In vitro recombinant vaginal mucosa model and construction method and application

Abstract

This application discloses an in vitro reconstructed vaginal mucosa model, its construction method, and its application. The construction method includes an improved culture medium: HVU medium is added to immortalized vaginal epithelial cell line VK2 / E6E7, which is then seeded into Transwell chambers and cultured. The HVU medium on the surface of the culture in the Transwell chambers is aspirated, and the Transwell chambers are raised to the air-liquid interface for air-liquid surface culture. The culture medium is then replaced with HVA medium to obtain the vaginal mucosa model. This method improves the stability of cells during in vitro culture and constructs an in vitro reconstructed vaginal mucosa model with a tissue structure highly similar to human vaginal mucosa (possessing a complete stratified squamous epithelial structure and normal intercellular connections), thereby enhancing the model's physiological functional simulation.

Description

Technical Field

[0001] This application relates to the fields of tissue engineering technology and pathogenic microorganism infection model construction technology, and in particular to an in vitro recombinant vaginal mucosa model, its construction method and application. Background Technology

[0002] Vaginitis is a common infectious disease of the female reproductive system, mainly including bacterial vaginosis, fungal vaginitis, and aerobic vaginitis. Its pathogenesis is closely related to vaginal microecological imbalance and pathogenic microorganism invasion. In order to further study the pathogenesis of vaginitis and develop targeted therapeutic drugs, constructing an in vitro model similar to the physiological structure of the human vaginal mucosa has become a key technical means.

[0003] Currently, the construction of in vitro vaginal mucosal models mainly relies on two cell sources: immortalized epithelial cell lines, such as A431 cells (human epidermal cancer cell line); and primary human vaginal epithelial cells. The construction method typically involves seeding these cells onto a matrix material and inducing cell differentiation by simulating the physiological environment within the human vagina (such as temperature, humidity, gaseous environment, and nutritional conditions) to form an in vitro mucosal model with a specific tissue structure.

[0004] Although existing technologies have enabled the construction of in vitro vaginal mucosal models and the translational application of vaginitis models, the following drawbacks remain: Defects in seed cell selection: When using A431 cells (human epidermal cancer cell line) to construct models, A431 cells originate from epidermal tissue and are not vaginal-specific epithelial cells. Their biological characteristics (such as differentiation capacity, surface antigen expression, and sensitivity to pathogens) differ significantly from human vaginal epithelial cells, resulting in mucosal models that cannot realistically simulate the physiological structure and function of the human vaginal mucosa, thus affecting the reliability of vaginitis models. When using primary human vaginal epithelial cells, there are issues of scarce cell sources, complex acquisition processes, and strict ethical restrictions. Furthermore, primary cells have limited in vitro proliferation capacity and few passages, and are prone to differentiation abnormalities during culture, leading to poor reproducibility, high costs, and difficulty in large-scale application. Low similarity between model tissue structure and human tissue: Existing technologies construct mucosal models that are mostly single-layered or thinly stratified structures, lacking the stratification characteristic of the human vaginal mucosa. The model lacks a complete squamous epithelial structure (such as the basal layer, spinous layer, granular layer, and stratum corneum), and the intercellular connections (such as tight junctions and desmosome junctions) in the model are incomplete. This makes it impossible to simulate the barrier function of the human vaginal mucosa, resulting in significant differences between the pathogen infection process and the actual pathological process in vivo, affecting the accuracy of experimental results. The model has a limited scope of application: existing models are mostly designed for single types of vaginitis (such as only applicable to bacterial vaginitis or only applicable to fungal vaginitis), and have poor compatibility with pathogens. They cannot simultaneously meet the research needs of different types of vaginitis, resulting in weak model versatility, increased research costs, and experimental cycles. The construction process is complex and the stability is poor: when constructing models with primary cells, the culture period is as long as 21-28 days, and the requirements for the culture environment (such as oxygen concentration and nutrient composition) are stringent. Even slight fluctuations can lead to model construction failure. Although the A431 cell model has a shorter culture period, the physiological function simulation of the model is low, and it cannot provide a reliable platform for subsequent infection experiments and drug evaluation. Summary of the Invention

[0005] Therefore, it is necessary to provide an in vitro reconstructed vaginal mucosa model, its construction method, and its application.

[0006] The first aspect of this application provides a method for constructing an in vitro recombinant vaginal mucosa model, the method comprising the following steps: Immortalized vaginal epithelial cell line VK2 / E6E7 cells were added to HVU medium, seeded into Transwell chambers, cultured, and the HVU medium on the surface of the culture in the Transwell chambers was aspirated. The Transwell chambers were then raised to the air-liquid interface for air-liquid surface culture. The medium was replaced with HVA medium to obtain a vaginal mucosa model. The HVU culture medium includes DMEM / F12 mixed medium, bovine pituitary extract, epidermal growth factor, hydrocortisone, insulin, adenine, L-glutamine, transferrin, calcium chloride, β-estradiol, and Y-27632. The HVA medium consists of HVU medium supplemented with vitamin C, fibroblast growth factor, triiodothyronine, and Wnt-3a.

[0007] In some embodiments, the HVU culture medium comprises DMEM / F12 mixed medium, bovine pituitary extract 10-30 μg / mL, epidermal growth factor 1-4 ng / mL, hydrocortisone 0.1-0.2 μg / mL, insulin 5-10 μg / mL, adenine 5-25 μg / mL, L-glutamine 0.4-2 mM, transferrin 5-10 ng / mL, calcium chloride 0.2-0.4 mM, β-estradiol 10-100 nmol / L, and Y-27632 5-10 μM.

[0008] In some embodiments, the concentration of the bovine pituitary extract is 10 μg / mL, 12 μg / mL, 14 μg / mL, 16 μg / mL, 18 μg / mL, 20 μg / mL, 22 μg / mL, 24 μg / mL, 26 μg / mL, 28 μg / mL, 30 μg / mL, or any range or value between two such values.

[0009] In some implementations, the epidermal growth factor concentration is 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, or any range or value between two values.

[0010] In some embodiments, the hydrocortisone concentration is 0.1 μg / mL, 0.11 μg / mL, 0.12 μg / mL, 0.13 μg / mL, 0.14 μg / mL, 0.15 μg / mL, 0.16 μg / mL, 0.17 μg / mL, 0.18 μg / mL, 0.19 μg / mL, 0.20 μg / mL, or any range or value between two values.

[0011] In some embodiments, the insulin concentration is 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, 7 μg / mL, 7.5 μg / mL, 8 μg / mL, 8.5 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, or a range or value between any two values.

[0012] In some embodiments, the adenine concentration is 5 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 15 μg / mL, 17 μg / mL, 20 μg / mL, 22 μg / mL, 25 μg / mL, or a range or value between any two values.

[0013] In some embodiments, the L-glutamine concentration is 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 2.0 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3.0 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4.0 mM, or a range or value between any two values.

[0014] In some embodiments, the transferrin concentration is 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 μg / mL, 8 ng / mL, 8.5 ng / mL, 9 ng / mL, 9.5 ng / mL, 10 ng / mL, or any range or value between two values.

[0015] In some embodiments, the calcium chloride concentration is 0.2 mM, 0.22 mM, 0.25 mM, 0.27 mM, 0.3 mM, 0.32 mM, 0.35 mM, 0.37 mM, 0.4 mM, or any range or value between two values.

[0016] In some embodiments, the β-estradiol concentration is 10 nmol / L, 20 nmol / L, 30 nmol / L, 40 nmol / L, 50 nmol / L, 60 nmol / L, 70 nmol / L, 80 nmol / L, 90 nmol / L, 100 nmol / L, or a range or value between any two values.

