In vitro model and method for evaluating anti-inflammatory performance of oral care product
By inducing THP-1 cells to differentiate into M0 macrophages on well plates and establishing a co-culture model with gingival epithelial cells in the cell embedding dish, the problem of the inability to establish a co-culture system for immune cells and gingival epithelial cells in the prior art is solved, and a deep study of the interaction between the two cells and an effective evaluation of the anti-inflammatory performance of oral care products is achieved.
Patent Information
- Application Number
- CN202311068229.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-23
- Publication Date
- 2025-03-04
AI Technical Summary
The existing methods of simulated periodontitis in vitro and evaluating the anti-inflammatory effect of gingival cannot establish a relatively independent and interconnected co-culture system between immune cells and gingival epithelial cells, limiting the in-depth study of the interaction between the two cells.
By inducing THP-1 cells to differentiate into M0 macrophages on well plates and establishing a co-culture model with gingival epithelial cells in the cell embedding dish, the design of induction layer and detection layer is used to achieve material exchange between gingival epithelial cells and macrophages without contacting each other.
This method allows for the simulation of the interaction between gingival epithelial cells and macrophages in vitro, meeting the needs of studying gingival immune barrier homeostasis and exploring deep pathological and pharmacological mechanisms of cells, while providing an effective in vitro model for evaluating the anti-inflammatory performance of oral care products.
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Figure CN119552938A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of establishing gingival epithelial barrier function in vitro, and specifically relates to an in vitro model and method for evaluating the anti-inflammatory performance of oral care products. Background Art
[0002] Periodontal disease is an inflammatory disease associated with bacterial biofilms, caused by the transfer of local microorganisms, which can cause gradual destruction of periodontal tissues and eventually lead to tooth loss. At present, the methods for simulating periodontitis in vitro and evaluating the anti-inflammatory effects of samples on gums mainly rely on single immune cells, animal experiments and human experiments. Invention patent CN110907372A discloses an in vitro evaluation model and method for the anti-inflammatory properties of toothpaste. The optimal concentration of toothpaste samples is determined by cell culture and activity detection, and then cells in the logarithmic growth phase are taken and LPS is added to induce the inflammation of HEGCs in the cells. The above-mentioned optimal concentrations of experimental samples and control samples are added respectively, and the content of pro-inflammatory cytokines secreted by them is continued to be cultured and detected, and the anti-inflammatory properties of toothpaste are evaluated by changes in content.
[0003] However, in the process of LPS-induced gingival epithelial cells (HEGCs), the glycolipid substances in the outer membrane of Gram-negative bacteria directly act on periodontal tissue cells, stimulate immune cells such as monocytes and macrophages, induce inflammatory responses and cause periodontal tissue destruction. Under this culture system, it is impossible to conduct relatively independent and interconnected studies on immune cells and gingival epithelial cells. Therefore, it is urgent to establish a co-culture system of the two cells so that there is both material exchange between the two cells without contacting each other. This can meet the needs of researchers to explore the deeper pathological and pharmacological mechanisms of cells based on the interaction between the two cells. In addition, the in vitro model of the present invention also provides strong support for related studies on the homeostasis of the gingival immune barrier. Summary of the invention
[0004] The present invention provides an in vitro model and method for evaluating the anti-inflammatory properties of oral care products, which solves the problem that it is impossible to conduct relatively independent research on two cells in a co-culture system of immune cells and gingival epithelial cells.
[0005] In order to solve the above-mentioned technical problems, the present invention is achieved as follows:
[0006] In a first aspect, the present invention provides an in vitro model for evaluating the anti-inflammatory properties of an oral care product, wherein the method for constructing the in vitro model comprises the following steps:
[0007] ① Induce THP-1 cells to differentiate into M0 macrophages in a well plate;
[0008] ② Gingival epithelial cells were inoculated into cell embedding dishes to prepare gingival epithelial cell monolayers;
[0009] ③ Establishing the in vitro co-culture model of the gingival epithelial cell monolayer and the M0 macrophages, comprising the steps of:
[0010] 1) Establishing a co-culture system: suspending the cell embedding dish (induction layer) in which the gingival epithelial cell monolayer prepared in step ② on the well plate (detection layer) inoculated with the M0 macrophages to form the in vitro model for co-culture, adding the test substance and 0.8 mL of conditioned medium to the induction layer, and adding 2 mL of RIPM 1640 medium containing 10% fetal bovine serum to the detection layer;
[0011] 2) Inflammation induction: replacing the conditioned medium in the induction layer with the induction medium to induce the M0 macrophages to differentiate into M1 macrophages;
[0012] ④ respectively detecting the evaluation factors in the in vitro model described in step ③;
[0013] Wherein, the in vitro model consists of the induction layer and the detection layer.
