Construction method of radioactive esophageal injury organ-like model

Abstract

The invention provides a construction method of a radioactive esophageal injury organ-like model. The construction method comprises the following steps: performing X-ray ionizing radiation on esophageal organs; wherein the esophageal organ comprises a rat esophageal organ, a mouse esophageal organ or a human esophageal organ; the radiation dosage is 0.5 to 4 Gy, and the radiation dosage rate is 1.5 to 2 Gy / min. Experimental results show that along with increase of irradiation dose, connection around spheres of human esophageal organs, mouse esophageal organs and rat esophageal organs is loose, cell debris is increased, and the sizes are smaller than those of normal organs at the same time. The construction of the radioactive esophageal injury organ-like model is of great significance for deeply understanding the injury mechanism of ionizing radiation to the esophagus, searching for effective protection and treatment measures and promoting the development of personalized radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and particularly relates to a method for constructing a radioactive esophageal injury organoid model. Background Art

[0002] Ionizing radiation is the general term for radiation such as X-rays, α and β charged particles, γ rays, and ultraviolet rays that can easily ionize substances. Ionizing radiation has two major sources. One is natural radiation (mainly cosmic and terrestrial radiation), and the other is artificial radiation (mainly applied in the military, economic, and medical fields). With the deepening of the research on ionizing radiation technology, while it benefits mankind, it also brings potential nuclear radiation risks to the current world. In the medical field, the widespread application of radiotherapy can kill tumor cells while also causing local and / or systemic adverse reactions in patients. The most common ones are damage to the hematopoietic system and digestive system. Therefore, how to prevent and treat the body damage caused by ionizing radiation has become a key concern worldwide.

[0003] The research on radioactive esophageal injury caused by ionizing radiation usually relies on the construction of cell and animal models. The cell model is to culture esophageal epithelial cells in vitro, expose them to different doses of ionizing radiation, and observe processes such as cell viability, DNA damage, and repair. The animal model is to perform local or whole-body radiation exposure on experimental animals (such as mice or rats) to simulate common complications such as radioactive esophagitis in clinical practice, in order to study its pathophysiological changes and treatment methods. However, the cell model lacks the complexity of tissue structure and cannot fully reflect the in-vivo environment; although the animal model can provide a more physiological response closer to that of humans, there are species differences and ethical issues. Summary of the Invention

[0004] Aiming at the problems of the prior art, the present invention provides a method for constructing a radioactive esophageal injury organoid model. The construction of this radioactive esophageal injury organoid model is of great significance for deeply understanding the injury mechanism of ionizing radiation to the esophagus, finding effective protection and treatment measures, and promoting the development of personalized radiotherapy.

[0005] A method for constructing a radioactive esophageal injury organoid model includes performing X-ray ionizing radiation on esophageal organoids; wherein, the esophageal organoids include rat esophageal organoids, mouse esophageal organoids, or human esophageal organoids;

[0006] The radiation dose is 0.5 - 4 Gy, and the radiation dose rate is 1.5 - 2 Gy / min.

[0007] According to the experimental results, in human esophageal organoids, mouse esophageal organoids, and rat esophageal organoids, as the radiation dose increases, the connections around the organoid spheres become loose, the cell debris increases, and the volume is also smaller than that of normal organoids at the same time. The construction of this organoid model of radiation-induced esophageal injury is of great significance for deeply understanding the injury mechanism of ionizing radiation on the esophagus, finding effective protective and treatment measures, and promoting the development of personalized radiotherapy.

[0008] Organoids are three-dimensional microstructures cultured from stem cells or progenitor cells, which can simulate the complexity and functional characteristics of real esophageal tissues in vitro. Through the organoid model, researchers can more precisely reproduce the effects of ionizing radiation on the esophagus, conduct detailed mechanism studies, drug screening, and personalized treatment evaluations. In addition, the application of the organoid model can reduce the dependence on experimental animals, comply with ethical norms, and be more cost-effective. Therefore, the construction of the organoid model of radiation-induced esophageal injury not only makes up for the deficiencies of existing models but also provides an innovative and powerful platform for in-depth research and clinical application of radiation injury.