[0017] In some embodiments, the concentration of Y-27632 is 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, or any range or value between two values.

[0018] In some embodiments, the HVA medium is based on HVU medium with the addition of vitamin C 20-50 ng / mL, fibroblast growth factor 5-20 ng / mL, triiodothyronine 0.1-0.4 nmol / mL, Wnt-3a 20-100 ng / mL, and the concentration of calcium chloride is adjusted to 1.0-1.5 mM.

[0019] In some embodiments, the concentration of vitamin C is 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, or any range or value between two values.

[0020] In some embodiments, the concentration of the fibroblast growth factor is 5 ng / mL, 8 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 17 ng / mL, 20 ng / mL, 22 ng / mL, 25 ng / mL, 27 ng / mL, 30 ng / mL, or any range or value between two values.

[0021] In some embodiments, the triiodothyronine concentration is 0.1 nmol / mL, 0.12 nmol / mL, 0.15 nmol / mL, 0.20 nmol / mL, 0.22 nmol / mL, 0.25 nmol / mL, 0.27 nmol / mL, 0.30 nmol / mL, 0.32 nmol / mL, 0.35 nmol / mL, 0.37 nmol / mL, 0.4 nmol / mL, or any range or value between two values.

[0022] In some embodiments, the Wnt-3a concentration is 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, or any range or value between two values.

[0023] In some embodiments, the concentration of calcium chloride is 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, or any range or value between two values.

[0024] In some implementations, the VK2 / E6E7 cells are adjusted to a density of 2.5 × 10⁻⁶ cells using HVU medium before being seeded into the Transwell chambers. 6 ~5×10 6 Cell suspension of cells / mL.

[0025] In some embodiments, the density of the cell suspension is 2.5 × 10⁻⁶. 6 2.6×10 6 2.7×10 6 2.8×10 6 2.9×10 6 3.0×10 6 3.1×10 6 3.2×10 6 3.3×10 6 3.4×10 6 3.5×10 6 3.8×10 6 4.0×10 6 4.2×10 6 4.5×10 6 4.8×10 6 5.0×10 6 , or a range or value between any two values.

[0026] In some implementations, one or more of the following features are satisfied: (1) Change the culture medium daily during any culture process; (2) Immersion culture is performed using HVU medium for 2 to 3 days; in some embodiments, the culture time is 2 days, 2.5 days, 3 days, or any range or value between two values.

[0027] (3) Replace HVU medium with HVA medium and culture for 6 to 9 days; in some embodiments, the culture time is 6 days, 7 days, 8 days, 9 days, or any range or value between two values.

[0028] In some embodiments, the immortalized vaginal epithelial cell line VK2 / E6E7 cell culture process includes: adding recombinant human epidermal growth factor, bovine pituitary extract, and calcium chloride to a serum-free keratinocyte culture medium, culturing, changing the medium, passage, and obtaining the immortalized vaginal epithelial cell line VK2 / E6E7 cells.

[0029] In some implementations, one or more of the following features are satisfied: (1) The concentration of recombinant human epidermal growth factor is 0.1~0.3 ng / mL; in some embodiments, the concentration of recombinant human epidermal growth factor is 0.1 ng / mL, 0.12 ng / mL, 0.14 ng / mL, 0.16 ng / mL, 0.18 ng / mL, 0.20 ng / mL, 0.22 ng / mL, 0.24 ng / mL, 0.26 ng / mL, 0.28 ng / mL, 0.30 ng / mL, or any range or value between two values.

[0030] (2) The concentration of bovine pituitary extract is 0.05~0.2 mg / mL; in some embodiments, the concentration of bovine pituitary extract is 0.05 mg / mL, 0.08 mg / mL, 0.10 mg / mL, 0.12 mg / mL, 0.15 mg / mL, 0.17 mg / mL, 0.20 mg / mL, or any range or value between two values.

[0031] (3) The final concentration of calcium chloride is 0.2~0.5 mM; in some embodiments, the final concentration of calcium chloride is 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, or any range or value between two values.

[0032] (4) The immortalized vaginal epithelial cell line VK2 / E6E7 needs to be passaged when the cells grow to a plating rate of 70%-80%; in some embodiments, the plating rate is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or any range or value between two values.

[0033] (5) After the third generation of the subculture.

[0034] The second aspect of this application provides an in vitro reconstructed vaginal mucosa model, which is constructed using the method for constructing an in vitro reconstructed vaginal mucosa model described in the first aspect of this application.

[0035] The third aspect of this application provides an aerobic vaginitis model and a fungal vaginitis model, and inoculates and cultures the in vitro recombinant vaginal mucosa model provided in the second aspect of this application.

[0036] In some embodiments, the process of constructing the aerobic vaginitis model includes: inoculating Staphylococcus aureus into nutrient broth, culturing once, collecting the bacterial cells by centrifugation, washing with PBS buffer, adjusting the bacterial cell concentration, inoculating the bacterial solution into the upper chamber of the vaginal mucosa model, adding HVA medium to the lower chamber, and culturing a second time to obtain the aerobic vaginitis model.

[0037] In some embodiments, the Staphylococcus aureus can be replaced with a clinical isolate of Staphylococcus aureus.

[0038] In some embodiments, the process of constructing the aerobic vaginitis model includes one or more of the following features: (1) The initial culture conditions were 37℃ with shaking culture for 24 hours; (2) Wash with PBS buffer 2-3 times; (3) The adjusted bacterial concentration is 0.5~2×10⁻⁶. 8 CFU / mL; in some embodiments, the bacterial concentration is 0.5 × 10⁻⁶. 8 CFU / mL, 0.6×10 8 CFU / mL, 0.7×10 8 CFU / mL, 0.8×10 8 CFU / mL, 0.9×10 8 CFU / mL, 1.0×10 8 CFU / mL, 1.1×10 8 CFU / mL, 1.2×10 8 CFU / mL, 1.3×10 8 CFU / mL, 1.4×10 8 CFU / mL, 1.5×10 8 CFU / mL, 1.6×10 8 CFU / mL, 1.7×10 8 CFU / mL, 1.8×10 8 CFU / mL, 1.9×10 8 CFU / mL, 2×10 8 CFU / mL, or any range or value between two values.

[0039] (4) The conditions for secondary culture are 37°C and 5% CO2 for 16 to 24 hours; in some embodiments, the culture time is 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or any range or value between two values.

[0040] In some embodiments, the process of constructing the fungal vaginitis model includes: inoculating Candida albicans into Sabouraud dextrose liquid medium, culturing once, collecting the bacterial cells by centrifugation, washing with PBS buffer, adjusting the bacterial cell concentration, inoculating the bacterial solution into the upper chamber of an in vitro recombinant vaginal mucosa model, adding HVA medium to the lower chamber, and culturing a second time to obtain the fungal vaginitis model.

[0041] In some embodiments, the Candida albicans can be replaced with a clinical isolate of Candida albicans.

[0042] In some embodiments, the process of constructing the fungal vaginitis model includes one or more of the following features: (1) The conditions for one culture were 28℃ shaking culture for 48 hours; (2) Wash with PBS buffer 2-3 times; (3) The adjusted bacterial concentration is 1-3×10⁻⁶. 8 CFU / mL; in some embodiments, the bacterial concentration is 1×10⁻⁶. 8 CFU / mL, 1.2×10 8 CFU / mL, 1.4×10 8 CFU / mL, 1.6×10 8 CFU / mL, 1.8×10 8 CFU / mL, 2.0×10 8 CFU / mL, 2.2×10 8 CFU / mL, 2.4×10 8 CFU / mL, 2.6×10 8 CFU / mL, 2.8×10 8 CFU / mL, 3.0×10 8 CFU / mL, or any range or value between two values.

