Screening method for cell-embedded death agonists or antagonists and use thereof

Abstract

This invention relates to a method for screening cell-embedded cell death agonists or antagonists and its application. The screening method includes: using adherent HEK293T cells as a screening model, co-culturing them with candidate substances, calculating the ratio of the number of cells undergoing embedded cell death to the total number of cells, and screening for cell-embedded cell death agonists or antagonists. This invention creatively uses the adherent HEK293T cell line instead of the conventional suspension breast cancer cell line MCF-7 as a cell model for embedded cell death research. This allows for easy observation under a microscope and requires a very short time; observation can be performed approximately 1 hour after the candidate substances are added, making it suitable for high-throughput drug screening. This invention has also used this screening method to screen a series of novel cell-embedded cell death agonists and antagonists.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a method for screening cell-embedded cell death agonists or antagonists and their applications. Background Technology

[0002] Cell-intercalation death is a phenomenon where a living cell enters the cytoplasm of another living cell. Scientists first discovered this phenomenon in cancer cells as early as 1891, but due to its extremely low occurrence rate, it was often overlooked, leading to a lag in research. In intercalation death, the inner cell is called the inner cell, and the outer cell is called the outer cell. Normally, the inner cell is digested and absorbed by the outer cell, hence the term "cell cannibalism." In tumor research, it has been found that the probability of intercalation death increases as cells approach the late stage. Studying the mechanism of this phenomenon will provide insights for cancer drug development. In the past decade, researchers have discovered that the MCF7 breast cancer cell line undergoes extensive intercalation death in suspension culture. Using this research model, the Rho-ROCK pathway and the cell junction protein cadherin have been confirmed to be related to intercalation death. Small molecule chemotherapy drugs such as doxorubicin and nintedanib can increase the probability of cell-intercalation death. Small molecules such as nocodazole and paplitaxel fix cells in the mid-stage; their round shape makes it easier for cells to enter another cell, thus promoting intercalation death. Additionally, the AMPK inhibitor compound C can reduce the probability of intercalation death.

[0003] Although there is a certain research foundation for cell embedded cell death, there is still room for technological advancement and the research approach needs improvement. First, there is a lack of a simple and rapid screening method. In the field of embedded cell death research, the suspension breast cancer cell line MCF-7 is often used as an experimental subject, but suspension cells are not suitable for microscopic observation, which limits the speed of drug screening. Second, the types of cell embedded cell death agonists or antagonists discovered so far are relatively few. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for screening cell-embedded cell death agonists or antagonists and their applications.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for screening cell-embedded death agonists or antagonists, the screening method comprising: using adherent HEK293T cells as a screening model, co-culturing them with candidate substances, calculating the ratio of the number of cells undergoing embedded death to the total number of cells, and screening to obtain cell-embedded death agonists or antagonists.

[0007] In previous studies of embedded cell death, the suspended breast cancer cell line MCF-7 was commonly used as the experimental subject. However, suspended cells are not suitable for microscopic observation, limiting the speed of drug screening. This invention creatively uses the adherent HEK293T cell line (human embryonic kidney cell 293) instead. HEK293T cells themselves do not undergo embedded cell death, but HEK293T cells overexpressing protein kinase B undergo massive adherent embedded cell death under the stimulation of an agonist. Ordinary cells enter the cells overexpressing protein kinase B, which is easily observed under a microscope under the tracking of fluorescent proteins, and can be used for high-throughput drug screening.

[0008] Preferably, the screening method includes the following steps:

[0009] (1) Mix ordinary HEK293T cells with HEK293T cells overexpressing protein kinase B and culture until they adhere to the culture vessel;

[0010] (2) The candidate material is then mixed with the adherent cells obtained in step (1) and co-incubated.

[0011] (3) After co-incubation, observe with a fluorescence microscope and calculate the ratio of the number of cells that undergo embedded death to the total number of cells (i.e., embedded death rate = (a+b) / (a+b+c)%, where a = number of phagocytes, b = number of phagocytes, c = number of all other cells, a+b+c>200), and screen for cell embedded death agonists or antagonists.