[0009] Furthermore, the radiation dose is 1 - 3 Gy. The inventors initially explored six dose concentrations of 0, 4, 8, 12, 16, and 20 Gy and found that the esophageal organoids were significantly inhibited and did not grow at 4 Gy. Subsequently, the doses explored were 0, 0.5, 1, 2, and 4 G. Esophageal organoids are solid spherical structures. After irradiation, some esophageal organoids are damaged and show hollow structures, and the connections around the organoid spheres become loose, with an increase in cell debris. Preferably, irradiation at 1 - 3 Gy is more suitable for subsequent experiments. For example, 1 Gy, 2 Gy, 3 Gy.

[0010] Furthermore, the esophageal organoids are esophageal organoids after culturing to the P3 generation.

[0011] Furthermore, the esophageal organoids are obtained through the following steps of culturing:

[0012] Take the adjacent tissue of human esophageal squamous cell carcinoma, the esophagus of 6 - 8-week-old male mice, or the esophagus of 4 - 6-week-old male rats, and wash them with PBS solution;

[0013] Cut the washed esophageal tissue into pieces;

[0014] Digest the cut esophageal tissue with the first digestive solution for the first digestion;

[0015] Add the second digestive solution to the esophageal tissue after the first digestion for the second digestion to obtain single esophageal tissue cells, terminate the digestion, filter, wash, centrifuge, resuspend the precipitated cells, and perform cell culture to obtain esophageal organoids after culturing to the P3 generation.

[0016] Furthermore, the first digestive solution includes Collagenase TypeⅠ, PBS, and Antibiotic - Antimycotic.

[0017] Furthermore, the second digestive solution includes TrypLE TM Express.

[0018] Furthermore, every 50 mL of mouse or rat esophageal organoid culture medium includes:

[0019] 0.8 mL - 1.2 mL of B27 additive diluted 40 - 60 times;

[0020] 40 - 60 μL of epidermal growth factor at 40 - 60 ng / ml;

[0021] 12 - 13 μL of R - spondin 1 at 200 - 300 ng / ml;

[0022] 200 - 300 μL of fibroblast growth factor 10 at 400 - 600 ng / ml;

[0023] 40 - 60 μL of Y - 27632 molecule at 8 - 12 μM;

[0024] 400 - 600 μL of GlutaMAX diluted 80 - 120 times TM Medium supplement;

[0025] 40 - 60 μL of leucine gastrin at 0.8 - 1.2 nM;

[0026] 80 - 120 μL of N - acetylcysteine at 0.8 - 1.2 mM;

[0027] 40 - 60 μL of noggin at 80 - 120 ng / ml;

[0028] 40 - 60 μL of A83 - 01 inhibitor at 150 - 250 nM;

[0029] 400 - 600 μL of nicotinamide at 8 - 12 mM;

[0030] 200 - 300 μL of Wnt - 3a protein at 40 - 60 ng / ml;

[0031] 400 - 600 μL of N2 medium diluted 80 - 120 times;

[0032] 400 - 600 μL of antibiotic - antimycotic mixture diluted 80 - 120 times;

[0033] 400 - 600 μL of 8 - 12 mM HEPES buffer;

[0034] 15 - 20 μL of 8 - 12 μM SB202190 inhibitor.

[0035] The present invention provides a culture medium for culturing and / or passaging esophageal organoids of mice and rats. This culture medium is used to culture esophageal organoids of mice and rats, and has the advantages of high cell survival rate and multiple passages. Both N2 and FGF10 are essential. On the existing basis, esophageal organoids of mice and rats can be passaged within 15 generations.

