CK19 (P<0.05).

[0041] The above results show that 13S-HODE produced by linoleic acid through ALOX15 enzyme metabolism can explain most of the linoleic acid-mediated keratinization regulation, so we believe that 13S-HODE is an important downstream target of linoleic acid-mediated mucosal epithelial cell keratinization phenotype transformation.

[0042] In summary, linoleic acid is metabolized by ALOX15 enzyme oxidation to produce 13S-HODE, which further mediates the regulation of keratinization-related indicators of mucosal epithelial cells.

[0043] For the mechanism of PPAR signaling pathway in the process of linoleic acid action, such as Figure 8As shown, the results of Western blotting (WB) analysis on the regulation of PPARα and PPARγ protein expression levels are presented. (a) is the WB grayscale image; (b)-(c) are bar charts (mean ± standard deviation) of the quantitative statistical analysis of the WB grayscale images, with the control group and internal reference protein (β-actin) as controls, n=3; (d)-(e) are the results of RT-qPCR analysis on the regulation of PPARα and PPARγ transcription levels, with 2... -△△Ct Statistical analysis was performed, with a control group and an internal reference protein (β-actin) as controls, n=3. Significant differences were expressed as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0044] like Figure 9 As shown, the results of Western blotting (WB) analysis on the regulation of PPARα and PPARγ protein expression levels by 13S-HODE are presented. (a) is the WB grayscale image; (b)-(c) are bar charts (mean ± standard deviation) of the quantitative statistical analysis of the WB grayscale images, with the control group and internal reference protein (β-actin) as controls, n=3; (d)-(e) are the results of RT-qPCR analysis on the regulation of PPARα and PPARγ transcription levels by 13S-HODE, with 2... -△△Ct Statistical analysis was performed, with a control group and an internal reference protein (β-actin) as controls, n=3. Significant differences were expressed as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0045] like Figure 11 The diagram illustrates the mechanism by which PPARs mediate the regulation of keratinization-related indicators in oral mucosal epithelial cells: the transcription of some target genes is directly regulated by PPARs (such as KRT10), while the transcription of others is indirectly regulated through PPARs-dependent mediating genes (such as Hgs and PRkcz genes).

[0046] like Figure 12 As shown, (a)-(d) are the results of Chip-seq analysis, indicating that Hgs and PRkcz genes are PPARγ-dependent genes, and their expression is directly regulated by PPARγ; (e) is the result of the luciferase reporter gene assay, confirming that PPARγ can directly regulate KRT10 gene transcription through PPRES-mediated mechanisms. Significant differences are represented as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0047] Since both linoleic acid and its enzymatic metabolite 13S-HODE are endogenous activators of PPARs-related pathway, and PPARs-related signaling pathway has been proved to be an important pathway for epidermal barrier regulation, we further explored whether linoleic acid and 13S-HODE mediated the phenotype change of mucosal epithelial cells through activating PPARs-related pathway.

[0048] Linoleic acid ( Figure 8 ) and 13S-HODE ( Figure 9 ) both promoted the transcription and protein expression of PPARa and PPARy, suggesting that the activation level of PPARs downstream signaling pathway was increased.

[0049] To further clarify the mechanism of PPARs pathway activation in regulating the keratinization indicators of oral mucosal epithelial cells, Chip-seq experiment ( Figure 12 , a-d) confirmed that PPARy could bind to the PPRE region upstream of the Hgs (P<0.05) and PRkzc (P<0.05) gene promoters, i.e. PPARy could directly regulate the transcription of the above two genes. In previous studies, through affinity purification-mass spectrometry (AP-MS) analysis, it was found that the proteins encoded by Hgs and PRkzc genes could interact with keratin, specifically: HGS protein binds to mucosal keratin CK13, CK19, and PRkzc protein binds to epidermal keratin CK10. The above evidence collectively indicates that PPARy can indirectly regulate the expression of keratinization-related indicators (CK13, CK19, CK10) by regulating the expression of Hgs and PRkzc and other "intermediate genes", and thus affect the transformation process of mucosal epithelium to dry lip epithelium.