[0014] Optionally, during the inflammation induction process of step ③, the conditioned medium is discarded, 0.8 mL of induction medium is added, the in vitro model is placed in a 37° C., 5% CO 2 incubator and cultured for 48 hours, and the pro-inflammatory marker proteins on the surface of the M1 macrophages are detected.
[0015] Optionally, the pro-inflammatory marker proteins include one or more of CD80, CD86, and HLA-DR.
[0016] Optionally, in step ④, the evaluation factors include one or more of pro-inflammatory cytokines, relative cell viability of the gingival epithelial cell monolayer, and transmembrane resistance.
[0017] Optionally, the proinflammatory cytokines include IL-1β and TNF-α.
[0018] Optionally, the transmembrane resistance value = (TEEREC cell-TEERfilter) × S, wherein TEEREC cell is the transmembrane resistance value of the gingival epithelial cell monolayer in the cell-embedded dish, TEER-filter is the transmembrane resistance value of the blank cell-embedded dish, and S is the area of the cell-embedded dish; the relative cell viability = (OD value of the gingival epithelial cell monolayer / OD value of the gingival epithelial cell monolayer in the single culture control group) × 100%.
[0019] Optionally, in the in vitro model for evaluating the anti-inflammatory properties of oral care products described in any one of claims 1 to 6, the conditioned culture medium is a High-DMEM culture medium containing 10% fetal bovine serum, and the induction culture medium is a High-DMEM culture medium containing 10% fetal bovine serum and 35 μg / mL lipopolysaccharide (Pg.LPS) derived from Porphyromonas gingivalis.
[0020] In a second aspect, the present invention provides a method for evaluating the anti-inflammatory properties of an oral care product, wherein the anti-inflammatory properties of the test object are evaluated by detecting the changes in the content of the evaluation factor in the induction layer and the detection layer in the in vitro model. Further, the evaluation criteria include:
[0021] The decrease in the proinflammatory cytokine IL-1β and / or the decrease in the proinflammatory cytokine TNF-α and / or the increase in the relative cell viability and / or the increase in the transmembrane resistance value indicate that the anti-inflammatory effect of the test object is better.
[0022] The present invention provides an in vitro model and method for evaluating the anti-inflammatory properties of oral care products. The construction method of the in vitro model comprises the following steps: ① inducing THP-1 cells to differentiate into M0 macrophages in a well plate; ② inoculating gingival epithelial cells in a cell embedding dish to prepare a gingival epithelial cell monolayer; ③ establishing an in vitro co-culture model of gingival epithelial cell monolayer and M0 macrophages; ④ respectively detecting the evaluation factors in the in vitro model in step ③. The anti-inflammatory properties of oral care products can be evaluated by evaluating the changes in the content of the evaluation factors. Since the in vitro model of the present invention is composed of an induction layer and a detection layer, the gingival epithelial cells and the test object are located in the induction layer, and the M0 macrophages are located in the detection layer. There is both material exchange between the two cells and they do not contact each other. Therefore, it meets the needs of researchers to explore the deeper pathological and pharmacological mechanisms of cells based on the interaction between the two cells.
