Olfactory disorder in-vitro model constructed based on olfactory epithelium organoid and application of olfactory disorder in-vitro model

Abstract

The invention discloses an olfactory disorder in-vitro model constructed based on olfactory epithelium organs and a construction method of the olfactory disorder in-vitro model. The olfactory disorder in-vitro model comprises a chronic nasosinusitis in-vitro model, an acute nasosinusitis in-vitro model, a first olfactory epithelium organ in-vitro inflammation model, a second olfactory epithelium organ in-vitro inflammation model and a third olfactory epithelium organ in-vitro inflammation model. The olfactory disorder in-vitro model provided by the invention can be used for researching the pathogenesis of olfactory disorder, identifying pathogenic genes and preparing medicines for treating olfactory disorder. According to the method, the olfactory disorder in-vitro model is constructed through the olfactory epithelium organ for the first time, compared with a conventional animal model, the construction period is shortened, the model cost is reduced, operation is easy, and application and popularization are easy; in addition, the in-vitro model constructed by the olfactory epithelium organoid can be combined with nuclear transfection, patch clamp, calcium imaging, RNA sequencing technology and the like to be applied to research of olfactory disorder pathogenic genes, and a new thought is provided for clinical treatment of olfactory disorder and related diseases.

Description

Technical Field

[0001] The present invention relates to an in vitro model of olfactory impairment constructed based on olfactory epithelium organoids and application thereof. Background Art

[0002] The sense of smell is an essential sensory function in human life. It affects the quality of human life in all aspects. The sense of smell can affect the nutritional status of the human body through appetite and food preferences, and help people quickly identify dangers in the environment (such as natural gas leaks, fires, dangerous chemical gases and rotten food, etc.). Olfactory dysfunction not only affects people's physical health, reduces the quality of life, and increases individual mortality, but it is also the first symptom of many major neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.).

[0003] There are many pathogenic factors for olfactory dysfunction, among which important factors include chronic inflammation, basal cell inactivation and neuronal degeneration. The loss of olfactory function caused by chronic inflammation is mainly caused by chronic sinusitis, which is a common inflammatory disease affecting the nasal mucosa. The resulting hyposmia and dysfunction are caused by the obstruction of odor transmission caused by mucosal edema or polyps. Animal models and clinical studies have shown that inflammatory reactions in the olfactory epithelium can cause temporary and reversible disorders of odor recognition and olfactory perception, while long-term inflammation causes changes in the structure of the olfactory neuroepithelium and its replacement by the respiratory epithelium. Therefore, olfactory dysfunction caused by chronic sinusitis is an inflammation-induced disease related to sensory nerves.

[0004] Since the pathogenesis of olfactory disorders is unclear, there is still no targeted treatment. Commonly used methods include drug therapy based on corticosteroids, olfactory training and surgical treatment. There is still a lack of disease models for the study of the pathogenic mechanism of olfactory disorders, and they mainly rely on animal models, including lipopolysaccharide (LPS)-induced sinusitis models, methyl indole-induced olfactory damage models, and transgenic mice related to neuronal transduction defects and inflammatory factors. There are still no reports of in vitro models of olfactory disorders. If an effective cell model is established to replace the animal model, it can not only greatly reduce the use of animals to improve animal welfare, but its low cost, easy maintenance and convenient experimental operation will definitely accelerate the research progress of the pathogenesis of olfactory disorders.

[0005] Organoids are three-dimensional structures developed from stem cells that accurately reflect tissue structure and cell composition. Organoids can be derived from two types of stem cells, including pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells) and organ-specific adult stem cells. Using the above two types of cells, scientists have successfully established organoids of many important organs such as brain, stomach, intestine, lung, liver and kidney. Organoids are usually derived from healthy or diseased tissues and are good models for studying tissue homeostasis and regeneration as well as disease mechanisms. The olfactory epithelium contains tissue-specific stem cells, which are ideal tissue sources for in vitro cultivation of organoids. Organoids can maintain genetic stability while being stably propagated, and the development of their technology has opened up new avenues for testing and developing preclinical treatment methods. At present, organoids have been used in a variety of clinical applications, including disease modeling, drug screening, host-microbe interactions and regenerative therapy. Patient-derived organoids make it possible to achieve personalized precision medicine. The use of patient organoids for high-throughput drug screening has become a trend in current tumor research. In most cases, the test results of patient-derived organoids can predict the patient's response to treatment. In addition, studies have found that using molecular technologies such as lentiviral expression systems and CRISPR / Cas9, genes can be manipulated in organoids, making disease modeling and targeted gene therapy possible. For example, cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In patient-derived intestinal organoids, homologous recombination using the CRISPR / Cas9 system corrected the D508 mutation at the CFTR site, and the corrected CFTR channel restored normal physiological function. Therefore, organoids can serve as an ideal model for studying disease pathogenesis and formulating treatment plans.

