Method and system for researching three-dimensional model of oral soft tissue invaded and infected by candida albicans

Abstract

The invention provides a research method and system for a three-dimensional model of oral soft tissue invaded and infected by candida albicans, and belongs to the field of biomedicine.The embodiment of the invention discloses the research method for the three-dimensional model of the oral soft tissue invaded and infected by the candida albicans, and the method comprises the steps that the three-dimensional model of the oral soft tissue is established, the oral soft tissue three-dimensional model comprises oral fibroblasts and epithelial cells; co-culturing candida albicans and the oral soft tissue three-dimensional model; during the co-culture period, collecting a culture system every preset duration, making tissue slices, and performing HE dyeing and PAS dyeing; according to HE dyeing and PAS dyeing, a plurality of stage processes of interaction between the oral soft tissue three-dimensional model and candida albicans are researched. In the embodiment of the invention, the dynamic process that the candida albicans invades epithelium and interstitial substance can be observed in vitro through the three-dimensional model. And more real and reliable data can be obtained by researching the interaction between the host and the microorganisms and performing combined analysis with in-vivo experimental results.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a research method and system for a three-dimensional culture system of oral soft tissue infected by Candida albicans. Background Art

[0002] Candida albicans is one of the most common opportunistic fungi, especially in the oral soft tissues. Candida albicans infection is considered a risk factor for tumor occurrence and development. Oral leukoplakia (OLK) is one of the most common oral potential malignant diseases occurring in the oral soft tissues. It is more susceptible to infection with Candida albicans, and concomitant Candida infection is one of the most dangerous factors for its carcinogenesis. In the past, direct co-culture and indirect co-culture methods of two-dimensional culture models were mostly used to observe whether the host cells (epithelial cells and fibroblasts, etc.) had changed in their ability to resist Candida albicans. However, this method is relatively limited and cannot truly simulate the response of the interstitial microenvironment in vivo.

[0003] Therefore, a new method for studying Candida invasive infection is urgently needed. Summary of the invention

[0004] In order to solve the problems existing in the above-mentioned background technology, the present invention provides a research method and system for a three-dimensional culture system of oral soft tissues infected by Candida albicans.

[0005] In a first aspect, the present invention provides a method for studying a three-dimensional model of oral soft tissue infection by Candida albicans, the method comprising:

[0006] Establishing a three-dimensional model of oral soft tissue, wherein the three-dimensional model of oral soft tissue comprises oral fibroblasts and epithelial cells;

[0007] co-culturing Candida albicans with the oral soft tissue three-dimensional model;

[0008] During the co-culture period, the culture system was collected at preset intervals, tissue sections were made, and HE and PAS staining were performed;

[0009] Based on HE staining and PAS staining, the multi-stage process of the interaction between the three-dimensional model of cavity soft tissue and Candida albicans was studied.

[0010] Optionally, establishing the oral soft tissue three-dimensional model includes:

[0011] Primary culture of oral soft tissue blocks was performed to obtain adherent primary oral fibroblasts;

[0012] Preparation of tissue matrix based on adherent primary oral fibroblasts;

[0013] An epithelial cell suspension is inoculated on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

[0014] Optionally, primary culture of oral soft tissue pieces is performed, including:

[0015] Soak tissue blocks in 3 ml of double antibody solution for 5 minutes, repeat three times;

[0016] Cut the interstitial layer into tissue blocks with a diameter of about 1 mm in a culture dish;

[0017] Use a probe to transfer the tissue blocks to the bottom of the bottle, so that the tissue blocks are evenly dispersed and attached to the bottom of the bottle. Place 9-12 tissue blocks in each bottle, add complete culture medium, tighten the bottle cap and invert it in a 37°C, 5% CO2 incubator for 2 hours. After the tissue blocks at the bottom of the bottle are completely attached to the wall, turn the culture bottle over to make it upright, and ensure that the culture medium can cover each tissue block. Continue to culture. After 2 to 3 weeks, adherent primary oral fibroblasts are obtained.

[0018] Optionally, a tissue matrix is ​​prepared based on adherent primary oral fibroblasts, comprising:

[0019] Mix type I collagen, Matrigel, fetal bovine serum, 10-fold basal DMEM medium, and DMEM complete medium containing 10% fetal bovine serum in a ratio of 3.5:3.5:1:1:1 to prepare a matrix gel;

[0020] The adherent oral primary fibroblasts were digested with trypsin and centrifuged to obtain a cell pellet, which was then mixed with the matrix gel to adjust the density to 5×10 5 cells / ml, and transferred into a 96-well plate. After the matrix gel solidified, 100 μl of complete culture medium was added to each well, and culture was continued for 24 hours to obtain the tissue matrix.

[0021] Optionally, epithelial cell suspension is inoculated on the tissue matrix to obtain a three-dimensional model of oral soft tissue, comprising:

[0022] The epithelial cells were digested with trypsin and centrifuged to obtain an epithelial cell pellet, which was resuspended in complete medium and the cell density was adjusted to 2×10 5 cells / ml, and epithelial cell suspension was obtained;

[0023] Inoculating 100 μl of the epithelial cell suspension in each well of the tissue matrix, and continuing to culture for 24 hours to implant the epithelial cells on the tissue matrix;

[0024] Its tissue matrix seeded with epithelial cells is placed on a collagen-coated nylon membrane supported by a metal grid;

[0025] The nylon membrane was placed in a culture dish, and 8 ml to 10 ml of complete culture medium was added so that the liquid surface just touched the bottom of the nylon membrane; the dish was placed in a 37° C., 5% CO 2 incubator and cultured for 8 to 10 days, with the culture medium changed every other day to obtain a three-dimensional model of oral soft tissue.

[0026] Optionally, co-culturing Candida albicans with the oral soft tissue three-dimensional model comprises:

[0027] The activated Candida albicans standard strain SC5314 was centrifuged to obtain a precipitate, which was resuspended in sterile PBS and adjusted to a density of 1×10 6 CFU / ml, and a suspension of Candida albicans was obtained;

[0028] The oral soft tissue three-dimensional model was taken out, and 20ul of the Candida albicans suspension was added to the surface of the epithelial cell layer of the oral soft tissue three-dimensional model; the DMEM culture medium was replaced and the culture was continued.