[0012] Specifically, the screening method for the cell-embedded death agonist includes the following steps:

[0013] (S1) Mix ordinary HEK293T cells with HEK293T cells overexpressing protein kinase B and culture until they adhere to the culture vessel;

[0014] (S2) Then mix the cell-embedded death agonist candidate with the adherent cells obtained in step (1) and co-incubate them;

[0015] (S3) After co-incubation, observe with a fluorescence microscope and calculate the ratio of the number of cells that undergo embedded death to the total number of cells (i.e., embedded death rate = (a+b) / (a+b+c)%), where a = number of phagocytes, b = number of phagocytes, c = number of all other cells, a+b+c>200), and screen for cell embedded death agonists.

[0016] The judgment criterion is: if the above ratio exceeds that of the control group (the group without co-incubation with candidate substances), then the candidate substance can be considered to be a cell-embedded death agonist.

[0017] Specifically, the screening method for the cell-embedded cell death antagonist includes the following steps:

[0018] (T1) Mix ordinary HEK293T cells with HEK293T cells overexpressing protein kinase B and culture until they adhere to the culture vessel;

[0019] (T2) Then mix the cell-embedded death agonist with the adherent cells obtained in step (1) and culture for 20-30 min. Then mix the cell-embedded death antagonist candidate with it and co-incubate.

[0020] (T3) After co-incubation, the cells were observed with a fluorescence microscope, and the ratio of the number of cells that underwent embedded death to the total number of cells was calculated (i.e., embedded death rate = (a+b) / (a+b+c)%), where a = number of phagocytes, b = number of phagocytes, c = number of all other cells, and a+b+c>200). Cell embedded death agonists were then screened.

[0021] The judgment criterion is: if the above ratio is lower than that of the control group (the group that was only incubated with the agonist mixture without the candidate substance), then the candidate substance can be considered to be a cell-embedded death antagonist.

[0022] Preferably, the ratio of ordinary HEK293T cells to HEK293T cells overexpressing protein kinase B in step (1) is 1:10-10:1, such as 1:10, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1 or 10:1, etc. Any specific point value within the above range can be selected, and will not be elaborated here.

[0023] Here, ordinary HEK293T cells (with or without fluorescent protein labeling) are co-cultured with HEK293T cells that overexpress protein kinase B and are labeled with fluorescent protein. When cell inlay death occurs, the cells forming hollow structures can be identified under a microscope and with the naked eye and a camera, that is, the phenomenon of cell inlay death is observed.

[0024] Preferably, the HEK293T cells overexpressing protein kinase B are labeled with a fluorescent protein.

[0025] Preferably, the fluorescent protein includes any fluorescent protein, including GFP.

[0026] Preferably, the protein kinase B includes AKT1, AKT2, or AKT3, with AKT1 being the most preferred.

[0027] Preferably, the mixed culture time in step (1) is 12-48h, such as 12h, 16h, 20h, 24h, 28h, 32h, 42h or 48h, etc. Any specific point value within the above range can be selected, and will not be described in detail here.

[0028] Preferably, the screening concentration of the candidate substance in step (2) is 1nM-5mM, such as 1nM, 10nM, 50nM, 100nM, 500nM, 1μM, 10μM, 50μM, 100μM, 500μM, 1mM, 2mM, 3mM, 4mM or 5mM, etc. Any specific point value within the above range can be selected, and will not be described in detail here.

[0029] Preferably, the co-incubation time in step (2) is 20-90 min, such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 60 min or 90 min, etc. Any specific point value within the above range can be selected, and will not be described in detail here.

[0030] Preferably, in order to further ensure the accuracy of the experiment, the cells will be fixed and stained after the co-incubation in step (3).

[0031] Preferably, the fixation is performed using 4-10% (e.g., 4%, 6%, 8% or 10%) of formalin.