[0036] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0037] The present invention provides a method for constructing a radioactive esophageal injury organoid model, which includes performing X - ray ionizing radiation on esophageal organoids. Among them, the esophageal organoids include rat esophageal organoids, mouse esophageal organoids or human esophageal organoids; the radiation dose is 0.5 - 4 Gy, and the radiation dose rate is 1.5 - 2 Gy / min. From the experimental results, it can be seen that for human esophageal organoids, mouse esophageal organoids and rat esophageal organoids, as the irradiation dose increases, the connection around the organoid spheres becomes loose, the cell debris increases, and the volume is also smaller than that of normal organoids at the same time. The construction of this radioactive esophageal injury organoid model is of great significance for deeply understanding the injury mechanism of ionizing radiation on the esophagus, finding effective protection and treatment measures, and promoting the development of personalized radiotherapy. Brief Description of the Drawings

[0038] Figure 1 It is a diagram showing the growth of mouse esophageal organoids.

[0039] Figure 2 It is a diagram showing the growth of mouse esophageal organoids after ionizing radiation.

[0040] Figure 3 It is a diagram showing the growth of rat esophageal organoids.

[0041] Figure 4 It is a diagram showing the growth of rat esophageal organoids after ionizing radiation.

[0042] Figure 5 It is a diagram showing the growth of human esophageal organoids.

[0043] Figure 6 It is a diagram showing the growth of human esophageal organoids after ionizing radiation. Detailed Embodiments

[0044] The following will describe the present invention in detail with reference to the drawings.

[0045] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] The specific description of the materials in the embodiments is as follows:

[0047] All the experimental reagents used in this embodiment are prior art and are commercially available products.

[0048] Specifically, the C57BL / 6 and SD rats are conventional experimental animals, which are commercially available products and are purchased from Chengdu Dashuo Experimental Animal Co., Ltd.; the human esophageal organoid medium is purchased from bioGenous, and the product number is K2040-HE; TrypLE TM Express is purchased from Thermo Fisher Scientific, and the product number is 12605-010; Collagenase TypeⅠ is purchased from Thermo Fisher Scientific, and the product number is 17100-017.

[0049] Specifically, a medium for culturing and / or passaging esophageal organoids of mice and rats in this embodiment consists of Advanced DMEM / F12 medium, B27 (B27 additive), EGF (epidermal growth factor), R-spondin1 (R-cell protein 1), FGF10 (fibroblast growth factor 10), Y-27632 (Y-27632 molecule), GlutaMAX (GlutaMAX TMCulture medium supplement), LeuGastrin, N-acetyl-l-cystein, Noggin, A8301 (A8301 inhibitor), Nicotinamide, Wnt-3a (Wnt-3a protein), N2 (N2 medium), Antibiotic-Antimycotic, HEPES buffer and SB202190 (SB202190 inhibitor). Specifically, Advanced DMEM / F-12 is a widely used basal medium that can culture mammalian cells with reduced fetal bovine serum supplementation. It is purchased from Thermo Fisher Scientific, with the product number 12634-010; B27 is an optimized serum-free additive, purchased from Thermo Fisher Scientific, with the product number 17504-044; Epidermal growth factor EGF is a growth factor that can promote the growth of organoids. It is purchased from bioGenous, with the product number 568-EGF-0100; R-spondin1 is a secreted protein that, when used in combination with Noggin and EGF, maintains the proliferation of stem cells and the growth of organoids. It is purchased from bioGenous, with the product number 861-RS1-1000; FGF10 is an important factor for culturing many organoids and can promote the proliferation of epithelial and mesenchymal cells. It is purchased from bioGenous, with the product number 816-FGF-0100; Y-27632 is a commonly used ROCK inhibitor that can inhibit stress-induced cell death and maintain cell proliferation ability. It is purchased from Sigma-Aldrich, with the product number Y0503; Glutamax is a substitute for L-glutamine with better stability and can improve cell health. It is purchased from Thermo Fisher Scientific, with the product number 35050-061; LeuGastrin is a synthetic analogue of gastrin that can promote the proliferation of gastric mucosal cells, maintain the integrity of the gastric wall structure to improve stability or alter biological activity. It is purchased from Sigma-Aldrich, with the product number G9145; N-acetyl-l-cystein is an acetylated form of cysteine that enhances the antioxidant capacity of cells, supports cell survival and normal growth. It is purchased from Sigma-Aldrich, with the product number A5099;Noggin is a secreted glycoprotein, often used in combination with Wnt activators (such as R-spondin) and other factors to regulate the differentiation and proliferation of stem cells, help maintain the three-dimensional structure and functional characteristics of organoids, purchased from bioGenous, product number 807-NOG-0100; A83-01 is an ALK5 inhibitor, which can be used in combination with Wnt, Noggin and R-spondin to maintain the proliferative state of intestinal epithelial stem cells and avoid differentiation, purchased from Tocris, product number 2939; Nicotinamide is a form of vitamin B3 or niacin, purchased from Sigma-Aldrich, product number N0636; Wnt-3a is a secreted glycoprotein that stimulates the proliferation of stem cells, enhances the expansion of stem cells and maintains the undifferentiated state, purchased from bioGenous, product number RWL003-0010; N2 is a basal culture medium containing various vitamins, minerals, amino acids and other factors that help cell growth, often used as a supplementary culture medium, purchased from Thermo Fisher Scientific, product number 17502-048; Antibiotic-Antimycotic is an antibiotic mixture commonly used in cell culture to inhibit the growth of bacteria, fungi and other microorganisms, help maintain a sterile environment and prevent microbial contamination during cell culture, purchased from Thermo Fisher Scientific, product number 15240-062; HEPES buffer is a pH stabilizer, usually used to maintain the pH value of the culture medium or solution to ensure that cells and other biological samples grow and react within the optimal pH range, purchased from Thermo Fisher Scientific, product number 15630-106; SB202190 is a selective p38 MAPK inhibitor, purchased from Sigma-Aldrich, product number S7067.;