[0029] Optionally, the method further comprises:

[0030] After 10 days of culture, the oral soft tissue three-dimensional model was separated from the nylon membrane, fixed, dehydrated, embedded, and sliced;

[0031] The three-dimensional model tissue sections were stained using a hematoxylin and eosin staining kit, and the structure of the three-dimensional model was observed under an upright microscope;

[0032] Immunohistochemical staining was used to identify the components of the three-dimensional model, and the detection indicators were Pan-CK, a marker of epithelial cells, and Vimentin, a marker of mesenchymal cells.

[0033] Optionally, the method further comprises:

[0034] The function of oral primary fibroblasts in resisting Candida albicans was studied by quantitatively detecting the infection area of ​​Candida albicans in a three-dimensional model.

[0035] In a second aspect, the present invention provides a method for constructing a three-dimensional model of oral soft tissue, characterized in that the method comprises:

[0036] Oral soft tissue pieces were cultured to obtain adherent primary oral fibroblasts;

[0037] Preparation of tissue matrix based on adherent primary oral fibroblasts;

[0038] An epithelial cell suspension is inoculated on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

[0039] In a third aspect, the present invention provides a system for studying a three-dimensional model of oral soft tissues infected by Candida albicans, the system comprising:

[0040] A three-dimensional model building subsystem is used to build a three-dimensional model of oral soft tissue, wherein the three-dimensional model of oral soft tissue includes fibroblasts and epithelial cells;

[0041] a co-culture subsystem, used for co-culturing Candida albicans with the oral soft tissue three-dimensional model;

[0042] The staining subsystem is used to collect the culture system at preset time intervals during the co-culture period, prepare tissue sections, and perform HE staining and PAS staining;

[0043] The observation subsystem is used to study the multi-stage process of the interaction between the three-dimensional model of cavity soft tissue and Candida albicans based on HE staining and PAS staining.

[0044] Optionally, the three-dimensional model building subsystem includes:

[0045] A primary culture device is used to culture oral soft tissue blocks to obtain adherent oral primary fibroblasts;

[0046] A matrix preparation device for preparing tissue matrix based on adherent primary oral fibroblasts;

[0047] The culture device is used to inoculate epithelial cell suspension on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

[0048] Optionally, the primary culture device is specifically used for:

[0049] Soak tissue blocks in 3 ml of double antibody for 5 minutes, repeat three times;

[0050] Cut the interstitial layer into tissue blocks with a diameter of about 1 mm in a culture dish;

[0051] Use a probe to transfer the tissue blocks to the bottom of the bottle, so that the tissue blocks are evenly dispersed and attached to the bottom of the bottle. Place about 9-12 tissue blocks in each bottle, add complete culture medium, tighten the bottle cap and invert it in a 37°C, 5% CO2 incubator for 2 hours. After the tissue blocks at the bottom of the bottle are completely attached to the wall, turn the culture bottle over to make it upright, and ensure that the culture medium can cover each tissue block. Continue to culture. After 2 to 3 weeks, adherent primary oral fibroblasts are obtained.

[0052] Optionally, the matrix preparation device is specifically used for:

[0053] Type I collagen, Matrigel, fetal bovine serum, 10× basic DMEM medium, and DMEM complete medium containing 10% fetal bovine serum were mixed in a ratio of 3.5:3.5:1:1:1 to prepare a matrix gel;

[0054] The adherent oral primary fibroblasts were digested with trypsin and centrifuged to obtain a cell pellet, which was then mixed with the matrix gel to adjust the density to 5×10 5 cells / ml, and transferred into a 96-well plate. After the matrix gel solidified, 100 μl of complete culture medium was added to each well, and culture was continued for 24 hours to obtain the tissue matrix.

[0055] Optionally, the culture device is specifically used for:

[0056] The epithelial cells were digested with trypsin and centrifuged to obtain an epithelial cell pellet, which was resuspended in complete medium and the cell density was adjusted to 2×10 5 cells / ml, and epithelial cell suspension was obtained;

[0057] Inoculating 100 μl of the epithelial cell suspension in each well of the tissue matrix, and continuing to culture for 24 hours to implant the epithelial cells on the tissue matrix;

[0058] Its tissue matrix seeded with epithelial cells is placed on a collagen-coated nylon membrane supported by a metal grid;

[0059] The nylon membrane was placed in a culture dish, and 8 ml to 10 ml of complete culture medium was added so that the liquid surface just touched the bottom of the nylon membrane; the dish was placed in a 37° C., 5% CO 2 incubator and cultured for 8 to 10 days, with the culture medium changed every other day to obtain a three-dimensional model of oral soft tissue.

[0060] Optionally, the co-culture subsystem is specifically used for:

[0061] The activated Candida albicans standard strain SC5314 was centrifuged to obtain a precipitate, which was resuspended in sterile PBS and adjusted to a density of 1×10 6 CFU / ml, and a suspension of Candida albicans was obtained;

[0062] The oral soft tissue three-dimensional model was taken out, and 20ul of the Candida albicans suspension was added to the surface of the epithelial cell layer of the oral soft tissue three-dimensional model; the DMEM culture medium was replaced and the culture was continued.

[0063] Optionally, the system further comprises:

[0064] A slicing device is used to separate the oral soft tissue three-dimensional model from the nylon membrane after 10 days of culture, and embed and slice it after fixation and dehydration;

[0065] A staining device, used to stain the three-dimensional model tissue sections using a hematoxylin and eosin staining kit, and observe the structure of the three-dimensional model under an upright microscope;

[0066] The identification device is used to identify the components of the three-dimensional model using immunohistochemical staining, and the detection indicators are Pan-CK, a marker of epithelial cells, and Vimentin, a marker of mesenchymal cells.

[0067] Optionally, the system further comprises:

[0068] The research subsystem is used to study the function of oral primary fibroblasts in resisting Candida albicans by quantitatively detecting the infection area of ​​Candida albicans in a three-dimensional model.