[0032] Preferably, the staining agents used include nuclear staining reagents such as HOECHST 33342.

[0033] According to the screening method for cell-embedded death agonists or antagonists described in the first aspect, the present invention screened a large number of candidate cell-embedded death agonists or antagonists, and screened out the novel cell-embedded death agonists and cell-embedded death antagonists described below.

[0034] In a second aspect, the present invention provides the use of nocodazole or its salts, mebendazole or its salts, oligomycin or its salts, FCCP or its salts, CCCP or its salts, KX2-391 or its salts, GNE-2861 or its salts, ENMD-2076 or its salts, PF-3758309 or its salts, tetrandrine or its salts, 3-norcolchicine or its salts, octendaazole or its salts, PF-04554878 or its salts, PF-06409577 or its salts, compressorine phosphate, and compressorine disodium phosphate in the preparation of cell-embedded death agonists, wherein the cell-embedded death agonists are obtained by the screening method described in the first aspect.

[0035] Specifically, substances that can be used as cell-embedded cell death agonists include nocodazole, pharmaceutical salts of nocodazole, mebendazole, pharmaceutical salts of mebendazole, oligomycin, pharmaceutical salts of oligomycin, FCCP, pharmaceutical salts of FCCP, CCCP or pharmaceutical salts of CCCP, KX2-391 or pharmaceutical salts thereof, GNE-2861 or pharmaceutical salts thereof, ENMD-2076 or pharmaceutical salts thereof, PF-3758309 or pharmaceutical salts thereof, tetrandrine or pharmaceutical salts thereof, 3-norcolchicine or pharmaceutical salts thereof, ocbendazole or pharmaceutical salts thereof, PF-04554878 or pharmaceutical salts thereof, PF-06409577 or pharmaceutical salts thereof, compressoritine phosphate, and compressoritine disodium phosphate.

[0036] CCCP, short for Carbonyl cyanide 3-chlorophenylhydrazone, is a proton carrier and a potent uncoupling agent for mitochondrial oxidative phosphorylation. It increases the permeability of the mitochondrial inner membrane to H+, leading to the loss of membrane potential across the mitochondrial inner membrane and inducing apoptosis. Its molecular formula is C9H5ClN4, and its molecular weight is 204.62.

[0037] FCCP stands for Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, a mitochondrial oxidative phosphorylation uncoupling agent. It can damage the inner mitochondrial membrane, causing mitochondrial membrane depolarization and proton leakage, thereby increasing oxygen consumption without affecting cellular phosphorylation and significantly reducing ATP production.

[0038] Preferably, the cell-embedded death agonist further includes pharmaceutical excipients.

[0039] Preferably, the pharmaceutical excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer. The combination of at least two components includes, for example, a combination of carrier and diluent, a combination of excipient and filler, a combination of binder and wetting agent, etc. Other combinations are also possible and will not be elaborated further here.

[0040] The cell-embedded cell death agonist described in this invention can be administered alone or in combination with excipients to form an appropriate dosage form. When the dosage form is a tablet, it may contain excipients such as microcrystalline cellulose, starch, or calcium carbonate; it may also contain disintegrants such as croscarmellose sodium. When the dosage form is a capsule, it can be prepared as a hard capsule or a soft capsule, and the aforementioned cell-embedded cell death agonist and excipients can be prepared as powder or granules and filled into the capsule. When the dosage form is a suspension, flavoring agents, suspending agents, etc., can be added to adjust the taste and mouthfeel. When the dosage form is an emulsion, emulsifiers and solubilizers can be added appropriately to adjust the solubility and emulsification degree for administration.