[0006] Therefore, in view of the problems of long construction cycle, high cost, difficulty in maintenance and inconvenient operation of animal models, it is necessary to develop an olfactory epithelium organoid model to make it an ideal tool to replace the olfactory disorder mouse model, so as to explore the pathogenesis of olfactory disorders, identify pathogenic genes and establish new methods for the treatment of olfactory disorders. Summary of the invention

[0007] The purpose of the present invention is to establish an olfactory epithelium organoid model, making it an ideal tool to replace the olfactory disorder mouse model, for exploring the pathogenesis of olfactory disorders, identifying pathogenic genes and establishing new methods for the treatment of olfactory disorders.

[0008] The technical solution adopted by the present invention is:

[0009] A method for constructing an in vitro model of olfactory impairment based on olfactory epithelium organoids, the method comprising one of the following:

[0010] (I) Chronic sinusitis model: 11-week-old C57 / BL mice were given LPS instillations into the nasal cavity three times a week at a dosage of 1 μg / 10 g. The mice were killed 21 days after the first administration, and the olfactory epithelium of the mice was obtained for organoid culture to obtain an in vitro model of chronic sinusitis.

[0011] (II) Acute sinusitis model: 11-week-old C57 / BL mice were given LPS drips into the nasal cavity for 7 consecutive days at a drip dose of 1 μg / 10 g. The mice were killed 7 days later and the olfactory epithelium of the mice was taken for organoid culture to obtain an in vitro model of acute sinusitis.

[0012] (III) The olfactory epithelium of 6-week-old Cyp2g1-rtTA / TRE TNF-α mice was cultured for organoids. After the organoids were mature, doxycycline was added to the culture medium at a dosage of 1 to 10 μg / ml. The production of TNF-α was controlled by the tetracycline response element (TRE). After 24 hours of treatment with doxycycline, the first in vitro inflammation model of olfactory epithelium organoids was obtained:

[0013] (IV) During the growth of 6-8 week old Cyp2g1-rtTA / TRE TNF-α mice, doxycycline diet was used for 5 months with a dietary dosage of 0.625 g / kg. The olfactory epithelium of the mice was taken for organoid culture to obtain the second in vitro inflammation model of olfactory epithelium organoids:

[0014] (V) The olfactory epithelium of 6-week-old wild-type mice was taken for organoid culture, and exogenous TNF-α was directly added to the culture medium. The amount of TNF-α added was 5-10 ng / ml. The treatment was carried out for 72 hours to obtain the third in vitro inflammation model of olfactory epithelium organoids.

[0015] In the method, the components of the culture medium used for the organoid culture are as follows:

[0016] The following components were added to the DMEM / F12 medium at final concentrations: 200 ng / mL R-spondin-1, 100 ng / mL Noggin, 50 ng / mL Wnt3a, 10 μM Y27632, 1% N2 additive, 2% B27 additive, 10 mM HEPES, 50 ng / mL penicillin-streptomycin mixture, and 50 ng / mL human epidermal growth factor.

[0017] Primary organoids should also be supplemented with 100 μg / ml of the primary antibiotic Primocin.

[0018] In the method (III) or (IV), the Cyp2g1-rtTA / TRE TNF-α mouse is constructed according to the following method:

[0019] CRISPR / Cas9 technology was used to establish Cyp2g1 gene knockin mice, and CRISPR / Cas9 technology was used to construct TIGRE site TNF-α gene fragment knockin mice. The two genotypes of mice were hybridized to produce Cyp2g1-rtTA / TRE TNF-α mice.

[0020] The present invention also provides an in vitro model of olfactory disorder constructed by the above method, including an in vitro model of chronic sinusitis, an in vitro model of acute sinusitis, a first in vitro inflammation model of olfactory epithelium organoids, a second in vitro inflammation model of olfactory epithelium organoids, and a third in vitro inflammation model of olfactory epithelium organoids.