[0023] Figure Description
[0024] Figure 1 A flow chart for the construction of the in vitro model of the present invention;
[0025] Figure 2 It is a schematic diagram of the co-culture of the induction layer and the detection layer of the in vitro model of the present invention;
[0026] Figure 3 It is a bar graph showing the proportion of CD80 positive cells in the co-culture control group and the induced group in the embodiment of the present invention;
[0027] Figure 4 It is a bar graph of TNF-α concentration in the supernatant of macrophages in the co-culture control group and the induced group in the embodiment of the present invention;
[0028] Figure 5 It is a bar graph of IL-1β concentration in the supernatant of macrophages in the co-culture control group and the induced group in the embodiment of the present invention;
[0029] Figure 6 The transmembrane resistance bar graph of the gingival epithelial cell monolayer in the cell embedding dish of the single culture group and the co-culture group in the embodiment of the present invention;
[0030] Figure 7 This is a bar graph of the relative cell viability of the gingival epithelial cell monolayer in the cell-embedded dish of the co-culture group and the single culture group in the embodiment of the present invention. DETAILED DESCRIPTION
[0031] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments. Under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form new embodiments. Any improvement or replacement based on the basic spirit of this embodiment still falls within the scope of protection required by the claims of the present invention.
[0032] It should be understood that the references to "a" or "an" throughout the specification mean that the specific features, structures or characteristics associated with the references are included in at least one of the present invention. Therefore, the references to "in one" or "in one" appearing throughout the specification do not necessarily refer to the same. In addition, the specific features, structures or characteristics may be combined in one or more in any suitable manner.
[0033] The specific embodiments are:
[0034] 1. Materials, instruments and software
[0035] (1) Experimental materials: Human monocytic THP-1 cells and gingival epithelial cell lines. THP-1 cells and gingival epithelial cells were purchased from ATCC and used in strict accordance with the instructions.
[0036] (2) Main experimental equipment: clean bench, CO2 incubator, 12-well plate, cell embedding dish (PET material, 0.4 μm), pipette, quantitative PCR instrument, and microplate reader.
[0037] (3) Statistical and data analysis software: Prism 8.0 statistical analysis software was used for ELISA analysis software, cell relative viability, transmembrane resistance and other data analysis.
[0038] 2. In vitro model construction method
[0039] Construct an in vitro model for evaluating the anti-inflammatory properties of oral care products. The specific process is as follows: Figure 1 As shown, the specific steps are as follows:
[0040] (1) Inducing THP-1 cells to differentiate into M0 macrophages
[0041] ① Proliferation and proliferation of THP-1 (human monocytic leukemia) cells: THP-1 cells were proliferated and cultured using RPMI1640 culture medium containing 10% fetal bovine serum.
[0042] ② Induce THP-1 cells to differentiate into M0 macrophages: 5×10 5 THP-1 cells were inoculated in a 12-well plate, and 2 mL of RPMI1640 medium containing 10% fetal bovine serum and 150 ng / mL phorbol was added to each well. The cells were induced to differentiate for 72 hours in an incubator at 37°C and 5% CO2. After the induction of differentiation, the suspended THP-1 cells differentiated into immature M0 macrophages, which were adherent and extended pseudopodia. The above culture was removed and the cells were washed 3 times with sterile PBS to remove the residual phorbol on the cell surface.
[0043] (2) Cultivation of gingival epithelial cells and preparation of gingival epithelial cell monolayers
[0044] ① Proliferation and proliferation of gingival epithelial cells: In this example, gingival epithelial cells were purchased from ATCC. Gingival epithelial cells were routinely revived and subcultured using High-DMEM medium containing 10% fetal bovine serum. The cells were placed in a 75 cm 2 The cells were cultured in a culture flask at 5% CO2 and 37°C. Gingival epithelial cells were centrifuged at 800 Rpm for 5 min before cell passage. The fifth passage of gingival epithelial cells was used for the preparation of monolayer cells.
[0045] ② Preparation of gingival epithelial cell monolayer: hang the cell embedding dish adapted to 12-well plate on the 12-well plate, add 2 mL of High-DMEM medium containing 10% fetal bovine serum to the well plate, and add 0.8 mL of 2.4×10 4 The gingival epithelial cells were cultured in a High-DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator for 48 hours to form a gingival epithelial cell monolayer.