[0043] (4) The conditions for secondary culture are 37°C and 5% CO2 incubator for 24-48 hours; in some embodiments, the culture time is 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, or any range or value between two values.

[0044] The fourth aspect of this application provides the application of the in vitro recombinant vaginal mucosa model prepared by the in vitro recombinant vaginal mucosa model construction method described in the first aspect, the in vitro recombinant vaginal mucosa model described in the second aspect, or the aerobic vaginitis model and fungal vaginitis model described in the third aspect in vaginitis research.

[0045] The in vitro recombinant vaginal mucosa model provided in this application improves the culture medium formulation, adds cytokines adapted to the proliferation and differentiation of VK2 / E6E7 cells, enhances the stability of cells during in vitro culture, and constructs an in vitro recombinant vaginal mucosa model with a tissue structure highly similar to human vaginal mucosa (with a complete stratified squamous epithelial structure and normal intercellular connections), thereby improving the physiological function simulation of the model.

[0046] VK2 / E6E7 cells were used as an immortalized human vaginal epithelial cell line. Their biological characteristics are highly consistent with those of human vaginal epithelial cells. Compared with A431 cells (derived from the epithelium), they can more realistically simulate the physiological functions of the vaginal mucosa and its response to pathogenic microorganisms. Compared with primary human vaginal epithelial cells, VK2 / E6E7 cells have a stable source, strong proliferation capacity, and can be passaged multiple times. They do not require isolation from human tissue, thus avoiding ethical restrictions, reducing culture costs, and improving the reproducibility of model construction.

[0047] This application utilizes submersible culture and gas-liquid interface induction culture to differentiate VK2 / E6E7 cells into a mucosal model with a complete stratified squamous epithelial structure. The tight junctions and desmosomes between cells are intact, and the vaginal mucosal barrier function is close to the physiological state of the human body, providing a more reliable in vitro platform for subsequent infection experiments and drug evaluation.

[0048] Based on the same VK2 / E6E7 cell-derived mucosal model, Staphylococcus aureus and Candida albicans can be inoculated separately to construct both aerobic vaginitis and fungal vaginitis models. This overcomes the limitation of existing technologies where a single model is only suitable for studying one type of vaginitis and can meet the diverse needs of research on the pathogenesis of different types of vaginitis, drug screening, and other fields.

[0049] The model culture cycle of this application is only 9-12 days, which is significantly shorter than that of the primary cell model (21-28 days). The culture process requires mild environmental conditions (conventional 37℃, 5% CO2 incubator), does not require a special hypoxic environment, is easy to operate, has a high success rate in model construction, and is convenient for large-scale production and application.

[0050] The vaginitis model constructed in this application can realistically simulate the infection process and pathological changes of pathogenic microorganisms in vivo. It can replace traditional animal experiments, reduce ethical controversies, and has the advantages of controllable experimental conditions and good reproducibility of results. It can be widely used in basic research, drug screening, efficacy evaluation, vaccine development and other fields related to vaginitis, and has important clinical translational value. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0052] Figure 1The images show the appearance and histological morphology of the vaginal mucosa model of this application (where the left side is the appearance image and the right side is the histological morphology image). Figure 2 Image showing the morphology (H&E staining) of a vaginal mucosa model after stimulation with Staphylococcus aureus; Figure 3 A schematic diagram showing the bacterial distribution in a vaginal mucosa model stimulated with Staphylococcus aureus; Figure 4 A schematic diagram of the tissue morphology (H&E staining) of a vaginal mucosa model after stimulation with Candida albicans; Figure 5 A schematic diagram showing the distribution of fungi (PAS staining) in a vaginal mucosa model stimulated with Candida albicans; Detailed Implementation

[0053] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0055] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings: The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0056] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0057] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0058] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0059] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0060] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0061] The stromal layer (usually referring to the basal layer) is the deepest layer of stratified epithelium (such as the epidermis of the skin and the vaginal mucosa). It is closely attached to the basement membrane and consists of a single layer of columnar or cuboidal basal cells. These cells have an active capacity for division and proliferation, and are responsible for replenishing the epithelial cells that have sloughed off or been damaged from the surface layer; therefore, it is also called the germinal layer. In addition, melanocytes (in the skin) or other functional cells are often scattered throughout this layer, which are crucial for maintaining tissue homeostasis and regeneration.

[0062] The typical vaginal mucosa consists of an epithelial layer and a lamina propria. The epithelial layer is stratified squamous epithelium (unkeratinized), which is divided into the basal layer (i.e., the stroma), intermediate layer, and superficial layer from the base to the surface. The stroma (basal layer) is the bottom layer of the epithelium, with cells that are low columnar and have relatively dense nuclei. They are attached to the basement membrane through hemidesmosomes. The cells in this layer continuously undergo mitosis to produce new epithelial cells, which then move upward and differentiate into intermediate and superficial cells to replenish the normal shedding of vaginal epithelium (such as the cyclical changes regulated by hormones during the menstrual cycle). The lamina propria, located below the basement membrane, is composed of connective tissue rich in elastic fibers and blood vessels, providing nutrition and support.

[0063] Staphylococcus aureus is a common Gram-positive aerobic bacterium that can cause a variety of infectious diseases. In the female reproductive tract, its excessive proliferation can lead to aerobic vaginitis (AV). Staphylococcus aureus disrupts the acidic environment of the vagina by inhibiting the growth of lactobacilli (e.g., through the production of antimicrobial peptides or nutrient competition), creating a microecological environment conducive to the proliferation of aerobic bacteria. The inflammatory response further damages the mucosal barrier, forming a vicious cycle of "infection-inflammation-barrier disruption".

[0064] Candida albicans is the main pathogen causing fungal vaginitis (also known as candidal vaginitis, vulvovaginal candidiasis, VVC). Candida albicans transforms from its yeast phase (harmless) to its hyphal phase (highly invasive), penetrating vaginal epithelial cells and triggering an inflammatory response.

[0065] Replace "Staphylococcus aureus" with "Clinical isolate of Staphylococcus aureus (isolated from vaginal secretions of patients with aerobic vaginitis)" and "Candida albicans" with "Clinical isolate of Candida albicans (isolated from vaginal secretions of patients with fungal vaginitis)". The clinical isolate is closer to the actual clinical infection situation. After inoculation, it can normally colonize and proliferate on the mucosal model constructed by VK2 / E6E7 cells, causing mucosal tissue damage and forming a vaginitis model that is closer to the clinical pathological characteristics. This alternative can improve the clinical translational value of the model and still achieve the invention goal of "constructing an in vitro model of vaginitis for research and drug evaluation".

[0066] This application achieves compatible infection of multiple pathogenic microorganisms using the same basic mucosal model, and simultaneously constructs aerobic vaginitis and fungal vaginitis models, thus broadening the applicability of the model.

[0067] Air-liquid interface stratified culture is the core technology for constructing three-dimensional skin models. The key lies in artificially creating an air-liquid interface. Cells are seeded in a special chamber with a permeable membrane. The bottom of the chamber is immersed in the culture medium (liquid surface), while the cells at the top of the chamber are directly exposed to the air (air surface), which can form a complete barrier that closely resembles real skin.