[0021] The present invention also provides the use of the in vitro model of olfactory disorder in studying the pathogenesis of olfactory disorder, identifying pathogenic genes, and preparing drugs for treating olfactory disorder.

[0022] Furthermore, the chronic sinusitis in vitro model or the acute sinusitis in vitro model can be used to study the effect of basal cell proliferation on olfactory dysfunction.

[0023] The first in vitro inflammation model of olfactory epithelium organoids can be used to study the effects of neuronal cells, cellular inflammatory responses, and cell proliferation capacity on olfactory disorders.

[0024] The second in vitro inflammation model of olfactory epithelium organoids can be used to study the impact of olfactory neuronal cells on olfactory disorders.

[0025] The third in vitro inflammation model of olfactory epithelium organoids can be used to study the impact of the ability to differentiate into mature olfactory sensory neurons in olfactory epithelium organoids on olfactory disorders.

[0026] Compared with the prior art, the present invention has at least the following beneficial effects:

[0027] The present invention proposes for the first time to construct an in vitro model of olfactory disorder using olfactory epithelium organoids. Compared with conventional animal models, this greatly reduces the construction cycle, reduces the model cost, is simple to operate, and is easy to promote and apply. In addition, the in vitro model constructed with olfactory epithelium organoids can be combined with nuclear transfection, patch clamp, calcium imaging, RNA sequencing technology, etc. to study pathogenic genes of olfactory disorders, providing new ideas for the clinical treatment of olfactory disorders and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the number of OMP+ neurons, ICAM1+ horizontal basal cells, and IL33+ supporting cells in the olfactory epithelium of the LPS-induced mouse model.

[0029] Figure 2 The changes in the number of CD45+ inflammatory cells and F4 / 80+ macrophages in the olfactory epithelium of the LPS-induced mouse model.

[0030] Figure 3 Comparison of organoid morphology and quantity in LPS-induced mouse organoid model.

[0031] Figure 4 It is the change in the number of Ki67+ proliferating cells in the LPS organoid model.

[0032] Figure 5 The changes in the number of OMP+ neurons, ICAM1+ horizontal basal cells, and IL33+ supporting cells in the LPS organoid model.

[0033] Figure 6 are the changes in organoid morphology in the doxycycline (DOX)-induced and non-induced groups.

[0034] Figure 7 is the differential expression of TNFα in the organoid model between the doxycycline (DOX)-induced and non-induced groups.

[0035] Figure 8 is the difference in the number of Ki67+ cells in the organoid model between the doxycycline (DOX)-induced and non-induced groups.

[0036] Fig. 9 It is the change in the number and morphology of OMP+ neurons, ICAM1+ horizontal basal cells and IL33+ supporting cells in the organoid model induced by doxycycline (DOX) and not induced.

[0037] Fig.10 The changes in the number of OMP+ neurons, ICAM1+ basal cells, IL33+ supporting cells, Ki67+ proliferating cells and F4 / 80+ macrophages in the olfactory epithelium of mice fed with doxycycline (DOX) for 5 months.

[0038] Fig.11 The changes in organoid morphology in the induced and uninduced groups at different time points after 5 months of doxycycline (DOX) induction in animals.

[0039] Fig.12 It is the change in the number of TNFα+ cells in the olfactory epithelium organoid model cultured 5 months after doxycycline (DOX) induced in animals.

[0040] Fig.13 It is the change in the number of Ki67+ cells in the olfactory epithelium organoid model cultured 5 months after doxycycline (DOX) induction in animals.

[0041] Fig.14The changes in the number of OMP+ neurons, ICAM1+ horizontal basal cells and IL33+ supporting cells in the olfactory epithelium organoid model cultured 5 months after doxycycline (DOX) induction in animals.

[0042] Fig.15 The changes in organoid morphology after adding 1, 5, and 10 ng / ml TNF-α to olfactory epithelial organoids and culturing for 24, 48, and 72 hours.

[0043] Fig.16 The changes in the number of Ki67-positive cells were detected after the olfactory epithelial organoids were added with 1, 5, and 10 ng / ml TNF-α and cultured for 24, 48, and 72 hours.