[0050] In some embodiments, every 50 mL of mouse or rat esophageal organoid culture medium comprises: 0.8 mL to 1.2 mL of 40-60-fold diluted B27 additive; 40-60 μL of 40-60 ng / ml epidermal growth factor; 12-13 μL of 200-300 ng / ml R-spondin 1; 200-300 μL of 400-600 ng / ml fibroblast growth factor 10; 40-60 μL of 8-12 μM Y-27632 molecule; 400-600 μL of 80-120-fold diluted GlutaMAX TMCulture medium supplement; 40 - 60 μL of 0.8 - 1.2 nM leucine gastrin; 80 - 120 μL of 0.8 - 1.2 mM N-acetylcysteine; 40 - 60 μL of 80 - 120 ng / ml noggin protein; 40 - 60 μL of 150 - 250 nM A8301 inhibitor; 400 - 600 μL of 8 - 12 mM nicotinamide; 200 - 300 μL of 40 - 60 ng / ml Wnt-3a protein; 400 - 600 μL of N2 medium diluted 80 - 120 times; 400 - 600 μL of antibiotic-antifungal mixture diluted 80 - 120 times; 400 - 600 μL of 8 - 12 mM HEPES buffer; 15 - 20 μL of 8 - 12 μM SB202190 inhibitor.

[0051] Example 1

[0052] For the construction of a radioactive mouse esophageal organoid model, the specific steps are as follows:

[0053] Two male C57BL / 6 mice were sacrificed by cervical dislocation. The whole body was sprayed with alcohol, the limbs were fixed, and the esophageal tissue was taken out and placed in 2% double-antibody PBS, rinsed repeatedly several times to wash away the blood;

[0054] The washed esophageal tissue was minced and added to a digestive solution prepared from Collagenase TypeⅠ(1 mg / ml), PBS, and Antibiotic-Antimycotic (1% vol / vol). It was shaken and digested at 37°C on a shaker at 100 rpm for 60 min, and then centrifuged at 2000 rpm for 5 min;

[0055] 5 mL of TrypLE TM Express was added to resuspend the precipitate, and it was shaken and digested at 37°C on a shaker at 100 rpm for 15 min;

[0056] An equal volume of Advanced DMEM / F12 containing FBS (10% vol / vol) was used to neutralize TrypLE TM Express to terminate digestion. The tissue suspension was filtered through a 70 μm filter twice and centrifuged at 2000 rpm for 5 min;