[0068] Example: Construction of a vaginal mucosa model: This application provides a method for constructing a vaginal mucosa model, which is achieved through the following technical solution: Step 1. Immortalized vaginal epithelial cell line VK2 / E6E7 cell culture: Keratinocyte-SFM medium was used, supplemented with 0.1 ng / mL recombinant human epidermal growth factor (rhEGF), 0.05 mg / mL bovine pituitary extract (BPE), and 44.1 mg / L calcium chloride (CaCl2, final concentration 0.4 mM). The cells were cultured at 37°C in a 5% CO2 incubator, with the medium changed every 24 hours. When the cells reached 80% plating rate, they were passaged. After the third passage, the cells were used for model construction. Step 2. Seeding of vaginal epithelial cells: Aspirate the culture medium from the surface of the matrix layer, and adjust the density of the immortalized vaginal epithelial cell line VK2 / E6E7 obtained in Step 1 to 2.5 × 10⁻⁶ cells using HVU medium. 6 Cell suspensions of cells per mL were seeded into Transwell chambers and cultured in HVU medium for immersion culture for 2 days at 37°C in a 5% CO2 incubator, with the medium changed daily. The HVU medium described above was prepared by adding bovine pituitary extract 20 μg / mL, epidermal growth factor 3 ng / mL, hydrocortisone 0.1 μg / mL, insulin 5 μg / mL, adenine 10 μg / mL, L-glutamine 1 mM, transferrin 5 ng / mL, calcium chloride 0.2 mM, β-estradiol 100 nmol / L, and Y-27632 5 μM to DMEM / F12 medium (volume ratio 1~3:1). Step 3. Cultivation of vaginal mucosa model: Remove the HVU culture medium from the surface of the culture in the Transwell chamber, raise the Transwell chamber to the air-liquid interface for air-liquid surface culture, replace the HVU culture medium used in Step 2 with HVA culture medium, and incubate at 37℃ and 5% CO2 for 7 days, changing the medium daily to obtain the vaginal mucosa model. The HVA medium was prepared by adding vitamin C 50 ng / mL, fibroblast growth factor (FGF) 10 ng / mL, triiodothyronine (T3) 0.2 nmol / mL, Wnt-3a 50 ng / mL to the HVU medium, and adjusting the calcium chloride concentration to 1.5 mM.

[0069] Construction of aerobic vaginitis models and fungal vaginitis models: Aerobic vaginitis model: Staphylococcus aureus was inoculated into nutrient broth and cultured at 37°C with shaking for 24 hours. The bacterial cells were collected by centrifugation, washed twice with PBS buffer, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL; 10 μL of the bacterial culture was inoculated into the upper chamber of the above-mentioned in vitro recombinant vaginal mucosa model, and HVA medium was added to the lower chamber. The model was then incubated at 37℃ in a 5% CO2 incubator for 24 hours to obtain an aerobic vaginitis model. After 24 hours of incubation, TEER value (mucosal barrier function assessment) was first performed: using an epithelial cell resistance meter (purchased from Millipore), the electrodes were placed in the upper and lower chambers of the Transwell chamber, respectively, ensuring that the electrodes were in full contact with the culture medium. After the value stabilized, the TEER value (unit: Ω·cm²) was recorded. Each sample was tested 3 times, and the average value was taken. 200 μL of the supernatant of the culture medium in the lower chamber of the in vitro recombinant vaginal mucosa model was carefully aspirated and placed in a sterile centrifuge tube and frozen at -80℃ for later use. Subsequently, the polycarbonate membrane of the Transwell chamber was taken, fixed, dehydrated, embedded, and sectioned for HE staining and Gram staining. The mucosal tissue damage and bacterial colonization were observed under a microscope to confirm the successful construction of the model.

[0070] Fungal vaginitis model: Candida albicans was inoculated into Sabouraud dextrose liquid medium and cultured at 28°C with shaking for 48 hours. The bacterial cells were collected by centrifugation, washed twice with PBS buffer, and the bacterial concentration was adjusted to 2 × 10⁻⁶. 8CFU / mL; 10 μL of the bacterial culture was inoculated into the upper chamber of the in vitro reconstructed vaginal mucosa model, and HVA medium was added to the lower chamber. The model was then incubated at 37℃ in a 5% CO2 incubator for 48 hours to obtain a fungal vaginitis model. After 48 hours of incubation, TEER (mucosal barrier function assessment) value was first detected: using an epithelial cell resistance meter (purchased from Millipore), the electrodes were placed in the upper and lower chambers of the Transwell chamber, respectively, ensuring that the electrodes were in full contact with the culture medium. After the value stabilized, the TEER value (unit: Ω·cm²) was recorded. Each sample was tested 3 times, and the average value was taken. 200 μL of the culture medium supernatant from the lower chamber of the in vitro reconstructed vaginal mucosa model was carefully aspirated and placed in a sterile centrifuge tube and frozen at -80℃ for later use. Subsequently, the polycarbonate membrane of the Transwell chamber was taken, fixed, dehydrated, embedded, and sectioned for H&E staining and periodic acid-Schiff (PAS) staining. The mucosal tissue damage, fungal colonization, and hyphal formation were observed under a microscope to confirm the successful construction of the model.

[0071] Characterization of the vaginal mucosa model H&E staining of tissue sections: The vaginal mucosal model was fixed in 4% paraformaldehyde solution, followed by paraffin embedding and section staining for histological examination. ① Paraffin embedding and sectioning of tissue: After fixing the tissue for more than 24 hours, the tissue was sequentially immersed in 75%, 85%, and 95% anhydrous ethanol for 60 minutes each, followed by two 100% anhydrous ethanol solutions for dehydration. After clearing, the tissue was sequentially immersed in xylene I and xylene II for 6 minutes each. After clearing, the tissue block was sequentially immersed in: soft wax I (30 minutes), soft wax II (30 minutes), and hard wax (1 hour). After paraffin infiltration, the molten wax was poured into the embedding cassette, and the tissue block was quickly picked up with forceps, cut side down, and placed in the center of the cassette. It was removed after the paraffin had completely solidified and hardened. Ten sections were prepared from each specimen, with a section thickness of approximately 5 μm. The sections were placed on glass slides and baked in a slide oven at 60°C for 60 minutes. Before staining, place the sections in a 37℃ oven overnight or bake at 60℃ for 2 hours. ② H&E staining of tissue sections: Immerse the sections sequentially in xylene I for 10 min; xylene II for 10 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; 95% ethanol for 5 min; 90% ethanol for 5 min; 80% ethanol for 5 min; 70% ethanol for 5 min; wash with distilled water. Stain the sections with hematoxylin for 3-8 min, then rinse with tap water. Stain the sections with eosin for 1-3 min, then rinse with tap water. Dehydrate and clear the sections sequentially in 95% ethanol I for 5 min; 95% ethanol II for 5 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; xylene I for 5 min; xylene II for 5 min. Remove the sections from xylene II and allow them to air dry slightly. Mount with neutral resin.

[0072] like Figure 1 As shown, the recombinant human vaginal mucosa model constructed in vitro using VK2 / E6E7 cells appears milky white (left); according to the tissue section (right), the model structure is complete and the layers are clear, the epithelial cells are neatly arranged and have regular morphology, with a complete stratified squamous epithelial structure, and the tight junctions and desmosomes between cells are intact, indicating that the model was successfully constructed.

[0073] Gram staining of an aerobic vaginitis model: a. Take a section of the model and dewax it to water as usual; b. Cover the section with crystal violet staining solution, stain for 1 minute, and rinse with tap water; c. Mordant with Lugol's iodine solution for 1 minute, and rinse with tap water; d. Decolorize with 95% ethanol for 30 seconds, and rinse with tap water; e. Counterstain with safranin solution for 30 seconds, and rinse with tap water; f. Dehydrate in a gradient (70%, 80%, 90%, 100% ethanol), clear (xylene), and mount with neutral resin; g. Observe under an optical microscope. Staphylococcus aureus appears purple (Gram positive). Record the colonization location and number of bacteria.