[0069] In the embodiments of the present invention, the dynamic process of Candida albicans invading epithelium and stroma can be observed in vitro through a three-dimensional model. The interaction between host and microorganisms can be studied and combined with the in vivo experimental results to obtain more real and reliable data. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 A flowchart showing the steps of a method for studying a three-dimensional culture system for oral soft tissues infected by Candida albicans according to an embodiment of the present invention;

[0071] Figure 2 The results of morphological observation of oral primary fibroblast culture in the embodiment of the present invention are shown;

[0072] Figure 3 The results of immunocytochemical staining for identifying primary fibroblasts in the embodiment of the present invention are shown;

[0073] Figure 4A A general schematic diagram of a three-dimensional model established in an embodiment of the present invention is shown;

[0074] Figure 4B The figure shows the result of the establishment and verification test of the three-dimensional model in the embodiment of the present invention;

[0075] Figure 5 The infection result diagram of the three-dimensional model of oral soft tissue infected by Candida albicans in the embodiment of the present invention is shown;

[0076] Figure 6 The results of infection area analysis of the three-dimensional model of oral soft tissue infected by Candida albicans in an embodiment of the present invention are shown. DETAILED DESCRIPTION

[0077] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0078] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased on the market.

[0079] In related research, in vitro experiments on the interaction between Candida albicans and the host or host cells mostly use two-dimensional culture models. Although the operation is simple and the effect is intuitive and obvious, the multiple stages of the host-microorganism interaction can be observed through the design of different models, but it cannot truly simulate the response of the interstitial microenvironment in vivo. Research based on two-dimensional culture models can only explore why OLK is susceptible to Candida albicans from the perspective of epithelium. However, the oral mucosa is a unified whole composed of epithelial and interstitial microenvironments, and the extensive interstitial tissue is also an important place for Candida albicans to interact with the host.

[0080] The three-dimensional model can provide a microenvironment that is closer to in vivo conditions, better simulate the physiological state, study the interaction between the host and microorganisms, and combine analysis with in vivo experimental results to obtain more realistic and reliable data.

[0081] Therefore, the embodiment of the present invention proposes: In order to achieve mutual communication between the epithelium and the matrix layer in an in vitro environment and simulate the in vivo interstitial microenvironment as much as possible, a three-dimensional culture model of oral mucosa infected with Candida albicans is established to further study the interaction between host cells and microorganisms in oral soft tissues. Specifically, in the embodiment of the present invention, in view of the fact that the dynamic interaction between epithelial cells and fibroblasts is crucial to maintaining the physical barrier function, therefore, in the embodiment of the present invention, a three-dimensional model of soft tissue containing fibroblasts and epithelial cells is first established. The dynamic process of Candida albicans invading the epithelium and interstitium was observed through a three-dimensional model, and it was found that in the early stage of Candida albicans infection (12 hours), spores and a small amount of hyphae were observed to be distributed on the surface and inside of the epithelium, and an obvious morphological transformation process was observed during the invasion of Candida albicans. In the late stage of infection (24 hours), it was found that the infected Candida in the epithelium and interstitial layer were almost all hyphae.

[0082] Specifically, the present invention proposes a method for studying Candida albicans infection in a three-dimensional culture system of oral soft tissue. Figure 1 As shown, Figure 1 A flowchart showing the steps of a method for studying a three-dimensional culture system for oral soft tissues infected by Candida albicans according to an embodiment of the present invention is shown. Specifically, the method comprises the following steps:

[0083] S11, establish a three-dimensional model of oral soft tissue.

[0084] In an embodiment of the present invention, the three-dimensional model of oral soft tissue includes oral fibroblasts and epithelial cells.

[0085] In the embodiments of the present invention, oral fibroblasts and epithelial cells may be derived from primary cultured cells or cell lines.

[0086] Specifically, step S11 includes the following sub-steps:

[0087] S111, performing primary culture on the oral soft tissue blocks to obtain adherent primary oral fibroblasts.

[0088] Specifically, in the embodiment of the present invention, S111 includes:

[0089] Soak the tissue specimen in 3 ml of double antibody solution for 5 minutes, repeat three times.

[0090] The interstitial layer was cut into tissue pieces with a diameter of about 1 mm in a culture dish.

[0091] In the embodiment of the present invention, the epithelium and stroma of the tissue specimen can be completely separated in the clean bench, the epithelium can be discarded, the stroma layer can be obtained, and the tissue block can be obtained based on the stroma layer.

[0092] Use a probe to transfer the tissue blocks to the bottom of the bottle, so that the tissue blocks are evenly dispersed and attached to the bottom of the bottle. Place 9-12 tissue blocks in each bottle, add complete culture medium, tighten the bottle cap and invert it in a 37°C, 5% CO2 incubator for 2 hours. After the tissue blocks at the bottom of the bottle are completely attached to the wall, turn the culture bottle over to make it upright, and ensure that the culture medium can cover each tissue block. Continue to culture. After 2 to 3 weeks, adherent primary oral fibroblasts are obtained.

[0093] S112, preparation of tissue matrix based on adherent primary oral fibroblasts.

[0094] Specifically, in the embodiment of the present invention, S112 includes:

[0095] Type I collagen, Matrigel, fetal bovine serum, 10-fold basal DMEM medium, and DMEM complete medium containing 10% fetal bovine serum were mixed in a ratio of 3.5:3.5:1:1:1 to prepare a matrix gel.

[0096] The adherent oral primary fibroblasts were digested with trypsin and centrifuged to obtain a cell pellet, which was then mixed with the matrix gel to adjust the density to 5×10 5 cells / ml, and transferred into a 96-well plate. After the matrix gel solidified, 100 μl of complete culture medium was added to each well, and culture was continued for 24 hours to obtain the tissue matrix.