[0044] Fig.17 The changes in the number of OMP-positive cells were detected after adding 1, 5, and 10 ng / ml TNF-α to the olfactory epithelial organoids and culturing for 24, 48, and 72 hours.

[0045] Fig.18 The changes in the number of ICAM1-positive cells were detected after adding 1, 5, and 10 ng / ml TNF-α to the olfactory epithelial organoids and culturing for 24, 48, and 72 hours. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is described in detail below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0047] Example 1

[0048] (1) Sinusitis model:

[0049] A. Chronic sinusitis model: 11-week-old C57 / BL mice were given LPS (1 μg / 10 g body weight) by nasal instillation three times a week. The mice were killed 21 days after the first administration, and the olfactory epithelium of the mice was obtained for organoid culture, which was recorded as LPS-21d.

[0050] B. Acute sinusitis model: 11-week-old C57 / BL mice were given LPS (1 μg / 10 g body weight) by nasal instillation for 7 consecutive days. The mice were killed after 7 days, and the olfactory epithelium of the mice was obtained for organoid culture, which was recorded as LPS-7d.

[0051] (2) Inflammatory response model:

[0052] In nasal inflammatory diseases, TNF-α is an inflammatory factor closely related to infection and damage of many cell types. Therefore, we used three methods to construct a TNF-α-induced olfactory epithelial organoid inflammation model:

[0053] A. Transgenic mice that can continuously produce TNF-α in the olfactory epithelium were used to simulate the occurrence of nasal inflammation. Cyp2g1-rtTA / TRE TNF-α mice were constructed as follows:

[0054] CRISPR / Cas9 technology was used to establish Cyp2g1 gene knockin mice, and CRISPR / Cas9 technology was also used to construct TIGRE site TNF-α gene fragment knockin mice. The two genotypes of mice were crossbred to produce Cyp2g1-rtTA / TRE TNF-α mice. Through the "Tet-On" system, the production of TNF-α was initiated in the olfactory epithelium under the control of the promoter of the specific gene Cyp2g1. The olfactory epithelium of Cyp2g1-rtTA / TRE TNF-α mice (6 weeks old) was taken. After the organoid culture matured, doxycycline (1ug / ml, 5ug / ml, 10ug / ml) was added to the culture medium for 24 hours to control the production of TNF-α through the tetracycline response element (TRE).

[0055] B. Transgenic mice that can continuously produce TNF-α in the olfactory epithelium were used to simulate the occurrence of nasal inflammation. Cyp2g1-rtTA / TRE TNF-α mice aged 6-8 weeks were fed a doxycycline diet (0.625 g / kg) for 5 months, and the olfactory epithelium of the mice was harvested for organoid culture.

[0056] C. Directly simulate olfactory epithelial organoids in an inflammatory response environment in vitro: culture olfactory epithelial organoids from 6-week-old wild-type mice, and directly add exogenous TNF-α (1ng / ml, 5ng / ml, 10ng / ml) to the organoid culture medium for 72 hours to create an inflammatory response environment.

[0057] In all the above methods, the organoid culture method used was as follows:

[0058] 1) The mice were intraperitoneally injected with 0.6 g of chloral hydrate (overdose). After the mice were anesthetized, their necks were dislocated and their heads were decapitated. The hair on their heads and their eyes were removed. The soft tissues along the nose were cut off, and unnecessary brain, bones, and soft tissues were removed. Only the upper jaw including the nasal passages was retained.

[0059] 2) Place the dissected palate in a 6 cm culture dish with an appropriate amount of sterile PBS and perform dissection under a stereo microscope;

[0060] 3) Dorsal side: use small scissors to cut along the inner edge of the eye socket to the front end; ventral side: use ophthalmic scissors to cut along the inner edge of the teeth to the front end. Use two forceps to separate the bone tissue on both sides of the mouse's nasal cavity, remove the bone tissue above the nasal cavity, expose the nasal mucosa and median cartilage, and place them in a new culture dish filled with sterile PBS;

[0061] 4) Separate the olfactory epithelium from the bony tissue and place it in a new small culture dish filled with sterile PBS;

[0062] 5) Add about 1 ml of 0.25% trypsin + EDTA to the olfactory epithelium of each mouse, cut the tissue into small pieces as much as possible, and digest it in a 37°C incubator for 30-60 minutes. During this period, observe under a microscope whether it is digested into single cells. The digestion time should not be too long to avoid affecting the cell state.