[0074] like Figure 2 As shown, the vaginal mucosa model after stimulation with Staphylococcus aureus (H&E staining) exhibits clear tissue layers, with regular and uniform epithelial cell arrangement and underlying matrix structure. No obvious inflammatory cell infiltration or tissue damage was observed, presenting an overall healthy and normal tissue morphology. Compared to the blank control group, the vaginal mucosa model stimulated with Staphylococcus aureus showed disordered epithelial cell arrangement, abnormal cell morphology in some areas, signs of cell edema or necrosis, and blurred tissue layer structure in some areas, indicating tissue damage and inflammatory response induced by Staphylococcus aureus infection. This demonstrates that Staphylococcus aureus can disrupt the normal structure of vaginal mucosa tissue, induce inflammatory responses, and has a significant damaging effect on the vaginal mucosa, providing intuitive morphological evidence for subsequent research on the mechanism of Staphylococcus aureus-induced vaginal mucosal inflammation and related intervention measures.

[0075] like Figure 3As shown, this illustrates the bacterial distribution (Gram staining) in a vaginal mucosa model stimulated with Staphylococcus aureus. In the blank control group, the tissue layers were clear, with regularly arranged and uniformly morphological epithelial cells and a homogeneous underlying matrix structure. No obvious inflammatory cell infiltration or tissue damage was observed, indicating a healthy and normal tissue morphology. Compared to the blank control group, after stimulation with Staphylococcus aureus, the vaginal mucosa model showed Staphylococcus aureus invading the surface layer (blue area in the figure), with some bacteria invading the lower layer, affecting the overall structure and physiological function of the model. This demonstrates that Staphylococcus aureus has a strong invasive ability, capable of penetrating the vaginal mucosa surface and invading deeper tissues. This is a crucial reason why it causes vaginal mucosal damage and inflammatory responses, further providing morphological support at the bacterial distribution level for elucidating the pathological mechanism of Staphylococcus aureus infection of the vaginal mucosa.

[0076] PAS staining of a fungal vaginitis model: a. Take model sections and dewax them routinely to water; b. Oxidize with 3% periodic acid solution for 10 minutes, then rinse with tap water; c. Add Schiff's reagent and stain at room temperature in the dark for 15 minutes, then rinse with tap water; d. Counterstain cell nuclei with hematoxylin for 5 minutes, rinse with tap water, differentiate with hydrochloric acid ethanol for 3 seconds, and rinse again with tap water to return to blue; e. Dehydrate in a gradient (70%, 80%, 90%, 100% ethanol), clear (xylene), and mount with neutral resin; f. Observe under an optical microscope. The cell walls and hyphae of Candida albicans appear purplish-red, and the cell nuclei appear blue. Record the location, number, and hyphal formation of the fungus.

[0077] like Figure 4 The image shows the morphology of a vaginal mucosa model after stimulation with Candida albicans (H&E staining). The blank control group model exhibits clear tissue layers, with regularly arranged and uniformly shaped epithelial cells and a homogeneous underlying matrix structure. No obvious inflammatory cell infiltration or tissue damage was observed, presenting an overall healthy and normal tissue morphology. In contrast, the vaginal mucosa model stimulated with Candida albicans showed disordered epithelial cell arrangement, abnormal cell morphology in some areas, signs of cell edema or necrosis, and blurred tissue layers in some areas, indicating tissue damage and inflammatory response induced by Candida albicans infection. Numerous hyphae were also visible on the surface. This demonstrates that Candida albicans can disrupt the integrity of the vaginal mucosal epithelium through adhesion, invasion, and the formation of hyphal structures, inducing local inflammatory responses and tissue damage. Its hyphal morphology may be a key factor enhancing its pathogenicity and exacerbating mucosal barrier damage, providing important morphological evidence for a deeper understanding of the pathological process of Candida albicans infection and the development of targeted prevention and control strategies.

[0078] like Figure 5As shown, this illustrates the fungal distribution (PAS staining) in a vaginal mucosa model stimulated with Candida albicans. In the blank control group, the tissue layers were clear, with regularly arranged and uniformly shaped epithelial cells and a homogeneous underlying matrix structure. No obvious inflammatory cell infiltration or tissue damage was observed, presenting an overall healthy and normal tissue morphology. Compared to the blank control group, the vaginal mucosa model stimulated with Candida albicans exhibited dense, dark purple-red hyphae / spore structures on the tissue surface. Due to pathogen colonization and inflammatory infiltration, the normal structure of the local tissue was disrupted, and the layers became blurred. This indicates that Candida albicans can colonize extensively on the vaginal mucosa surface and form hyphae / spore structures. Its colonization ability and hyphae / spore formation disrupt the integrity of the vaginal mucosal barrier and trigger local inflammatory responses. PAS staining clearly reveals its distribution characteristics, providing intuitive morphological evidence of fungal distribution layers for elucidating the colonization mechanism and pathogenic process of Candida albicans infection of the vaginal mucosa.

[0079] Comparative Example 1 The difference from the example is that in step 3, the HVU culture medium is not changed, and the same HVU culture medium as in step 2 is used. The rest of the process is the same as in the example.

[0080] Comparative Example 2 The difference from the example is that the culture medium used in step 2 is HVA culture medium, and the rest is the same as the example.

[0081] Comparative Example 3 The difference from the example is that the HVU medium in step 2 is based on DMEM / F12 medium (volume ratio 1~3:1) with the addition of bovine pituitary extract BPE 20 μg / ml, epidermal growth factor 3 ng / mL, hydrocortisone 0.1 μg / mL, insulin 5 μg / mL, adenine 10 μg / mL, L-glutamine 1mM, transferrin 5 ng / mL, calcium chloride 0.2mM, and β-estradiol 100 nmol / L; the rest is the same as the example.

[0082] Comparative Example 4 The difference from the example is that the HVA medium in step 3 is prepared by adding 50 ng / mL of vitamin C to the HVU medium and adjusting the concentration of calcium chloride to 1.5 mM.

[0083] Detection steps for culture medium supernatant: a. Detection of inflammatory factors: Take the frozen culture medium supernatant, thaw it at room temperature, and follow the instructions of the IL-6, IL-8, TNF-α ELISA kit (purchased from R&D Systems). Set up standard wells, blank wells, and sample wells, add the corresponding reagents, and incubate at 37℃ for 60 minutes. After washing, add enzyme-labeled HRP secondary antibody and incubate for 30 minutes. After washing again, add chromogenic solution and incubate in the dark for 15 minutes. Finally, add stop solution to terminate the reaction. Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Calculate the concentrations of IL-6, IL-8, and TNF-α in the supernatant based on the standard curve; b. LDH detection: Take the supernatant of the thawed culture medium and follow the instructions of the lactate dehydrogenase (LDH) detection kit (purchased from Nanjing Jiancheng Bioengineering Institute). Mix the supernatant with the reagents in the kit according to the ratio, incubate at 37°C for 30 minutes, and then measure the OD value at a wavelength of 490 nm using an ELISA reader. Calculate the LDH activity according to the standard curve to assess the degree of mucosal cell damage.