[0097] S113, inoculating an epithelial cell suspension on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

[0098] Specifically, in the embodiment of the present invention, S113 includes:

[0099] The epithelial cells were digested with trypsin and centrifuged to obtain an epithelial cell pellet, which was resuspended in complete medium and the cell density was adjusted to 2×10 5 cells / ml, and epithelial cell suspension was obtained;

[0100] Inoculating 100 μl of the epithelial cell suspension in each well of the tissue matrix, and continuing to culture for 24 hours to implant the epithelial cells on the tissue matrix;

[0101] Its tissue matrix seeded with epithelial cells is placed on a collagen-coated nylon membrane supported by a metal grid;

[0102] The nylon membrane was placed in a culture dish, and 8 ml to 10 ml of complete culture medium was added so that the liquid surface just touched the bottom of the nylon membrane; the dish was placed in a 37° C., 5% CO 2 incubator and cultured for 8 to 10 days, with the culture medium changed every other day to obtain a three-dimensional model of oral soft tissue.

[0103] S12, co-culturing Candida albicans with the oral soft tissue three-dimensional model.

[0104] Specifically, in the embodiment of the present invention, step S12 includes:

[0105] The activated Candida albicans standard strain SC5314 was centrifuged to obtain a precipitate, which was resuspended in sterile PBS and adjusted to a density of 1×10 6 CFU / ml, and obtain Candida albicans suspension.

[0106] The oral soft tissue three-dimensional model was taken out, and 20ul of the Candida albicans suspension was added to the surface of the epithelial cell layer of the oral soft tissue three-dimensional model; the DMEM culture medium was replaced and the culture was continued.

[0107] S13, at preset time intervals during the co-culture period, the culture system is collected, tissue sections are prepared, and HE staining and PAS staining are performed.

[0108] S14, HE staining and PAS staining were used to study the multi-stage process of the interaction between the three-dimensional model of cavity soft tissue and Candida albicans.

[0109] In an optional embodiment of the present invention, the obtained oral soft tissue three-dimensional model may also be identified, which specifically includes the following steps:

[0110] S21, after 10 days of culture, the oral soft tissue three-dimensional model is separated from the nylon membrane, fixed, dehydrated, embedded, and sliced.

[0111] S22, use the hematoxylin and eosin staining kit to stain the three-dimensional model tissue sections, and observe the structure of the three-dimensional model under an upright microscope.

[0112] S23, immunohistochemical staining was used to identify the components of the three-dimensional model, and the detection indicators were Pan-CK, a marker of epithelial cells, and Vimentin, a marker of mesenchymal cells.

[0113] In an optional embodiment of the present invention, the function of oral primary fibroblasts in resisting Candida albicans can also be studied, which specifically includes the following steps:

[0114] The function of oral primary fibroblasts in resisting Candida albicans was studied by quantitatively detecting the infection area of ​​Candida albicans in a three-dimensional model.

[0115] Based on the same inventive concept, in a second aspect, the present invention provides a method for constructing a three-dimensional model of oral soft tissue, the method comprising:

[0116] Primary culture of oral soft tissue blocks was performed to obtain adherent primary oral fibroblasts;

[0117] Preparation of tissue matrix based on adherent primary oral fibroblasts;

[0118] An epithelial cell suspension is inoculated on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

[0119] Specifically, the method for constructing the three-dimensional model of oral soft tissue is similar to steps S111 to S113 in the above embodiment, and the embodiment of the present invention will not be described in detail here.

[0120] Based on the same inventive concept, in a third aspect, the present invention provides a research system for a three-dimensional model of oral soft tissues infected by Candida albicans. Specifically, the system comprises:

[0121] The three-dimensional model building subsystem is used to build a three-dimensional model of oral soft tissue, wherein the three-dimensional model of oral soft tissue includes fibroblasts and epithelial cells.

[0122] In the embodiment of the present invention, the three-dimensional model building subsystem may actually include devices related to building a three-dimensional model of oral soft tissue, such as: a clean bench, a culture dish, a culture bottle, an incubator, etc.

[0123] The co-culture subsystem is used to co-culture Candida albicans with the oral soft tissue three-dimensional model.

[0124] In the embodiment of the present invention, the co-culture subsystem may actually include devices related to co-culture, such as a centrifuge, a culture dish, and the like.

[0125] The staining subsystem is used to collect the culture system at preset time intervals during the co-culture period, prepare tissue sections, and perform HE staining and PAS staining.

[0126] In the embodiment of the present invention, the dyeing subsystem may actually include devices related to dyeing.

[0127] The observation subsystem is used to study the multi-stage process of the interaction between the three-dimensional model of cavity soft tissue and Candida albicans based on HE staining and PAS staining.

[0128] In the embodiment of the present invention, the observation subsystem may actually include devices related to observation, such as a microscope.

[0129] Optionally, the three-dimensional model building subsystem includes:

[0130] A primary culture device is used to perform primary culture on oral soft tissue blocks to obtain adherent primary oral fibroblasts;

[0131] A matrix preparation device for preparing tissue matrix based on adherent primary oral fibroblasts;

[0132] The culture device is used to inoculate epithelial cell suspension on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

[0133] Optionally, the primary culture device is specifically used for:

[0134] Soak tissue blocks in 3 ml of double antibody for 5 minutes, repeat three times;

[0135] Cut the interstitial layer into tissue blocks with a diameter of about 1 mm in a culture dish;

[0136] Use a probe to transfer the tissue blocks to the bottom of the bottle, so that the tissue blocks are evenly dispersed and attached to the bottom of the bottle. Place about 9-12 tissue blocks in each bottle, add complete culture medium, tighten the bottle cap and invert it in a 37°C, 5% CO2 incubator for 2 hours. After the tissue blocks at the bottom of the bottle are completely attached to the wall, turn the culture bottle over to make it upright, and ensure that the culture medium can cover each tissue block. Continue to culture. After 2 to 3 weeks, adherent primary oral fibroblasts are obtained.

[0137] Optionally, the matrix preparation device is specifically used for:

[0138] Type I collagen, Matrigel, fetal bovine serum, 10× basic DMEM medium, and DMEM complete medium containing 10% fetal bovine serum were mixed in a ratio of 3.5:3.5:1:1:1 to prepare a matrix gel;

[0139] The adherent oral primary fibroblasts were digested with trypsin and centrifuged to obtain a cell pellet, which was then mixed with the matrix gel to adjust the density to 5×10 5 cells / ml, and transferred into a 96-well plate. After the matrix gel solidified, 100 μl of complete culture medium was added to each well, and culture was continued for 24 hours to obtain the tissue matrix.