[0063] 6) After digestion is completed, add an excess of DMEM / F12 medium containing 10% fetal bovine serum to terminate digestion, transfer the cell suspension to a 15 ml centrifuge tube, and centrifuge at 1500 g for 5 minutes;

[0064] 7) Remove the supernatant, resuspend the cells in DMEM / F12 containing 10% fetal bovine serum, pipette several times, filter with a 0.40 μm mesh to remove large pieces of tissue, retain the single cell suspension, and centrifuge at 1500 g for 5 minutes;

[0065] 8) Remove the supernatant and resuspend the cells with Matrigel (operate on ice to prevent Matrigel from solidifying), 40 μl of cell suspension per well of a 24-well plate, about 10,000 cells per well;

[0066] 9) After inoculating the cells into the culture plate, place it in a 37°C incubator for 30 minutes until Matrigel solidifies, and add 500-800ul complete culture medium to each well;

[0067] 10) Replace the culture medium every 2-3 days. Organoid cultures mature after about 7 days and can be passaged after 7-14 days.

[0068] The complete medium for culturing organoids in step (9) is prepared as follows: Add the following components to the DMEM / F12 medium at final concentrations: 200 ng / mL R-spondin-1, 100 ng / mL Noggin, 50 ng / mL Wnt3a, 10 μM Y27632, 1% N2 additive, 2% B27 additive, 10 mM HEPES, 50 ng / mL penicillin-streptomycin mixture, 50 ng / mL human epidermal growth factor. 100 μg / ml of primary antibiotic Primocin should also be added to the primary organoids.

[0069] After the olfactory epithelium organoid model is constructed by the above method, the constructed olfactory epithelium organoid model is detected by immunofluorescence staining technology to determine whether the olfactory epithelium organoid model is successfully cultivated. The specific operation is as follows:

[0070] Collect and culture organoids into 1.5 ml EP tubes, fix with 4% paraformaldehyde at room temperature for 30 minutes, wash with PBS, add 30% sucrose solution to completely submerge the organoids, and dehydrate at 2-8°C overnight. The next day, remove the 30% sucrose solution, add 20-50 μl of gelatin solution preheated in a 37°C water bath to each tube, and bathe at 37°C for 15 minutes. Embed the olfactory epithelium organoids in OCT and store at -80°C for slicing.

[0071] Organoids embedded in tissue cryogen were cut into 20 μm thick tissue slices using a freezing microtome, adsorbed on positively charged anti-slip slides, baked at 60°C for 30 minutes, washed three times with PBS for 5 minutes each time, and OCT was removed. Blocked with 5% fetal bovine serum and 0.5% Triton-X100 dissolved in PBS at room temperature for one hour. After blocking, incubate with primary antibodies at 2-8°C overnight, including Ki67, IL33, OMP, and ICAM1. Wash with PBST (0.3% Triton-X100 dissolved in PBS) three times for 5 minutes each time. After incubating with secondary antibodies at room temperature for one hour, wash the slides three times with PBST, counterstain the cell nuclei with DAPI, seal the slides with anti-fading sealing agents, and detect the fluorescence signal using a laser scanning confocal fluorescence microscope.

[0072] Experimental Results

[0073] For (1) sinusitis model, the experimental results of chronic sinusitis model LPS-21d and acute sinusitis model LPS-7d are as follows Figures 1 to 5 As shown,

[0074] Figure 1 is the number of OMP+ neurons, ICAM1+ horizontal basal cells, and IL33+ supporting cells in the olfactory epithelium of the LPS-induced mouse model.

[0075] Figure 2 The changes in the number of CD45+ inflammatory cells and F4 / 80+ macrophages in the olfactory epithelium of the LPS-induced mouse model.

[0076] Figure 3 Comparison of organoid morphology and quantity in LPS-induced mouse organoid model.

[0077] Figure 4 It is the change in the number of Ki67+ proliferating cells in the LPS organoid model.

[0078] Figure 5 The changes in the number of OMP+ neurons, ICAM1+ horizontal basal cells, and IL33+ supporting cells in the LPS organoid model.