[0057] Red blood cell lysate was added, and the red blood cells were lysed at 4°C and centrifuged at 2000 rpm for 5 min;

[0058] Pre-cooled DPBS was added to wash 1 - 2 times and centrifuged at 2000 rpm for 5 min;

[0059] The supernatant was discarded, and Matrigel was added to resuspend the precipitated cells on ice, trying to avoid air bubbles as much as possible. The cells were seeded at 50 μm / well in a low-attachment 24-well culture plate and left to stand in the incubator for 15 min;

[0060] Add 500 μL of pre-warmed self-prepared mouse esophageal organoid medium to each well, and then culture it in a cell incubator at 37 °C and 5% CO2. Replace the medium every 2 - 3 days. Organoids can be seen after 5 - 7 days of culture.

[0061] The growth of mouse esophageal organoids cultured according to the method provided in this example is as Figure 1 shown. Starting from a nearly single-cell state, it gradually grows larger and forms translucent spherical structures in about 3 days; by 4 - 9 days, the spheroids gradually grow into clearly visible solid sac-like structures, and the color gradually deepens. The growth state is good throughout the process.

[0062] Among them, every 50 mL of mouse or rat esophageal organoid medium contains 1 mL of 50-fold diluted B27; 50 μL of 50 ng / ml EGF; 12.5 μL of 250 ng / ml R-spondin1; 250 μL of 500 ng / ml FGF10; 50 μL of 10 μM Y-27632; 500 μL of 100-fold diluted GlutaMAX; 50 μL of 1 nM LeuGastrin; 100 μL of 1 mM N-acetyl-l-cystein; 50 μL of 100 ng / ml Noggin; 50 μL of 200 nM A8301; 500 μL of 10 mM Nicotinamide; 250 μL of 50 ng / ml Wnt-3a; 500 μL of 100-fold diluted N2; 500 μL of 100-fold diluted Antibiotic-Antimycotic; 500 μL of 10 mM HEPES; 17 μL of 10 μM SB202190.

[0063] After successfully culturing the P3 generation of mouse esophageal organoids, the organoids on the first day after passage were randomly divided into 0.5, 1, 2, 4 Gy irradiation groups and a control group (0 Gy). Among them, the organoids in the irradiation groups were placed in a biological X-ray irradiator KUBTEC XCELL320 with a dose rate of 1.7 Gy / min and doses of 0.5, 1, 2, 4 Gy, and the control group was sham-irradiated (the organoid culture plates were brought to the irradiation room but not irradiated).

[0064] Observe the morphological changes of the cell spheres under a microscope and take pictures at 2, 4, 6 d after irradiation.

[0065] The growth of mouse esophageal organoids after ionizing radiation cultured according to the method provided in this example is as Figure 2 shown. With the increase of the irradiation dose, the connections around the mouse esophageal organoid spheres become loose, the cell debris increases, and the volume is also smaller than that of normal organoids at the same time.

[0066] Example 2

[0067] For the construction of a radioactive rat esophageal organoid model, the specific steps are as follows:

[0068] 1) Sacrifice 2 male SD rats by cervical dislocation, spray the whole body with alcohol, fix the four limbs, put the esophageal tissue into 2% double-antibody PBS after taking it out, rinse several times repeatedly, and wash away the blood;

[0069] 2) Cut the washed esophageal tissue into pieces, add digestive juice prepared from Collagenase TypeⅠ(1mg / ml), PBS and Antibiotic-Antimycotic(1% vol / vol), shake in a 37℃ shaker at 100rpm for 60min, and centrifuge at 2000rpm for 5min;

[0070] 3) Add 5mL TrypLE TM Express to resuspend the precipitate, shake in a 37℃ shaker at 100rpm for 15min;

[0071] 4) Neutralize TrypLE TM Express with an equal volume of Advanced DMEM / F12 containing FBS(10% vol / vol) to terminate digestion, filter the tissue suspension through a 70μm filter twice, and centrifuge at 2000rpm for 5min;