[0140] Optionally, the culture device is specifically used for:

[0141] The epithelial cells were digested with trypsin and centrifuged to obtain an epithelial cell pellet, which was resuspended in complete medium and the cell density was adjusted to 2×10 5 cells / ml, and epithelial cell suspension was obtained;

[0142] Inoculating 100 μl of the epithelial cell suspension in each well of the tissue matrix, and continuing to culture for 24 hours to implant the epithelial cells on the tissue matrix;

[0143] Its tissue matrix seeded with epithelial cells is placed on a collagen-coated nylon membrane supported by a metal grid;

[0144] The nylon membrane was placed in a culture dish, and 8 ml to 10 ml of complete culture medium was added so that the liquid surface just touched the bottom of the nylon membrane; the dish was placed in a 37° C., 5% CO 2 incubator and cultured for 8 to 10 days, with the culture medium changed every other day to obtain a three-dimensional model of oral soft tissue.

[0145] Optionally, the co-culture subsystem is specifically used for:

[0146] The activated Candida albicans standard strain SC5314 was centrifuged to obtain a precipitate, which was resuspended in sterile PBS and adjusted to a density of 1×10 6 CFU / ml, and a suspension of Candida albicans was obtained;

[0147] The oral soft tissue three-dimensional model was taken out, and 20ul of the Candida albicans suspension was added to the surface of the epithelial cell layer of the oral soft tissue three-dimensional model; the DMEM culture medium was replaced and the culture was continued.

[0148] Optionally, the system further comprises:

[0149] A slicing device is used to separate the oral soft tissue three-dimensional model from the nylon membrane after 10 days of culture, and embed and slice it after fixation and dehydration;

[0150] A staining device, used to stain the three-dimensional model tissue sections using a hematoxylin and eosin staining kit, and observe the structure of the three-dimensional model under an upright microscope;

[0151] The identification device is used to identify the components of the three-dimensional model using immunohistochemical staining, and the detection indicators are Pan-CK, a marker of epithelial cells, and Vimentin, a marker of mesenchymal cells.

[0152] Optionally, the system further comprises:

[0153] The research subsystem is used to study the function of oral primary fibroblasts in resisting Candida albicans by quantitatively detecting the infection area of ​​Candida albicans in a three-dimensional model.

[0154] In order to enable those skilled in the art to better understand the present invention, the following specific examples are used to illustrate the research method of the three-dimensional model of oral soft tissue infected by Candida albicans provided by the present invention using oral leukoplakia-associated fibroblasts (OLKAFs).

[0155] Example 1: Primary culture and identification of oral leukoplakia-related fibroblasts:

[0156] The present invention explores and studies that found that compared with normal fibroblasts (NFs), OLKAFs have a reduced ability to resist the adhesion and invasion of Candida albicans. In the present invention, a three-dimensional co-culture model is proposed to detect the function of NFs, OLKAFs and carcinoma-associated fibroblasts (CAFs) in resisting Candida albicans.

[0157] Human OLK, oral squamous cell carcinoma (OSCC) and normal oral mucosal tissues were collected for primary culture of oral leukoplakia-associated fibroblasts (OLKAFs), cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs). The growth characteristics and morphology of the cultured cells were observed, and immunocytochemistry (ICC) staining was used to identify and screen the cells required for the next experiment.

[0158] 1 Materials and methods

[0159] 1.1 Source of tissue samples

[0160] In the embodiment of the present invention, oral leukoplakia tissues from resection surgery and normal tissues from tooth extraction or maxillofacial plastic surgery were collected with the approval of a hospital ethics committee and informed consent of the patients.

[0161] 1.2 Candida albicans detection

[0162] Use a sterile cotton swab to wipe the lesion site for biopsy 10 times clockwise, soak it in a centrifuge tube containing 1 ml of sterile PBS, and shake it thoroughly on a vortex shaker for 1 minute. Dilute the original sample 10 times in a biosafety cabinet. 1 times, 10 2 times, 10 3 times, 10 4 After the original sample and the diluted sample are shaken thoroughly, 50 μl of each sample is inoculated into a YPD solid culture dish, and evenly spread with a sterile coating stick. The culture dish is placed in a fungal culture incubator for 48 hours, and the results of Candida albicans culture detection are observed and recorded.

[0163] 1.3 Primary cell isolation and culture

[0164] In the clean bench, the epithelium and stroma of the tissue specimen were completely separated, the epithelium was discarded, and the stroma was cut into pieces of about 1 mm. 3 The tissue blocks were allowed to adhere to the bottom of the culture flask, 5 ml of complete DMEM medium was added, and the cells were cultured in an incubator at 37°C and 5% CO2.

[0165] 1.4 Immune cytochemical staining of primary cells

[0166] The third-generation OLKAFs, CAFs and NFs were plated at 5×10 4 The cells were seeded in a twelve-well plate and identified by ICC staining using a kit. The detection indicators were as follows: pan-cytokeratin (Pan-CK), vimentin (Vimentin), alpha smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1), fibroblast activation protein (FAP), etc.