[0046] (3) Establishment of an in vitro co-culture model of gingival epithelial cell monolayer and M0 macrophages
[0047] ① Establish a co-culture system: The prepared gingival epithelial cell monolayer was suspended in a cell embedding dish (induction layer) on a well plate inoculated with M0 macrophages (detection layer) to form an in vitro model for co-culture, such as Figure 2As shown, an in vitro co-culture model of gingival epithelial cell monolayer and M0 macrophages was established and marked as co-culture group, with a total of two groups, each with 3 parallel replicates. ② Inflammation induction: The in vitro model was divided into co-culture group and single culture group
[0048] In the co-culture group, it was divided into a co-culture control group and a co-culture induction group. The in vitro models of the two co-culture groups contained M0 macrophages and 2 mL of RPMI 1640 medium containing 10% fetal bovine serum in the well plate of the detection layer, and 0.8 mL of High-DMEM medium containing 10% fetal bovine serum was added to the cell embedding dish of the induction layer of the co-culture control group. The induction layer of the co-culture induction group was added with the test substance and 0.8 mL of High-DMEM medium containing 10% fetal bovine serum and 35 μg / mL Pg.LPS. The two groups were cultured in an incubator at 37°C, 5% CO2 for 48 hours to induce inflammation.
[0049] The single culture group was set as the reference group of the co-culture group. A new 12-well plate was taken and the prepared gingival epithelial cell monolayer cell embedding dish was suspended on the 12-well plate. There were two groups in total, with 3 parallel replicates in each group, 3 labeled single culture control group and 3 labeled single culture induction group. The well plates of the 6 single culture groups contained 2mL of RPMI1640 medium with 10% fetal bovine serum, the cell embedding dishes of the 3 single culture control groups were added with 0.8mL of High-DMEM medium containing 10% fetal bovine serum, and the cell embedding dishes of the 3 single culture induction groups were added with the test substance and 0.8mL of High-DMEM medium containing 10% fetal bovine serum and 35μg / mL Pg.LPS. The two groups were cultured in an incubator at 37°C and 5% CO2 for 48h to induce inflammation.
[0050] (4) Evaluation methods of in vitro co-culture models
[0051] ① Detection of CD80 marker on the surface of M1 macrophages after inflammation induction by flow cytometry
[0052] Take three parallel replicate samples of the co-culture control group and the co-culture induction group, move the two groups of cell embedding dishes to a new 12-well plate for standby, collect the M1 macrophage culture fluid into a new 1.5mL centrifuge tube and mark it for standby, and wash the M1 macrophages with PBS. Digest the M1 macrophages with 0.25% trypsin in a 37°C incubator for 5 minutes, terminate the digestion with RPMI medium containing serum, collect the cells into a 1.5mL centrifuge tube, and obtain the cell pellet by centrifugation. After blocking the nonspecific binding sites with 100 μL 10% GAMUNEX (human immunoglobulin solution) on ice for 10 minutes, incubate the cells with the primary antibody (CD80) in the dark for 30 minutes, remove the supernatant by low-temperature centrifugation, and wash the cell pellet twice with flow buffer (PBS containing 0.1% BSA), detect cell surface proteins by flow cytometry, and analyze the data with Flow Jo software. The results of flow cytometry showed that compared with the co-culture control group, the expression of M1 macrophage surface marker CD80 in the co-culture control group was 5.57±0.65%, and that in the co-culture induced group was 87.27±3.2%. The expression of M1 macrophage surface marker CD80 in the co-culture induced group was significantly increased. Figure 3 .
[0053] The expression of pro-inflammatory marker proteins such as CD80, CD86, and HLA-DR on the surface of M1 macrophages was positive, indicating that Pg.LPS successfully induced immature M0 macrophages to differentiate into M1 macrophages through gingival epithelial cells, and inflammation was successfully induced.
[0054] ② Use ELISA kit to detect the release of inflammatory factors such as TNF-α and IL-1β in the M1 macrophage culture medium in the in vitro model detection layer.
[0055] A co-culture control group and a co-culture induction group were set up, and the M1 macrophage culture fluid samples or standards of the co-culture control group and the co-culture induction group and 50 μL / well of the diluted biotinylated antibody were added to the sample wells or standard wells at 100 μL / well. Blank wells were set up to prepare standard curves, respectively. The plates were sealed with sealing membranes and incubated at room temperature for 3 h.