[0072] 5) Add red blood cell lysate, lyse red blood cells at 4℃, and centrifuge at 2000rpm for 5min;

[0073] 6) Add pre-cooled DPBS to wash 1-2 times, and centrifuge at 2000rpm for 5min;

[0074] 7) Discard the supernatant, add Matrigel to resuspend the precipitated cells on ice, try to avoid air bubbles as much as possible, inoculate at 50μm / well in a low-attachment 24-well culture plate, and place it in the incubator to stand for 15min;

[0075] 8) Add 500μL of self-prepared rat esophageal organoid medium preheated in advance to each well, and then culture in a cell culture incubator at 37℃ and 5% CO2. Replace the medium every 2-3 days, and organoids can be seen after culturing for 5-7 days.

[0076] The growth of rat esophageal organoids cultured according to the method provided in this example is as Figure 3 shown. Starting from a nearly single-cell state, it gradually grows and forms a translucent spherical structure in about 3 days; by 4-7 days, the small sphere gradually grows into a clearly visible solid cyst, and the color gradually deepens. The growth state is good throughout the process.

[0077] 9) After successfully culturing the P3-generation rat esophageal organoids, the organoids on the 1st day after subculture were randomly divided into 0.5, 1, 2, 4 Gy irradiation groups and a control group (0 Gy). Among them, the organoids in the irradiation groups were placed in a biological X-ray irradiator KUBTEC XCELL320 with a dose rate of 1.7 Gy / min and doses of 0.5, 1, 2, 4 Gy, and the control group was subjected to sham irradiation (the organoid culture plates were taken to the irradiation room but not irradiated).

[0078] 10) At 2, 4, 6 d after irradiation, the morphological changes of the cell spheres were observed under a microscope and photographed.

[0079] The growth of the rat esophageal organoids after ionizing radiation cultured according to the method provided in this example is as Figure 4 shown. With the increase of the irradiation dose, the connections around the spheres of the rat esophageal organoids became loose, the cell debris increased, and the volume was also smaller than that of the normal organoids at the same time.

[0080] Example 3

[0081] For the construction of a radioactive human esophageal organoid model, the specific steps are as follows:

[0082] 1) The adjacent tissue of human esophageal squamous cell carcinoma freshly resected in the operating room was placed in ice-precooled Advanced DMEM / F12 medium supplemented with double antibiotics (1% vol / vol) and quickly transported to the laboratory for subsequent operations;

[0083] 2) The washed esophageal tissue was minced and added to a digestive solution prepared from Collagenase TypeⅠ(1 mg / ml), PBS, and Antibiotic-Antimycotic (1% vol / vol). It was digested on a shaker at 37 °C with a shaking speed of 100 rpm for 60 min and then centrifuged at 2000 rpm for 5 min;

[0084] 3) 5 mL of TrypLE TM Express was added to resuspend the precipitate, and it was digested on a shaker at 37 °C with a shaking speed of 100 rpm for 15 min;

[0085] 4) An equal volume of Advanced DMEM / F12 containing FBS (10% vol / vol) was used to neutralize TrypLE TM Express to terminate digestion. The tissue suspension was filtered through a 70-μm filter twice and centrifuged at 2000 rpm for 5 min;

[0086] 5) The precipitate was washed 1-2 times with pre-cooled DPBS and centrifuged at 2000 rpm for 5 min;

[0087] 6) Discard the supernatant, add Matrigel to resuspend the precipitated cells on ice, try to avoid air bubbles, seed the cells at 50 μm / well in a low-attachment 24-well culture plate, and place it in the incubator for 15 min of static incubation;

[0088] 7) Add 500 μL of pre-warmed human esophageal organoid medium to each well, and then culture it in a cell culture incubator at 37 °C and 5% CO2. Replace the medium every 2 - 3 days. Organoids can be seen after 5 - 7 days of culture.

[0089] The growth of human esophageal organoids cultured according to the method provided in this example is as Figure 5 shown. Starting from a nearly single-cell state, it gradually grows larger to form a translucent spherical structure in about 3 days; by 4 - 9 days, the spheroid gradually grows into a clearly visible solid sac-like structure, and the color gradually deepens. The growth state is good throughout the process.