[0084] Table 1. Barrier function of the vaginal mucosa model after Staphylococcus aureus stimulation (24h)

[0085] In the control group of Example 1, the mean Teer value was 850.67, indicating tight epithelial cell junctions and intact barrier function. After stimulation with Staphylococcus aureus, the mean Teer value in the Example 1 model was 483.83, a significant decrease, indicating significant disruption of the epithelial barrier function. In the control group of Comparative Example 1, the mean Teer value was 229.40, indicating impaired barrier function compared to the Example 1 model. After adding bacteria, the mean Teer value in Comparative Example 1 model became 176.73, indicating significant disruption of the epithelial barrier function. In the control group of Comparative Example 2, the mean Teer value was 209.47, indicating impaired barrier function compared to the Example 1 model. After adding bacteria, the mean Teer value in Comparative Example 2 model became 194.13, indicating significant disruption of the epithelial barrier function. In the control group of Comparative Example 3, the mean Teer value was 532.17, indicating impaired barrier function compared to the Example 1 model. After adding bacteria to the Comparative Example 3 model, the mean Teer value became 444.03, indicating that the epithelial barrier function of the Comparative Example 3 model was significantly damaged. The mean Teer value of the blank control group in Comparative Example 4 was 511.33, indicating that the barrier function was incomplete compared to the Comparative Example model. After adding bacteria to the Comparative Example 4 model, the mean Teer value became 464.53, indicating that the epithelial barrier function of the Comparative Example 4 model was significantly damaged. It can be seen that the epithelial barrier function of the Comparative Example model was intact, and the barrier function decreased significantly after bacterial stimulation. The epithelial barrier function of the models in Comparative Examples 1-4 was incomplete, and the response to stimulation was abnormal.

[0086] Table 2. Cell damage level in a vaginal mucosa model after Staphylococcus aureus stimulation (24h)

[0087] The mean LDH level in the blank control group of Example 1 was 100.17, which was very low, indicating that the cells were in good condition with almost no damage or rupture. In Example 2, after stimulation by Staphylococcus aureus, the mean LDH level in the model increased sharply to 1127.72, indicating that a large number of cells were damaged and ruptured due to infection, leading to the release of intracellular LDH into the extracellular space. This demonstrates that Staphylococcus aureus infection causes severe cell damage. In Comparative Example 1, the mean LDH level in the blank control group was 686.82, indicating more severe cell damage and poor cell condition compared to the Example 1 model. After adding bacteria to Comparative Example 1, the mean LDH level increased to 886.53, indicating that Staphylococcus aureus caused cell damage, leading to elevated LDH levels. In Comparative Example 2, the mean LDH level in the blank control group was 694.65, indicating more severe cell damage and poor cell condition compared to the Example 1 model. After adding bacteria to Comparative Example 2, the mean LDH level... The mean LDH level of 832.26 indicates that Staphylococcus aureus caused cell damage, leading to elevated LDH. The mean LDH level in the blank control group of Comparative Example 3 was 286.06, indicating more severe cell damage and poor cell condition compared to the model in the Example 1. After adding bacteria, the mean LDH level in the Comparative Example 3 model was 566.65, indicating that Staphylococcus aureus caused cell damage and elevated LDH. The mean LDH level in the blank control group of Comparative Example 4 was 341.65, indicating more severe cell damage and poor cell condition compared to the model in the Example 1. After adding bacteria, the mean LDH level in the Comparative Example 4 model was 570.65, indicating that Staphylococcus aureus caused cell damage and elevated LDH. This shows that the cells in the Example 1 model were in good condition, with no obvious damage or rupture, and showed a significant response to Staphylococcus aureus damage. The cells in Comparative Examples 1-4 were in poor condition, resulting in higher LDH expression and abnormal responses to stimuli.

[0088] Table 3-5 Expression of inflammatory factors in a vaginal mucosa model stimulated with Staphylococcus aureus (24h) Table 3

[0089] In the control group of Example 1, the mean IL-6 level was 0.73. After Staphylococcus aureus stimulation, the mean IL-6 level in the Example 1 model was 8.27, indicating that Staphylococcus aureus infection significantly promotes the secretion of IL-6 inflammatory factors, thereby triggering a strong inflammatory response. In Comparative Example 1, the mean IL-6 level in the control group was 4.51, indicating that the cells were in a poorer state than the Example 1 model, exhibiting an inflammatory stress state. After the addition of Staphylococcus aureus to the Comparative Example 1 model, the mean IL-6 level was 8.36, indicating that Staphylococcus aureus further stimulated cell damage and the expression and secretion of inflammatory factors. In Comparative Example 2, the mean IL-6 level in the control group was 5.08, indicating that the cells were in a poorer state than the Example 1 model, exhibiting an inflammatory stress state. After the addition of Staphylococcus aureus to the Comparative Example 2 model, the mean IL-6 level was 8.50, indicating that Staphylococcus aureus further stimulated cell damage. The models in Comparative Example 1 and 4 showed better cell condition and inflammatory stress compared to the control group (3.14 IL-6). After bacterial addition, the model in Comparative Example 3 showed a mean IL-6 level of 5.34, indicating that Staphylococcus aureus further stimulated cell damage and led to the expression and secretion of inflammatory factors. Similarly, the control group in Comparative Example 4 showed a mean IL-6 level of 3.53 IL-6, indicating that the model in Comparative Example 4 showed better cell condition and inflammatory stress compared to the control group. After bacterial addition, the model in Comparative Example 4 showed a mean IL-6 level of 5.17 IL-6, indicating that Staphylococcus aureus further stimulated cell damage and led to the expression and secretion of inflammatory factors. This demonstrates that the model in the control group had a better cell condition, the mucosal model was in a normal physiological homeostasis, and the model had a strong immune response. In contrast, the models in Comparative Examples 1-4 showed severe cell damage, intense local inflammation, and abnormal immune responses.

[0090] Table 4

[0091] In the control group of Example 1, the mean IL-8 level was 7.49. After Staphylococcus aureus stimulation, the mean IL-8 level in the Example 1 model was 1533.02, indicating that Staphylococcus aureus infection significantly promoted the secretion of IL-8 inflammatory factors, thereby triggering a strong inflammatory response. In Comparative Example 1, the mean IL-8 level in the control group was 554.72, indicating that the cells were in a poorer state than in the Example 1 model, exhibiting an inflammatory stress state. After the addition of Staphylococcus aureus to the Comparative Example 1 model, the mean IL-8 level was 779.96, indicating that Staphylococcus aureus further stimulated cell damage and the expression and secretion of inflammatory factors. In Comparative Example 2, the mean IL-8 level in the control group was 567.84, indicating that the cells were in a poorer state than in the Example 1 model, exhibiting an inflammatory stress state. After the addition of Staphylococcus aureus to the Comparative Example 2 model, the mean IL-8 level was 726.39, indicating that Staphylococcus aureus further stimulated cell damage. The control group in Comparative Example 3 had a mean IL-8 level of 350.76, indicating that the cells in the model were in poor condition and exhibited an inflammatory stress state compared to the control group in the Example 3. After the addition of bacteria to the model in Comparative Example 3, the mean IL-8 level was 551.27, indicating that Staphylococcus aureus further stimulated cell damage and the expression and secretion of inflammatory factors. The control group in Comparative Example 4 had a mean IL-8 level of 373.30, indicating that the cells in the model were in poor condition and exhibited an inflammatory stress state compared to the control group in the Example 3. After the addition of bacteria to the model in Comparative Example 4, the mean IL-8 level was 563.38, indicating that Staphylococcus aureus further stimulated cell damage and the expression and secretion of inflammatory factors. It can be seen that the cells in the model in the Example 1 were in good condition, the mucosal model was in a normal physiological homeostasis, and the model had a strong immune response. The models in Comparative Examples 1-4 had severe cell damage, severe local inflammation, and abnormal immune response.