[0056] After washing the plate 3 times with 100 μL / well, 100 μL / well of diluted Streptavidin-HRP was added, the plate was covered with a sealing film, and incubated at room temperature for 45 min. After washing the plate 3 times, 100 μL / well of TMB was added, color was developed for 15 min, and stop solution was added to 100 μL / well. The values were read using a microplate reader at a wavelength of 450 nm, and the release of TNF-α and IL-1β in the culture medium of the co-culture control group and the co-culture induced group was calculated based on the standard curve. The results showed that compared with the co-culture control group, the TNF-α in the co-culture control group was 1177.41±1.88 pg / mL, and the TNF-α in the culture induced group was 5001.55±95.04 pg / mL. Figure 4 As shown; the IL-1β in the co-culture control group was 42.08±1.22pg / mL, and the IL-1β in the culture induced group was 76.32±3.61pg / mL. Figure 5 As shown, the release of TNF-α and IL-1β in the culture medium of the co-culture induction group was significantly increased.
[0057] ③ The transmembrane resistance of the gingival epithelial cell monolayer in the co-culture group and the single culture group was measured using a transmembrane resistance meter.
[0058] The cell embedding dishes of gingival epithelial cells in the co-culture group and the single culture group, a total of 4 groups, were moved to new 12-well plates, 2 mL of High-DMEM culture medium was added to each well of the well plate of each group, and 0.8 mL of High-DMEM culture medium was added to the cell embedding dish. The transmembrane resistance of the gingival epithelial cell monolayer was measured using a transmembrane resistance meter, and 6 transmembrane resistance values were collected for each cell embedding dish, including 3 values at the periphery and 3 values at the center.
[0059] The same method was used to measure the transmembrane resistance of a blank cell-embedded dish without cells. The transmembrane resistance of a single cell layer of gingival epithelial cells was calculated according to the following formula: TEEREC (Ω·cm2) = (TEEREC cell-TEERfilter) × S, where TEEREC cell is the transmembrane resistance of a single cell layer of gingival epithelial cells, TEERfilter is the transmembrane resistance of a blank cell-embedded dish, and S is the area of the cell-embedded dish.
[0060] The relative transmembrane resistance of the gingival epithelial cell monolayer was calculated according to the following formula: relative transmembrane resistance of the gingival epithelial cell monolayer = (TEEREC / TEEREC of the single culture group control group) × 100%. The results showed that the relative transmembrane resistance of the gingival epithelial cell monolayer in the single culture control group was 100±1.1%, and the relative transmembrane resistance of the gingival epithelial cell monolayer in the single culture induced group was 110.31±9.55%. Compared with the single culture control group, the transmembrane resistance of the gingival epithelial cell monolayer in the single culture induced group did not change significantly; the relative transmembrane resistance of the gingival epithelial cell monolayer in the co-culture control group was 121.83±9.43%, and the relative transmembrane resistance of the gingival epithelial cell monolayer in the co-culture induced group was 71.21±10.55%. Compared with the co-culture control group, the transmembrane resistance of the monolayer in the co-culture induced group was significantly reduced. Figure 6 shown.
[0061] ④MTT method was used to detect the relative viability of the gingival epithelial cell monolayer in the co-culture group and the single culture group.
[0062] The cell embedding dishes of the gingival epithelial cell monolayers of the co-culture group and the single culture group were moved to 12-well plates, and the excess liquid around the cell embedding dishes was sucked off with a cotton swab. 1.5 mL of 0.5 mg / mL MTT working solution was added to each well and incubated in an incubator (37°C, 5% CO2, 95% RH) for 4 h; after the incubation, the MTT working solution was discarded, 2 mL of DMSO was added to each well, the well was sealed, and shaken for 10 min. The DMSO organic solvent was transferred to a 96-well plate, and the OD value at 490 nm was read on a microplate reader.