[0090] 8) Randomly divide the organoids on the 1st day after passage of successfully cultured P3-generation human esophageal organoids into 0.5, 1, 2, 4 Gy irradiation groups and a control group (0 Gy). Among them, the organoids in the irradiation groups are placed in a biological X-ray irradiator KUBTEC XCELL320 with a dose rate of 1.7 Gy / min and doses of 0.5, 1, 2, 4 Gy, and the control group is subjected to sham irradiation (bringing the organoid culture plate to the irradiation room but not irradiating).

[0091] 9) Observe the morphological changes of the cell spheres under a microscope and take pictures at 2, 4, 6 d after irradiation.

[0092] The growth of human esophageal organoids after ionizing radiation cultured according to the method provided in this example is as Figure 6 shown. With the increase of the irradiation dose, the connections around the human esophageal organoid spheres become loose, the cell debris increases, and the volume is also smaller than that of normal organoids at the same time.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a radiation-induced esophageal injury organoid model, characterized in that: This involves subjecting the esophageal organoids to ionizing X-ray radiation; The esophageal organoids include rat esophageal organoids, mouse esophageal organoids, or human esophageal organoids; The radiation dose is 0.5-4Gy, and the radiation dose rate is 1.5-2Gy / min.

2. The method for constructing a radiation-induced esophageal injury organoid model according to claim 1, characterized in that: The radiation dose is 1-3 Gy.

3. The method for constructing a radiation-induced esophageal injury organoid model according to claim 1, characterized in that: Esophageal organoids are esophageal organoids cultured at the P3 generation.

4. The method for constructing a radiation-induced esophageal injury organoid model according to claim 3, characterized in that: Esophageal organoids were cultured using the following steps: The paracancerous tissue of human esophageal squamous cell carcinoma, the esophagus of 6-8 week-old male mice, or the esophagus of 4-6 week-old male rats were washed with PBS solution; Cut the cleaned esophageal tissue into small pieces; Performing a first digestion process on the chopped esophageal tissue using a first digestive fluid; A second digestion solution is added to the esophageal tissue after the first digestion treatment for a second digestion treatment to obtain single esophageal tissue cells, the digestion is terminated, the cells are filtered, washed, centrifuged, the precipitated cells are resuspended, and cell culture is performed to obtain esophageal organoids after the P3 generation of culture.

5. The method for constructing a radiation-induced esophageal injury organoid model according to claim 4, characterized in that: The first digestion solution includes Collagenase Type I, PBS and Antibiotic-Antimycotic.

6. The method for constructing a radiation-induced esophageal injury organoid model according to claim 4, characterized in that: The second digestion solution includes TrypLE TM Express.

7. The method for constructing a radiation-induced esophageal injury organoid model according to claim 4, characterized in that: Each 50 mL of mouse or rat esophageal organoid culture medium includes: 0.8mL to 1.2mL of 40-60 times diluted B27 additive; 40-60 μL of 40-60 ng / ml epidermal growth factor; 12-13 μL of 200-300 ng / ml R-cell protein 1; 200-300 μL of 400-600 ng / ml fibroblast growth factor 10; 40-60 μL of 8-12 μM Y-27632 molecules; 400-600 μL of 80-120-fold diluted GlutaMAX TM Culture medium supplement; 40-60 μL of 0.8-1.2 nM leucine gastrin; 80-120 μL of 0.8-1.2 mM N-acetylcysteine; 40-60 μL of 80-120 ng / ml Nogin protein; 40-60 μL of 150-250 nM A8301 inhibitor; 400-600 μL of 8-12 mM nicotinamide; 200-300 μL of 40-60 ng / ml Wnt-3a protein; 400-600 μL of 80-120-fold diluted N2 medium; 400-600 μL of 80-120 times diluted antibiotic-antifungal mixture; 400-600 μL of 8-12 mM HEPES buffer; 15-20 μL of 8-12 μM SB202190 inhibitor.

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