[0167] 2 Results

[0168] 2.1 Isolation and culture of oral leukoplakia-related fibroblasts

[0169] In the embodiment of the present invention, a total of 18 strains of OLKAFs were successfully isolated and cultured, of which 8 strains were not accompanied by epithelial abnormal hyperplasia, and 10 strains were accompanied by epithelial abnormal hyperplasia, which were respectively recorded as HLKAFs (OLKAFs with hyperplasia) or DLKAFs (OLKAFs with dysplasia). At the same time, 4 strains of CAFs and 6 strains of NFs were successfully isolated and cultured. All samples were tested and found to be free of Candida albicans infection. The growth characteristics and morphology of primary fibroblasts were observed under an inverted microscope, and it was found that around the 7th day of culture, a small number of cells could crawl out of the edge of the tissue block attached to the wall. At this time, the fibroblasts were mostly long spindle-shaped and scattered around the tissue block (such as Figure 2 As shown in part A, Figure 2 The morphological observation results of primary culture of oral leukoplakia-related fibroblasts in the embodiment of the present invention are shown. Figure 2 Part A in the middle shows the distribution of cells around the tissue block on the 7th day of primary culture, among which a small number of fibroblasts in the shape of long spindles crawl out from the edge of the dark tissue block. After culturing for about 2-3 weeks, the tissue block is covered with long spindle-shaped fibroblasts. At this time, the cells are densely arranged radially in all directions and connected to the cells crawling out of the adjacent tissue blocks to form a network. Occasionally, small, round or oval cells can be observed between the primary fibroblasts, which may be mixed epithelial cells (such as Figure 2 As shown in Part B, Figure 2 Part B shows the distribution of cells around the tissue block after 2-3 weeks of primary culture. Dense fibroblasts are arranged radially around, with occasional round or oval epithelial cells mixed in. After cell purification and passage 2-3 times, the remaining primary fibroblasts are long spindle-shaped and arranged in bundles (such as Figure 2 As shown in part C, Figure 2 Part C in the middle represents primary fibroblasts that have been purified and passaged 2-3 times. It can be seen that they are dividing vigorously, and the cells are long spindle-shaped and arranged in bundles).

[0170] 2.2 Identification and screening of primary fibroblasts by immunocytochemical staining

[0171] ICC staining was performed on the third generation primary fibroblasts, and the results were as follows: Pan-CK expression in the four groups of cells, NFs, HLKAFs, DLKAFs and CAFs, was negative, and Vimentin and FSP-1 expression were positive, indicating that the primary cultured cells were fibroblasts, and they were all of mesenchymal rather than epithelial origin. From NFs, HLKAFs to DLKAFs, CAFs, the expression levels of FAP and α-SMA in the four groups of cells gradually increased (e.g. Figure 3 As shown, Figure 3 Shown are the results of immunocytochemical staining to identify primary fibroblasts).

[0172] Example 2: Construction of a three-dimensional model of oral soft tissues infected by Candida albicans:

[0173] In order to achieve mutual communication between the epithelium and the matrix layer in an in vitro environment and simulate the in vivo interstitial microenvironment as much as possible, in the embodiment of the present invention, a three-dimensional culture model of oral mucosa infected with Candida albicans is intended to be established on the basis of two-dimensional culture to further study the interaction between oral leukoplakia host cells and microorganisms.

[0174] In the embodiment of the present invention, the process of establishing a three-dimensional co-culture model can be divided into three stages: the first stage is the preparation of interstitial tissue, in which fibroblasts are mixed with matrix components and cultured for 24 hours; the second stage is the preparation of epithelial tissue, in which Cal-27 cells are selected as epithelial cells, added on top of the matrix components, and cultured for another 24 hours; the third stage is the model growth stage, in which the tissue is placed on a pre-prepared nylon membrane, cultured for 10 days, and the medium is changed every other day. The three-dimensional model is obtained as shown in FIG. Figure 4A As shown, Figure 4A A general schematic diagram of a three-dimensional model established in an embodiment of the present invention is shown.

[0175] After 10 days of culture, the three-dimensional model tissue blocks were separated from the nylon membrane, fixed, dehydrated, embedded, and sliced. The three-dimensional model tissue sections were stained with a hematoxylin and eosin (HE) staining kit, and the structure of the three-dimensional model was observed under an upright microscope. Immunohistochemistry (IHC) staining was further used to identify the components of the three-dimensional model, and the detection indicators were Pan-CK, a marker of epithelial cells, and Vimentin, a marker of mesenchymal cells.

[0176] In an embodiment of the present invention, a modified three-dimensional co-culture model was successfully established to simulate the normal structure of the oral mucosal epithelium. HE staining and IHC staining were used to verify the structure and cellular components of the three-dimensional model. HE staining showed that the three-dimensional model was divided into two layers, the upper layer was a stratified squamous epithelium, and the lower layer was a stroma. Under a high-power microscope, the epithelial cells were round or polygonal and arranged closely; the collagen structure in the stroma showed pink waves of varying shades, interspersed with fibroblasts or their cross-sections that were consistent with or perpendicular to the direction of the cut surface, showing long spindles or irregular shapes, and the cell nuclei were stained blue. IHC staining showed that the epithelial-specific marker Pan-CK was mainly expressed in the epithelial layer of the model, and the stroma-specific marker Vimentin was mainly expressed in the stroma of the model (such as Figure 4B As shown, Figure 4B The figure shows the results of the establishment and verification test of the three-dimensional model in the embodiment of the present invention. Figure 4BHE staining showed that the three-dimensional model was divided into an epithelial layer and an interstitial layer. The epithelial layer was of moderate thickness, with closely arranged epithelial cells, and fibroblasts were scattered in the interstitial layer (100×, 400×). IHC staining showed that Pan-CK was mainly expressed in the epithelial layer (100×, 400×), and Vimentin was mainly expressed in the interstitial layer (100×, 400×).

[0177] On the 9th day of the three-dimensional model culture, Candida albicans suspension was added to the surface of the epithelial layer and continued to be cultured. In order to determine the optimal co-culture time, different time gradients were set up for preliminary experiments. After the culture was completed, the cells were fixed and embedded, and the fungal glycogen staining (periodic acid-schiff stain, PAS) kit was used for staining and observation. The PAS staining results show the time course of the three-dimensional model infected with Candida albicans. After 6 hours of Candida albicans infection in the three-dimensional model, the epithelial structure was intact and the morphology was clear; after 12 hours of infection, a small amount of spores and hyphae could be found in the epithelial layer and the interstitial layer, but the epithelial structure was not obviously damaged; after 24 hours of infection, a large number of hyphae could be seen in the epithelial layer, and they invaded the interstitial layer, and the integrity of the epithelium was destroyed; after 48 hours of infection, the epithelial layer and the interstitial layer were almost completely covered with hyphae, and the epithelial structure was obviously damaged; after 72 hours of infection, the epithelial and interstitial structures were completely destroyed and almost unrecognizable (such as Figure 5 As shown, Figure 5 FIG. 1 shows the infection result diagram of the three-dimensional model of oral mucosa infected by Candida albicans in the embodiment of the present invention, Figure 5 It can be seen that after 6 hours of Candida albicans infection in the three-dimensional model, the epithelial structure was intact; after 12 hours of infection, the epithelial layer structure was intact, and a small amount of spores and hyphae were visible in the epithelial and interstitial layers; after 24 hours of infection, the epithelial layer structure was destroyed, and the hyphae covered the epithelium and invaded the interstitium; after 48 hours and beyond, the hyphae covered the entire epithelium and interstitial layer).