[0050] In addition, luciferase reporter gene experiment ( Figure 12 , e) confirmed that PPARy could also directly regulate the transcription of KRT10 gene (encoding CK10 protein), suggesting that in addition to the indirect pathway, there is also a direct regulation pathway.

[0051] In summary, linoleic acid and its enzymatic metabolite 13S-HODE produced by ALOX15 can directly promote the transcription and expression of KRT10 gene by activating PPARy, and can also indirectly affect the expression of keratinization indicators such as CK10, CK13, and CK19 by promoting the expression of intermediate genes such as Hgs and PRkzc, ultimately realizing the transformation of mucosal epithelial cell phenotype to dry lip epithelial cell phenotype. Linoleic acid (LA) regulates the expression of keratinization-related indicators directly or indirectly through the LA / ALOX15 / 13S-HODE / PPARs pathway, and thus regulates the transformation of mucosal epithelial cells to dry lip epithelial cells ( Figure 11 ).

[0052] For tissue-level effectiveness evidence, such as Figure 10The results of the human tissue validation of the regulatory effect of linoleic acid on the lip mucosa barrier are shown: human lip mucosa full-thickness tissue was cultured on the upper layer of the Transwell chamber, and the mucosal epithelium was exposed to air-liquid interface by not adding culture medium to the upper layer to simulate postoperative air exposure, and the lower layer was given culture medium and group linoleic acid treatment. After 14 days of culture, the immunohistochemical staining of the CK10 index of the tissue was detected.

[0053] The results show that the newly generated epithelium of the linoleic acid treatment group of the human lip mucosa full-thickness tissue cultured on the upper layer of the Transwell chamber after 14 days of culture induced by the culture medium in the lower layer showed higher CK10 protein expression level and thicker epithelial thickness than the control group (0.1% DMSO) Figure 10 , indicating that linoleic acid treatment helps the barrier repair of the mucosa after air exposure.

[0054] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

[0055] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.

Claims

1. An application of linoleic acid in inducing the transformation of oral mucosal epithelium into a dry lip epithelium phenotype, characterized in that, By applying an appropriate concentration of linoleic acid to oral mucosal epithelial cells, the cell differentiation process is regulated, resulting in downregulation of specific markers in oral mucosal epithelial cells and upregulation of specific markers in dry lip epithelial cells, thereby achieving targeted transformation of cell phenotype.

2. The application according to claim 1, characterized in that, The appropriate concentration of linoleic acid is 10μM-20μM.

3. The application according to claim 1, characterized in that, The oral mucosal epithelial cells are derived from mouse and human lip mucosal tissues.

4. The application according to claim 1, characterized in that, The specific markers for oral mucosal epithelial cells are cytokeratin 13 and SPRR3; The specific markers for dry lip epithelial cells were cytokeratin 10, cytokeratin 16, involucrin, and loricrin. After treatment with linoleic acid, the expression of epidermal differentiation markers cytokeratin 10, early terminal differentiation markers Involucrin, and late terminal differentiation markers Loricrin in oral mucosal epithelial cells were all upregulated.

5. The application according to claim 1, characterized in that, After linoleic acid was applied to oral mucosal epithelial cells, the expression of SPRR3, a member of the oral mucosal epithelial differentiation complex, did not change significantly.

6. A method for inducing oral mucosal epithelial cells to transform into dry lip epithelial cell phenotypes, characterized in that... Includes the following steps: Step S1: Isolate and obtain oral mucosal epithelial cells, and seed them into a cell culture system for routine culture; Step S2: When the cell confluence reaches 60%-80%, add linoleic acid to the culture system to make the final concentration of linoleic acid in the system 10μM-20μM; Step S3: Continue culturing for 24-48 hours and confirm the phenotypic transformation effect by detecting the expression of cell markers.

7. The method according to claim 6, characterized in that, The cell culture system described in step S1 is HD-DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics, and the culture conditions are 37°C and 5% CO2.

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