[0079] In this model, the researchers first constructed acute and chronic sinusitis mouse models by nasal instillation of LPS in mice. For the acute sinusitis model, C57 / BL mice around 11 weeks of age were taken and LPS (1μg / 10g body weight) was instilled into the nasal cavity for 7 consecutive days. For the chronic sinusitis model, C57 / BL mice around 11 weeks of age were taken and LPS (1μg / 10g body weight) was instilled into the nasal cavity three times a week for three weeks. After the induction, the mice were killed and the olfactory epithelium of the mice was subjected to immunofluorescence staining to determine whether the two models were successfully constructed and the changes in the number of various types of cells in the olfactory epithelium after successful construction. Immunofluorescence results showed that in the chronic (LPS instillation three times a week for three weeks) and acute (LPS instillation for 7 consecutive days) sinusitis mouse models, the number of OMP-positive olfactory sensory neurons was significantly reduced in the olfactory epithelium of the acute model, the activated horizontal basal cells were significantly increased, and the number of IL33-positive supporting cells was significantly reduced ( Figure 1 In addition, the number of CD45-positive inflammatory cells and F4 / 80-positive macrophages in the olfactory epithelium of the sinusitis mouse model was also significantly increased ( Figure 2 ), indicating that the inflammatory response in the olfactory epithelium of the sinusitis mouse model constructed in this project was enhanced.

[0080] Next, the olfactory epithelium of the chronic and acute sinusitis mouse models was cultured into organoids. When cultured for about 7 days, it was observed under a microscope that the size and number of organoids in the sinusitis model mice were significantly reduced compared with the control group ( Figure 3 ), at the same time, immunostaining results showed that the number of Ki67-positive proliferating cells in the organoids of the acute sinusitis model was significantly reduced ( Figure 4 ), which indicates that the proliferation of organoid cells in the LPS-induced mouse sinusitis model is significantly slowed down. In addition, the immunostaining results also suggest that in the LPS-induced sinusitis model, there are no significant changes in OMP-positive neuronal cells and IL33-positive supporting cells, while ICAM1-positive horizontal basal cells are significantly reduced ( Figure 5 ), suggesting that this model primarily affects the proliferation capacity of organoids, especially the proliferation of basal cells.

[0081] For model A in (2) inflammatory response model, the experimental results of the nasal inflammation organoid model induced by different doses of doxycycline (DOX) are as follows: Figures 6 to 9 As shown,

[0082] Figure 6 are the changes in organoid morphology in the doxycycline (DOX)-induced and non-induced groups.

[0083] Figure 7 is the differential expression of TNFα in the organoid model between the doxycycline (DOX)-induced and non-induced groups.

[0084] Figure 8is the difference in the number of Ki67+ cells in the organoid model between the doxycycline (DOX)-induced and non-induced groups.

[0085] Fig. 9 It is the change in the number and morphology of OMP+ neurons, ICAM1+ horizontal basal cells and IL33+ supporting cells in the organoid model induced by doxycycline (DOX) and not induced.

[0086] In this model, olfactory epithelial organoids were cultured from 6-8 week old Cyp2g1-rtTA / TRE TNFα homozygous mice. After 7-9 days of culture, the organoids gradually matured and different concentrations of doxycycline (DOX, 1μg / ml, 5μg / ml, 10μg / ml) were added for 24 hours. After the treatment, the morphological changes of the organoids were recorded under a microscope, and the organoids were collected for immunofluorescence staining to clarify the effect of DOX-induced Cyp2g1-rtTA / TRE TNFα homozygous mice on cell proliferation and the number of each cell type. The experimental results showed that the higher the DOX induction concentration, the more the organoids gradually disintegrated, but the organoids of wild-type mice were not affected, proving that the changes in the organoids were not due to DOX toxicity ( Figure 6 In addition, the results of in situ hybridization showed that the expression of TNFα in organoids increased significantly after adding 1μg / ml, 5μg / ml and 10μg / ml doxycycline ( Figure 7 ), immunostaining results showed that the number of Ki67-positive proliferating cells was significantly reduced after the addition of doxycycline ( Figure 8 In addition, the number of OMP-positive neuronal cells decreased significantly when the concentration of doxycycline was 1 μg / ml, and the number of IL33-positive supporting cells increased significantly when 1 μg / ml and 5 μg / ml doxycycline were added, while the number of ICAM1-positive cells did not change significantly ( Fig. 9 ). This indicates that after adding doxycycline, the inflammatory response of olfactory epithelial cells is enhanced and the cell proliferation ability is weakened, which in turn affects the decrease in the number of neurons in the olfactory epithelium and the increase in the number of supporting cells, but doxycycline has no significant effect on basal cells.