[0092] Table 5

[0093] In the control group of Example 1, the mean TNF-α level was 1.62. After treatment with Staphylococcus aureus, the mean TNF-α level in the Example 1 model was 3.77, indicating that Staphylococcus aureus infection significantly promotes the secretion of TNF-α inflammatory factors, thereby triggering a strong inflammatory response. In Comparative Example 1, the mean TNF-α level in the control group was 5.82, indicating that the cells were in a poorer state than in the Example 1 model, exhibiting an inflammatory stress state. After the addition of Staphylococcus aureus to the Comparative Example 1 model, the mean TNF-α level was 7.45, indicating that Staphylococcus aureus further stimulated cell damage and the expression and secretion of inflammatory factors. In Comparative Example 2, the mean TNF-α level in the control group was 5.46, indicating that the cells were in a poorer state than in the Example 1 model, exhibiting an inflammatory stress state. After the addition of Staphylococcus aureus to the Comparative Example 2 model, the mean TNF-α level was 6.55, indicating that Staphylococcus aureus further stimulated cell damage. The cells in the control group of Comparative Example 3 showed a mean TNF-α level of 2.73, indicating that the cells were in poor condition and underwent inflammatory stress compared to the control group of Comparative Example 3. After the addition of bacteria, the mean TNF-α level in the model of Comparative Example 3 was 3.54, indicating that Staphylococcus aureus further stimulated cell damage and led to the expression and secretion of inflammatory factors. The mean TNF-α level in the control group of Comparative Example 4 was 2.54, indicating that the cells were in poor condition and underwent inflammatory stress compared to the control group of Comparative Example 4. After the addition of bacteria, the mean TNF-α level in the model of Comparative Example 4 was 3.17, indicating that Staphylococcus aureus further stimulated cell damage and led to the expression and secretion of inflammatory factors. It can be seen that the cells in the model of Comparative Example 1 were in good condition, the mucosal model was in a normal physiological homeostasis, and the immune response of the model was strong. The cells in the models of Comparative Examples 1-4 were severely damaged, with severe local inflammation and abnormal immune response.

[0094] Table 6 - Barrier function of vaginal mucosa model after Candida albicans stimulation (48h)

[0095] In the Example 1, the mean Teer value of the blank control group was relatively high at 772.00, indicating that the epithelial cells were tightly connected and the barrier function was intact. In the Example 1 model, after stimulation with Candida albicans, the mean Teer value was 329.83, a significant decrease, indicating that the epithelial barrier function of the model was significantly disrupted. In Comparative Example 1, the mean Teer value of the blank control group was 267.13, indicating that the barrier function was incomplete compared to the Example 1 model. After adding Candida albicans, the mean Teer value of Comparative Example 1 was 194.97, indicating that the epithelial barrier function of the model was significantly disrupted. In Comparative Example 2, the mean Teer value of the blank control group was 204.03, indicating that the barrier function was incomplete compared to the Example 1 model. After adding Candida albicans, the mean Teer value of Comparative Example 2 was 189.00, indicating that the epithelial barrier function of the model was significantly disrupted. The mean Teer value of the blank control group in Comparative Example 3 was 358.63, indicating that the barrier function of the model was incomplete compared to the example model. After adding bacteria, the mean Teer value of the model in Comparative Example 3 was 293.70, indicating that the epithelial barrier function of the model was significantly damaged. The mean Teer value of the blank control group in Comparative Example 4 was 358.23, indicating that the barrier function was incomplete compared to the example model. After adding bacteria, the mean Teer value of the model in Comparative Example 4 was 290.70, indicating that the epithelial barrier function of the model was significantly damaged. It can be seen that the epithelial barrier function of the example model was intact, and the barrier function decreased significantly after bacterial stimulation. The epithelial barrier function of the models in Comparative Examples 1-4 was incomplete, and the response to stimulation may be abnormal.

[0096] Table 7 - Degree of cell damage in a vaginal mucosa model after stimulation with Candida albicans (48h)

[0097] In the example, the mean LDH level of the blank control group was 100.00, which was very low, indicating that the cells were in good condition with almost no damage or rupture. In the example model, after stimulation by Candida albicans, the mean LDH level was 441.44, a sharp increase, indicating that a large number of cells were damaged and ruptured due to infection, leading to the release of intracellular LDH into the extracellular space. This demonstrates that Candida albicans infection causes severe cell damage. (Comparative Example 1: LDH in the blank control group) The mean LDH level of Comparative Example 1 was 443.07, indicating more severe cell damage and poor cell condition compared to the model in the Example 1. After adding bacteria, the mean LDH level of Comparative Example 2 was 589.69, indicating that Candida albicans caused cell damage, leading to elevated LDH. The mean LDH level of the blank control group in Comparative Example 2 was 436.88, indicating more severe cell damage and poor cell condition compared to the model in the Example 1. After adding bacteria, the mean LDH level of Comparative Example 2 was 541.67, indicating that Candida albicans caused cell damage, leading to elevated LDH. The mean LDH level of the blank control group in Comparative Example 3 was 335.98, indicating more severe cell damage and poor cell condition compared to the model in the Example 1. After adding bacteria to the model in Example 3, the mean LDH level was 468.81, indicating that Candida albicans caused cell damage, leading to elevated LDH. The mean LDH level in the blank control group in Example 4 was 304.27, indicating that the cell damage was more severe and the cell condition was poor compared to the model in Example 1. After adding bacteria to the model in Example 4, the mean LDH level was 392.91, indicating that Candida albicans caused cell damage, leading to elevated LDH. It can be seen that the cells in Example 1 were in better condition, with no obvious damage or rupture, and the response to Staphylococcus aureus damage was more obvious. The cells in the models in Examples 1-4 were in poor condition, resulting in higher LDH expression and possible abnormal responses to stimuli.

[0098] Table 8-9 Expression of inflammatory factors in a vaginal mucosa model stimulated by Candida albicans (48h) Table 8

[0099] In the control group of Example 1, the mean IL-8 level was 6.99. In the model of Example 1, after stimulation by Staphylococcus aureus, the mean IL-8 level was 64.28, indicating that Candida albicans infection significantly promotes the secretion of IL-8 inflammatory factors, thereby triggering a strong inflammatory response. In Comparative Example 1, the mean IL-8 level in the control group was 53.50, indicating that the cells were in a poorer state than in the model of Example 1, exhibiting an inflammatory stress state. After adding Candida albicans to the model of Comparative Example 1, the mean IL-8 level was 80.61, indicating that Candida albicans further stimulated cell damage and the expression and secretion of inflammatory factors. In Comparative Example 2, the mean IL-8 level in the control group was 51.71, indicating that the cells were in a poorer state than in the model of Example 1, exhibiting an inflammatory stress state. After adding Candida albicans to the model of Comparative Example 2, the mean IL-8 level was 77.70, indicating that Candida albicans further stimulated cell damage and the expression and secretion of inflammatory factors. The control group in Comparative Example 3 had a mean IL-8 level of 43.55, indicating that the cells in the model were in poor condition and exhibited an inflammatory stress state compared to the control group in the Example 1. After adding bacteria, the mean IL-8 level in the model in Comparative Example 3 was 54.18, indicating that Candida albicans further stimulated cell damage and expressed and secreted inflammatory factors. The control group in Comparative Example 4 had a mean IL-8 level of 40.20, indicating that the cells in the model were in poor condition and exhibited an inflammatory stress state compared to the control group in the Example 2. After adding bacteria, the mean IL-8 level in the model in Comparative Example 4 was 51.99, indicating that Candida albicans further stimulated cell damage and expressed and secreted inflammatory factors. It can be seen that the cells in the model in the Example 1 were in good condition, the mucosal model was in a normal physiological homeostasis, and the model had a strong immune response. The models in Comparative Examples 1-4 had severe cell damage, severe local inflammation, and abnormal immune response.