[0178] Example 3: Using a three-dimensional model to detect the function of OLKAFs in resisting Candida albicans:

[0179] Using the three-dimensional model of oral soft tissues infected by Candida albicans established in the above Example 2, this example intends to detect the function of OLKAFs in resisting Candida albicans by quantitatively detecting the infection area of ​​Candida albicans in the three-dimensional model.

[0180] Four groups of three-dimensional models were established using four types of fibroblasts (NFs, HLKAFs, DLKAFs, CAFs), respectively, and the specific experimental method was the same as in Example 2, and Candida albicans was added for co-culture. According to the experimental results of Experiment 3, tissue fixation, dehydration, embedding, sectioning and PAS staining observation were performed 24 hours after infection.

[0181] A three-dimensional model constructed by different primary fibroblasts was established to study its ability to resist Candida albicans infection. After 24 hours of Candida albicans stimulation, the results showed that the depth and area of ​​Candida albicans hyphae invasion gradually increased from NFs to CAFs (e.g. Figure 6 As shown, Figure 6 The results of infection area analysis of the three-dimensional model of oral soft tissue infected by Candida albicans in the embodiment of the present invention are shown. Figure 6 As shown in Figure 3, the percentage of Candida albicans infection area in the NFs or HLKAFs group was lower than that in the DLKAFs or CAFs group.

[0182] Based on the above embodiments, the embodiments of the present invention have determined that oral leukoplakia-related fibroblasts are a type of fibroblasts in an "activated" state.

[0183] Specifically, fibroblasts are the most important mesenchymal cells in the microenvironment. They are mainly derived from the primitive mesenchyme of the mesoderm and can undergo reversible and irreversible activation. Under normal circumstances, fibroblasts play an important role in maintaining the homeostasis of the extracellular matrix by synthesizing important matrix components such as collagen and matrix metalloproteinases. In addition, fibroblasts maintain normal communication and function between cells by secreting a variety of growth factors. When a disease occurs, tissue regeneration, inflammation, and matrix remodeling cause changes in the microenvironment, and fibroblasts in the microenvironment are activated. Studies have found that in cancer, activated fibroblasts undergo phenotypic transformation and become cancer-associated fibroblasts (CAFs) that express various markers (such as α-SMA, FSP-1, etc.). In recent years, CAFs, as the main component of the tumor microenvironment, have been shown to be involved in a variety of cellular processes and play a key role in the biological behavior of tumors.

[0184] In the embodiment of the present invention, oral leukoplakia-associated fibroblasts (OLKAFs) were found in the OLK stroma, and the biological characteristics of OLKAFs were found to be between NFs and CAFs, and were related to the degree of epithelial dysplasia. In view of the differences in genomic variation, risk of malignant transformation, microbial infection, disease management and prognosis in OLK with or without epithelial dysplasia, the embodiment of the present invention divides OLKAFs into HLKAFs (without epithelial dysplasia) or DLKAFs (with epithelial dysplasia). Pan-CK and Vimentin were used as markers to identify fibroblasts and epithelial cells, proving that the primary cells obtained in Example 1 were fibroblasts of interstitial origin. FAP and α-SMA are currently the most widely used markers for identifying CAFs. This experiment found that the FAP and α-SMA staining positivity of DLKAFs was strong, close to CAFs, while HLKAFs was weaker, close to NFs, suggesting that with the occurrence and development of OLK, the biological characteristics of fibroblasts have also changed.

[0185] The embodiments of the present invention also studied and determined that fibroblasts are closely involved in the host's anti-infection immunity.

[0186] Specifically, as one of the most important cell components in the microenvironment, when fibroblasts are stimulated by damage, inflammation, etc., static fibroblasts are activated, and their biological functions change in response to external signals, including obtaining a contractile phenotype, maintaining adjacent epithelial cell homeostasis, promoting angiogenesis, mediating immune responses, etc., wherein changes in immune function are gradually receiving attention. In the OLK study, the problem of being susceptible to Candida infection is closely related to immunity. In the embodiment of the present invention, the dynamic process of Candida albicans invading epithelium and interstitium was observed by a three-dimensional model, and it was found that spores and a small amount of hyphae were observed to be distributed on the surface and inside of the epithelial layer in the early stage of Candida albicans infection (12 hours), and obvious morphological conversion processes were observed during the invasion of Candida albicans. In the late stage of infection (24 hours and later), it can be found that the infection of the epithelium and interstitial layer of Candida is almost entirely hyphae phase. Similar to the detection results of the two-dimensional model, the infection progression process is accompanied by an increase in hypha length and number, and the overall performance is an increase in hypha density and an increase in infection area, and is accompanied by the destruction process of epithelial cells.

[0187] Based on the above research and exploration, the embodiments of the present invention propose the inventive concept: establishing a three-dimensional model of oral soft tissue, wherein the three-dimensional model of oral soft tissue comprises oral fibroblasts and epithelial cells; co-culturing Candida albicans with the three-dimensional model of oral soft tissue; collecting the culture system at preset time intervals during the co-culture period, making tissue sections, and performing HE staining and PAS staining; based on HE staining and PAS staining, studying the multi-stage process of the interaction between the three-dimensional model of oral soft tissue and Candida albicans.