[0087] The experimental results of model B in the inflammatory response model, i.e., the nasal inflammation organoid model induced by doxycycline diet (0.625 g / kg), are as follows Figures 10 to 14 As shown,

[0088] Fig.10 The changes in the number of OMP+ neurons, ICAM1+ basal cells, IL33+ supporting cells, Ki67+ proliferating cells and F4 / 80+ macrophages in the olfactory epithelium of mice fed with doxycycline (DOX) for 5 months.

[0089] Fig.11 The changes in organoid morphology in the induced and uninduced groups at different time points after 5 months of doxycycline (DOX) induction in animals.

[0090] Fig.12 It is the change in the number of TNFα+ cells in the olfactory epithelium organoid model cultured 5 months after doxycycline (DOX) induced in animals.

[0091] Fig.13 It is the change in the number of Ki67+ cells in the olfactory epithelium organoid model cultured 5 months after doxycycline (DOX) induction in animals.

[0092] Fig.14 The changes in the number of OMP+ neurons, ICAM1+ horizontal basal cells and IL33+ supporting cells in the olfactory epithelium organoid model cultured 5 months after doxycycline (DOX) induction in animals.

[0093] After Cyp2g1-rtTA / TRE TNFα mice were fed doxycycline (0.625 g / kg) for five months, the results of mouse olfactory epithelial staining showed that after doxycycline induction, the number of OMP-positive neuronal cells and ICAM1-positive basal cells decreased, F4 / 80-positive macrophages increased significantly, Ki67-positive proliferating cells decreased significantly, and IL33-positive supporting cells did not change significantly, indicating that after doxycycline induction, the inflammatory response of the olfactory epithelium was enhanced and the cell proliferation ability was weakened, thereby causing a decrease in the number of olfactory neurons and basal cells, but had no significant effect on supporting cells ( Fig.10 ). Next, the olfactory epithelial tissue was cultured into organoids, and the morphology of the organoids was observed under a microscope. The results showed that compared with the control group, the growth rate of the olfactory epithelial organoids of mice fed with DOX slowed down, and the size of the organoids was significantly smaller than that of the control group at the same time point ( Fig.11 ). In situ hybridization results showed that the expression level of TNFα in the organoids of the doxycycline group was increased ( Fig.12 ), while the number of Ki67-positive proliferating cells was significantly decreased ( Fig.13 In addition, compared with the control group, the number of OMP-positive mature olfactory neurons was significantly reduced, while the number of IL33-positive supporting cells and ICAM1-positive horizontal basal cells did not change significantly ( Fig.14 ), indicating that DOX-induced inflammation in mice mainly affects olfactory neurons.

[0094] The experimental results of model C in the inflammatory response model, i.e., the nasal inflammation organoid model induced by exogenous addition of TNFα, are as follows: Figures 15 to 18 As shown,

[0095] Fig.15The changes in organoid morphology after adding 1, 5, and 10 ng / ml TNF-α to olfactory epithelial organoids and culturing for 24, 48, and 72 hours.

[0096] Fig.16 The changes in the number of Ki67-positive cells were detected after the olfactory epithelial organoids were added with 1, 5, and 10 ng / ml TNF-α and cultured for 24, 48, and 72 hours.

[0097] Fig.17 The changes in the number of OMP-positive cells were detected after adding 1, 5, and 10 ng / ml TNF-α to the olfactory epithelial organoids and culturing for 24, 48, and 72 hours.

[0098] Fig.18 The changes in the number of ICAM1-positive cells were detected after adding 1, 5, and 10 ng / ml TNF-α to the olfactory epithelial organoids and culturing for 24, 48, and 72 hours.