[0063] The relative cell viability was calculated according to the following formula: Relative cell viability of gingival epithelial cell monolayer = (OD value of gingival epithelial cell monolayer / OD value of gingival epithelial cell monolayer in the single culture control group) × 100%. The cell viability results showed that the OD value of the gingival epithelial cell monolayer in the single culture control group was 100±1.48%, and the OD value of the gingival epithelial cell monolayer in the single culture induced group was 101.73±4.91%. Compared with the single culture control group, the cell viability of the single culture induced group did not change significantly; the OD value of the gingival epithelial cell monolayer in the co-culture control group was 103.02±0.68%, and the OD value of the gingival epithelial cell monolayer in the co-culture induced group was 81.67±0.8%. Compared with the co-culture control group, the cell viability of the co-culture induced group was significantly reduced. Figure 7 shown.
[0064] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. An in vitro model for evaluating the anti-inflammatory properties of oral care products, characterized in that The method for constructing the in vitro model comprises the following steps: ① Induce THP-1 cells to differentiate into M0 macrophages in a well plate; ② Gingival epithelial cells were inoculated into cell embedding dishes to prepare gingival epithelial cell monolayers; ③ Establishing the in vitro co-culture model of the gingival epithelial cell monolayer and the M0 macrophages, comprising the steps of: 1) Establishing a co-culture system: suspending the cell embedding dish (induction layer) in which the gingival epithelial cell monolayer prepared in step ② on the well plate (detection layer) inoculated with the M0 macrophages to form the in vitro model for co-culture, adding the test substance and 0.8 mL of conditioned medium to the induction layer, and adding 2 mL of RIPM 1640 medium containing 10% fetal bovine serum to the detection layer; 2) Inflammation induction: replacing the conditioned medium in the induction layer with the induction medium to induce the M0 macrophages to differentiate into M1 macrophages; ④ respectively detecting the evaluation factors in the in vitro model described in step ③; Wherein, the in vitro model consists of the induction layer and the detection layer.
2. The in vitro model for evaluating the anti-inflammatory properties of oral care products according to claim 1, characterized in that: During the inflammation induction process of step ③, the conditioned medium is discarded, 0.8 mL of induction medium is added, the in vitro model is placed in a 37° C., 5% CO 2 incubator and cultured for 48 hours, and the pro-inflammatory marker proteins on the surface of the M1 macrophages are detected.
3. The in vitro model for evaluating the anti-inflammatory properties of oral care products according to claim 2, characterized in that: The pro-inflammatory marker proteins include one or more of CD80, CD86, and HLA-DR.
4. The in vitro model for evaluating the anti-inflammatory properties of oral care products according to claim 1, characterized in that: In step ④, the evaluation factors include one or more of pro-inflammatory cytokines, relative cell viability of the gingival epithelial cell monolayer, and transmembrane resistance.
5. The in vitro model for evaluating the anti-inflammatory properties of oral care products according to claim 4, characterized in that: The pro-inflammatory cytokines include IL-1β and TNF-α.
6. The in vitro model for evaluating the anti-inflammatory properties of oral care products according to claim 4, characterized in that The transmembrane resistance value = (TEEREC cell - TEERfilter) × S, wherein TEEREC cell is the transmembrane resistance value of the gingival epithelial cell monolayer in the cell-embedded dish, TEER-filter is the transmembrane resistance value of the blank cell-embedded dish, and S is the area of the cell-embedded dish; the relative cell viability = (OD value of the gingival epithelial cell monolayer / OD value of the gingival epithelial cell monolayer in the single culture control group) × 100%.
7. The in vitro model for evaluating the anti-inflammatory properties of oral care products according to any one of claims 1 to 6, characterized in that: The conditioned medium is a High-DMEM medium containing 10% fetal bovine serum, and the induction medium is a High-DMEM medium containing 10% fetal bovine serum and 35 μg / mL lipopolysaccharide (Pg.LPS) derived from Porphyromonas gingivalis.
8. The method for evaluating the anti-inflammatory properties of an oral care product according to any one of claims 1 to 7, characterized in that: The anti-inflammatory property of the test object is evaluated by detecting the content change of the evaluation factor in the induction layer and the detection layer in the in vitro model.
9. The method for evaluating the anti-inflammatory properties of an oral care product according to claim 8, characterized in that: The decrease in the proinflammatory cytokine IL-1β and / or the decrease in the proinflammatory cytokine TNF-α and / or the increase in the relative cell viability and / or the increase in the transmembrane resistance value indicate that the anti-inflammatory effect of the test object is better.