[0188] The above is a detailed introduction to the research method and system of the three-dimensional culture system for oral soft tissue infection by Candida albicans provided by the present invention. Specific examples are used in this article to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea; at the same time, for general technicians in this field, according to the ideas of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A method for studying a three-dimensional model of oral soft tissue infection by Candida albicans, characterized in that: The method comprises: Establishing a three-dimensional model of oral soft tissue, wherein the three-dimensional model of oral soft tissue comprises oral fibroblasts and epithelial cells; co-culturing Candida albicans with the oral soft tissue three-dimensional model; During the co-culture period, the culture system was collected at preset intervals, tissue sections were made, and HE and PAS staining were performed; Based on HE staining and PAS staining, the multi-stage process of the interaction between the three-dimensional model of oral soft tissue and Candida albicans was studied.

2. The method for studying the three-dimensional model of oral soft tissue infection by Candida albicans according to claim 1, characterized in that: The method of establishing the oral soft tissue three-dimensional model comprises: Oral soft tissue pieces were cultured to obtain adherent primary oral fibroblasts; Preparation of tissue matrix based on adherent primary oral fibroblasts; An epithelial cell suspension is inoculated on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

3. The method for studying the three-dimensional model of oral soft tissue infection by Candida albicans according to claim 2, characterized in that: Primary culture of oral soft tissue pieces, including: Soak tissue blocks in 3 ml of double antibody solution for 5 minutes, repeat three times; Cut the interstitial layer into tissue blocks with a diameter of about 1 mm in a culture dish; Use a probe to transfer the tissue blocks to the bottom of the bottle, so that the tissue blocks are evenly dispersed and attached to the bottom of the bottle. Place 9-12 tissue blocks in each bottle, add complete culture medium, tighten the bottle cap and invert it in a 37°C, 5% CO2 incubator for 2 hours. After the tissue blocks at the bottom of the bottle are completely attached to the wall, turn the culture bottle over to make it upright, and ensure that the culture medium can cover each tissue block. Continue to culture. After 2 to 3 weeks, adherent primary oral fibroblasts are obtained.

4. The method for studying the three-dimensional model of oral soft tissue infection by Candida albicans according to claim 3, characterized in that: Prepare tissue matrix based on adherent primary oral fibroblasts, including: Type I collagen, Matrigel, fetal bovine serum, 10-fold basal DMEM medium, and DMEM complete medium containing 10% fetal bovine serum were mixed in a ratio of 3.5:3.5:1:1:1 to prepare a matrix gel; The adherent oral primary fibroblasts were digested with trypsin and centrifuged to obtain a cell pellet, which was then mixed with the matrix gel to adjust the density to 5×10 5 cells / ml, and transferred into a 96-well plate. After the matrix gel solidified, 100 μl of complete culture medium was added to each well, and culture was continued for 24 hours to obtain the tissue matrix.

5. The method for studying the three-dimensional model of oral soft tissue infection by Candida albicans according to claim 4, characterized in that: Inoculating epithelial cell suspension on the tissue matrix to obtain a three-dimensional model of oral soft tissue, comprising: The epithelial cells were digested with trypsin and centrifuged to obtain an epithelial cell pellet, which was resuspended in complete medium and the cell density was adjusted to 2×10 5 cells / ml, and epithelial cell suspension was obtained; Inoculating 100 μl of the epithelial cell suspension in each well of the tissue matrix, and continuing to culture for 24 hours to implant the epithelial cells on the tissue matrix; Its tissue matrix seeded with epithelial cells is placed on a collagen-coated nylon membrane supported by a metal grid; The nylon membrane was placed in a culture dish, and 8 ml to 10 ml of complete culture medium was added so that the liquid surface just touched the bottom of the nylon membrane; the dish was placed in a 37° C., 5% CO 2 incubator and cultured for 8 to 10 days, with the culture medium changed every other day to obtain a three-dimensional model of oral soft tissue.

6. The method for studying the three-dimensional model of oral soft tissue infection by Candida albicans according to claim 5, characterized in that: Co-culturing Candida albicans with the oral soft tissue three-dimensional model comprises: The activated Candida albicans standard strain SC5314 was centrifuged to obtain a precipitate, which was resuspended in sterile PBS and adjusted to a density of 1×10 6 CFU / ml, and a suspension of Candida albicans was obtained; The oral soft tissue three-dimensional model was taken out, and 20ul of the Candida albicans suspension was added to the surface of the epithelial cell layer of the oral soft tissue three-dimensional model; the DMEM culture medium was replaced and the culture was continued.

7. The method for studying the three-dimensional model of oral soft tissue infection by Candida albicans according to claim 5, characterized in that: The method further comprises: After 10 days of culture, the oral soft tissue three-dimensional model was separated from the nylon membrane, fixed, dehydrated, embedded, and sliced; The three-dimensional model tissue sections were stained using a hematoxylin and eosin staining kit, and the structure of the three-dimensional model was observed under an upright microscope; Immunohistochemical staining was used to identify the components of the three-dimensional model, and the detection indicators were Pan-CK, a marker of epithelial cells, and Vimentin, a marker of mesenchymal cells.

8. The method for studying the three-dimensional model of oral soft tissues infected by Candida albicans according to any one of claims 1 to 7, characterized in that: The method further comprises: The function of oral primary fibroblasts in resisting Candida albicans was studied by quantitatively detecting the infection area of ​​Candida albicans in a three-dimensional model.

9. A method for constructing a three-dimensional model of oral soft tissue, characterized in that: The method comprises: Oral soft tissue pieces were cultured to obtain adherent primary oral fibroblasts; Preparation of tissue matrix based on adherent primary oral fibroblasts; An epithelial cell suspension is inoculated on the tissue matrix to obtain a three-dimensional model of oral soft tissue.

10. A research system for a three-dimensional model of oral soft tissue infection by Candida albicans, characterized in that: The system comprises: A three-dimensional model building subsystem is used to build a three-dimensional model of oral soft tissue, wherein the three-dimensional model of oral soft tissue includes fibroblasts and epithelial cells; a co-culture subsystem, used for co-culturing Candida albicans with the oral soft tissue three-dimensional model; The staining subsystem is used to collect the culture system at preset time intervals during the co-culture period, prepare tissue sections, and perform HE staining and PAS staining; The observation subsystem is used to study the multi-stage process of the interaction between the three-dimensional model of cavity soft tissue and Candida albicans based on HE staining and PAS staining.

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