[0099] In this model, olfactory epithelial organoids were cultured from 6-8 week old wild-type C57 / BL mice. When the organoids were cultured for 7-9 days, the organoids gradually matured and different concentrations of TNF-α protein (1ng / ml, 5ng / ml, 10ng / ml) were added for 24, 48, and 72 hours. The culture medium containing the corresponding concentration of TNF-α was replaced every 24 hours. During the treatment process, photos were taken under a microscope to record the morphological changes of the organoids, and the organoids were collected for immunofluorescence staining to clarify the effect of TNF-α protein on the olfactory epithelial organoids of wild-type mice. The results showed that with the increase of TNF-α protein concentration and the extension of culture time, the olfactory epithelial organoids gradually showed a disintegration trend, which was most significant when treated with 10ng / ml for 72 hours ( Fig.15 In addition, immunofluorescence staining showed that compared with the untreated control group, there was no significant change in Ki67-positive proliferating cells in the organoids after adding TNF-α protein for 24 and 48 hours, but the number gradually decreased after 72 hours of treatment, with the highest difference at 10 ng / ml ( Fig.16 In addition, there was no significant change in OMP-positive olfactory neurons in organoids treated with TNFα protein for 24 and 48 hours, and the number of OMP-positive neurons was significantly reduced after 72 hours of treatment ( Fig.17 ), indicating that the addition of TNFα weakened the ability of olfactory epithelial organoids to differentiate into mature olfactory neurons, which is one of the important signs of olfactory disorders. After 24, 48, and 72 hours of TNFα treatment, the number of ICAM1-positive horizontal basal cells (HBCs) in the organoids did not change significantly ( Fig.18 ), indicating that TNFα does not affect dormant stem cells.

[0100] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A method for constructing an in vitro model of olfactory impairment based on olfactory epithelium organoids, characterized in that The method comprises one of the following: (I) Chronic sinusitis model: 11-week-old C57 / BL mice were given LPS instillations into the nasal cavity three times a week at a dosage of 1 μg / 10 g. The mice were killed 21 days after the first administration, and the olfactory epithelium of the mice was obtained for organoid culture to obtain an in vitro model of chronic sinusitis. (II) Acute sinusitis model: 11-week-old C57 / BL mice were given LPS drips into the nasal cavity for 7 consecutive days at a drip dose of 1 μg / 10 g. The mice were killed 7 days later and the olfactory epithelium of the mice was taken for organoid culture to obtain an in vitro model of acute sinusitis. (III) The olfactory epithelium of 6-week-old Cyp2g1-rtTA / TRE TNF-α mice was cultured for organoids. After the organoids were mature, doxycycline was added to the culture medium at a dosage of 1 to 10 μg / ml. After 24 hours of doxycycline treatment, the first in vitro inflammation model of olfactory epithelium organoids was obtained: (IV) During the growth of 6-8 week old Cyp2g1-rtTA / TRE TNF-α mice, doxycycline diet was used for 5 months with a dietary dosage of 0.625 g / kg. The olfactory epithelium of the mice was taken for organoid culture to obtain the second in vitro inflammation model of olfactory epithelium organoids: (V) The olfactory epithelium of 6-week-old wild-type mice was taken for organoid culture, and exogenous TNF-α was directly added to the culture medium. The amount of TNF-α added was 5-10 ng / ml. The treatment was carried out for 72 hours to obtain the third in vitro inflammation model of olfactory epithelium organoids.

2. The method according to claim 1, characterized in that In the method, the components of the culture medium used for the organoid culture are as follows: The following components were added to the DMEM / F12 medium at final concentrations: 200 ng / mL R-spondin-1, 100 ng / mL Noggin, 50 ng / mL Wnt3a, 10 μM Y27632, 1% N2 additive, 2% B27 additive, 10 mM HEPES, 50 ng / mL penicillin-streptomycin mixture, and 50 ng / mL human epidermal growth factor.

3. The method according to claim 1, characterized in that In the method (III) or (IV), the Cyp2g1-rtTA / TRE TNF-α mouse is constructed according to the following method: CRISPR / Cas9 technology was used to establish Cyp2g1 gene knockin mice, and CRISPR / Cas9 technology was used to construct TIGRE site TNF-α gene fragment knockin mice. The two genotypes of mice were hybridized to produce Cyp2g1-rtTA / TRETNF-α mice.

4. The in vitro model of olfactory impairment constructed according to the method of claim 1 comprises an in vitro model of chronic sinusitis, an in vitro model of acute sinusitis, a first in vitro inflammation model of olfactory epithelium organoids, a second in vitro inflammation model of olfactory epithelium organoids, and a third in vitro inflammation model of olfactory epithelium organoids.

5. Use of the in vitro model of olfactory impairment as claimed in claim 4 in studying the pathogenesis of olfactory impairment, identifying pathogenic genes, and preparing drugs for treating olfactory impairment.

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