[0100] Table 9

[0101] In the control group of Example 1, the mean TNF-α level was 1.62. In the model of Example 1, after stimulation by Staphylococcus aureus, the mean TNF-α level was 1.96, indicating that Candida albicans infection significantly promotes the secretion of TNF-α inflammatory factors by cells, thereby triggering a strong inflammatory response. In Comparative Example 1, the mean TNF-α level in the control group was 3.69, indicating that the cells were in a poorer state than in the model of Example 1, exhibiting an inflammatory stress state. After adding Candida albicans to the model of Comparative Example 1, the mean TNF-α level was 4.57, indicating that Candida albicans further stimulated cell damage and the expression and secretion of inflammatory factors. In Comparative Example 2, the mean TNF-α level in the control group was 3.73, indicating that the cells were in a poorer state than in the model of Example 1, exhibiting an inflammatory stress state. After adding Candida albicans to the model of Comparative Example 2, the mean TNF-α level was 4.79, indicating that Candida albicans further stimulated cell damage. The models expressed and secreted inflammatory factors. In Comparative Example 3, the mean TNF-α level in the blank control group was 2.69, indicating that the cell state was poor compared to the model in the Example, showing an inflammatory stress state. After adding bacteria to the Comparative Example 3 model, the mean TNF-α level was 3.42, indicating that Candida albicans further stimulated cell damage and expressed and secreted inflammatory factors. In Comparative Example 4, the mean TNF-α level in the blank control group was 2.43, indicating that the cell state was poor compared to the model in the Example, showing an inflammatory stress state. After adding bacteria to the Comparative Example 4 model, the mean TNF-α level was 3.36, indicating that Candida albicans further stimulated cell damage and expressed and secreted inflammatory factors. It can be seen that the cell state of the Example model was better, the mucosal model was in a normal physiological homeostasis, and the model had a strong immune response. The models in Comparative Examples 1-4 had severe cell damage, severe local inflammation, and abnormal immune response.

[0102] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing an in vitro reconstructed vaginal mucosa model, characterized in that, The construction method includes the following steps: Immortalized vaginal epithelial cell line VK2 / E6E7 cells were added to HVU medium, seeded into Transwell chambers, cultured, and the HVU medium on the surface of the culture in the Transwell chambers was aspirated. The Transwell chambers were then raised to the air-liquid interface for air-liquid surface culture. The medium was replaced with HVA medium to obtain a vaginal mucosa model. The HVU culture medium includes DMEM / F12 mixed medium, bovine pituitary extract, epidermal growth factor, hydrocortisone, insulin, adenine, L-glutamine, transferrin, calcium chloride, β-estradiol, and Y-27632. The HVA medium consists of HVU medium supplemented with vitamin C, fibroblast growth factor, triiodothyronine, and Wnt-3a.

2. The method for constructing an in vitro reconstituted vaginal mucosa model according to claim 1, characterized in that, The HVU culture medium includes DMEM / F12 mixed medium, bovine pituitary extract 10-30 μg / mL, epidermal growth factor 1-4 ng / mL, hydrocortisone 0.1-0.2 μg / mL, insulin 5-10 μg / mL, adenine 5-25 μg / mL, L-glutamine 0.4-2 mM, transferrin 5-10 ng / mL, calcium chloride 0.2-0.4 mM, β-estradiol 10-100 nmol / L, and Y-27632 5-10 μM.

3. The method for constructing an in vitro reconstituted vaginal mucosa model according to claim 1, characterized in that, The HVA medium is based on HVU medium, with the addition of vitamin C 20-50 ng / mL, fibroblast growth factor 5-20 ng / mL, triiodothyronine 0.1-0.4 nmol / mL, Wnt-3a 20-100 ng / mL, and the calcium chloride concentration adjusted to 1.0-1.5 mM.

4. The method for constructing an in vitro reconstituted vaginal mucosa model according to claim 1, characterized in that, Before seeding the VK2 / E6E7 cells into the Transwell chambers, the VK2 / E6E7 cells need to be adjusted to a density of 2.5 × 10⁻⁶ cells using HVU medium. 6 ~5×10 6 Cell suspension of cells / mL; Optionally, one or more of the following characteristics must be satisfied: (1) Change the culture medium daily during any culture process; (2) Use HVU medium for immersion culture for 2-3 days; (3) Replace HVU medium with HVA medium and culture for 6 to 9 days.

5. The method for constructing an in vitro reconstituted vaginal mucosa model according to claim 1, characterized in that, The immortalized vaginal epithelial cell line VK2 / E6E7 cell culture process includes: adding recombinant human epidermal growth factor, bovine pituitary extract, and calcium chloride to a serum-free keratinocyte culture medium, culturing, changing the medium, passage, and obtaining the immortalized vaginal epithelial cell line VK2 / E6E7 cells. Optionally, one or more of the following characteristics must be satisfied: (1) The concentration of recombinant human epidermal growth factor was 0.1~0.3 ng / mL; (2) The concentration of bovine pituitary extract was 0.05~0.2 mg / mL; (3) The final concentration of calcium chloride is 0.2~0.5 mM; (4) Immortalized vaginal epithelial cell line VK2 / E6E7 cells need to be passaged when the cells reach a plating rate of 70%-80%; (5) After the third generation of the subculture.

6. An in vitro reconstructed vaginal mucosa model, characterized in that, The in vitro reconstituted vaginal mucosa model was constructed using the method described in any one of claims 1 to 5.

7. An aerobic vaginitis model and a fungal vaginitis model, characterized in that, The in vitro recombinant vaginal mucosa model is obtained by inoculating and culturing the in vitro recombinant vaginal mucosa model constructed by the method of any one of claims 1 to 5 or the in vitro recombinant vaginal mucosa model of claim 6.

8. The aerobic vaginitis model according to claim 7, characterized in that, The construction process includes: inoculating Staphylococcus aureus into nutrient broth, culturing once, collecting the bacterial cells by centrifugation, washing with PBS buffer, adjusting the bacterial cell concentration, inoculating the bacterial solution into the upper chamber of the vaginal mucosa model, adding HVA medium to the lower chamber, culturing a second time, and obtaining an aerobic vaginitis model. Optionally, the Staphylococcus aureus can be replaced with a clinical isolate of Staphylococcus aureus; Optionally, the process of constructing the aerobic vaginitis model includes one or more of the following features: (1) Wash with PBS buffer 2-3 times; (2) The adjusted bacterial concentration is 0.5~2×10⁻⁶. 8 CFU / mL; (3) The conditions for secondary culture are 16 to 24 hours of culture.

9. The fungal vaginitis model according to claim 7, characterized in that, The construction process includes: inoculating Candida albicans into Sabouraud dextrose medium, culturing once, collecting the bacterial cells by centrifugation, washing with PBS buffer, adjusting the bacterial cell concentration, inoculating the bacterial solution into the upper chamber of an in vitro recombinant vaginal mucosa model, adding HVA medium to the lower chamber, culturing a second time, and obtaining a fungal vaginitis model. Optionally, the Candida albicans can be replaced with a clinical isolate of Candida albicans; Optionally, the process of constructing the aerobic vaginitis model includes one or more of the following features: (1) Wash with PBS buffer 2-3 times; (2) The adjusted bacterial concentration is 1-3×10⁻⁶. 8 CFU / mL; (3) The conditions for secondary culture are to culture in an incubator for 24-48 hours.

10. The application of the in vitro recombinant vaginal mucosa model constructed by the construction method according to any one of claims 1 to 5, the in vitro recombinant vaginal mucosa model according to claim 6, or the aerobic vaginitis model and fungal vaginitis model according to any one of claims 7 to 9 in vaginitis research.

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