[0041] Thirdly, the present invention also provides a method for promoting embedded cell death, the method comprising: administering to cells any one or a combination of at least two of the following drugs: nocodazole or a salt thereof, mebendazole or a salt thereof, oligomycin or a salt thereof, FCCP or a salt thereof, CCCP or a salt thereof, KX2-391 or a salt thereof, GNE-2861 or a salt thereof, ENMD-2076 or a salt thereof, PF-3758309 or a salt thereof, tetrandrine or a salt thereof, 3-norcolchicine or a salt thereof, ocbendazole or a salt thereof, PF-04554878 or a salt thereof, PF-06409577 or a salt thereof, compressoritine phosphate, and compressoritine disodium phosphate.

[0042] The above method is not used for the diagnosis or treatment of diseases. It is used to explore the mechanism of embedded cell death and to provide a basis for the development of tumor drugs.

[0043] Fourthly, the present invention provides the use of cysteine ​​or its salts, glutathione or its salts, acetylcysteine ​​or its salts, paclitaxel or its salts in the preparation of cell-embedded death antagonists, wherein the cell-embedded death antagonists are obtained by the screening method described in the first aspect.

[0044] Specifically, cell-embedded death antagonists include cysteine, pharmaceutical salts of cysteine, glutathione, pharmaceutical salts of glutathione, acetylcysteine, pharmaceutical salts of acetylcysteine, paclitaxel, or pharmaceutical salts of paclitaxel.

[0045] Preferably, the cell-embedded death antagonist further includes pharmaceutical excipients.

[0046] Preferably, the pharmaceutical excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer. The combination of at least two components includes, for example, a combination of carrier and diluent, a combination of excipient and filler, a combination of binder and wetting agent, etc. Other combinations are also possible and will not be elaborated further here.

[0047] The cell-embedded cell death antagonist described in this invention can be administered alone or in combination with excipients to form an appropriate dosage form. When the dosage form is a tablet, it may contain excipients such as microcrystalline cellulose, starch, or calcium carbonate; it may also contain disintegrants such as croscarmellose sodium. When the dosage form is a capsule, it can be prepared as a hard capsule or a soft capsule, and the aforementioned cell-embedded cell death antagonist and excipients can be prepared as powder or granules and filled into the capsule. When the dosage form is a suspension, flavoring agents, suspending agents, etc., can be added to adjust the taste and mouthfeel. When the dosage form is an emulsion, emulsifiers and solubilizers can be added appropriately to adjust the solubility and emulsification degree for administration.

[0048] Fifthly, the present invention also provides a method for inhibiting embedded cell death, characterized in that the method comprises: administering to cells any one or a combination of at least two of the following drugs: cysteine ​​or a salt thereof, glutathione or a salt thereof, acetylcysteine ​​or a salt thereof, paclitaxel or a salt thereof.

[0049] The above method is not used for the diagnosis or treatment of diseases. It is used to explore the mechanism of embedded cell death and to provide a basis for the development of tumor drugs.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This invention creatively uses the adherent HEK293T cell line (human embryonic kidney cells 293) instead of the conventional suspension breast cancer cell line MCF-7 as a cell model for embedded cell death research. This model is very easy to observe under a microscope and requires very little time; observation can be performed approximately 1 hour after the candidate substance is added, making it suitable for high-throughput drug screening. This invention also uses this screening method to identify a series of novel embedded cell death agonists and antagonists. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the cell reaction process in Example 1;

[0053] Figure 2 These are fluorescence microscope field-of-view images of the DMSO and CCCP groups;

[0054] Figure 3 yes Figure 2 The corresponding magnified view. Detailed Implementation

[0055] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0056] The HEK293T cell line used in the following preparation examples or embodiments was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0057] Preparation Example 1

[0058] This preparation example prepares ① HEK293T cells that simultaneously overexpress GFP and AKT1, ② HEK293T cells that simultaneously overexpress GFP and AKT2, and ③ HEK293T cells that simultaneously overexpress GFP and AKT3. The specific preparation methods are as follows:

[0059] (1) AKT1, AKT2 and AKT3 cDNA were cloned into plasmid pSLenti-CMV-EGFP-PGK-puro (restriction sites BsrGI and XbaI) to obtain pSLenti-CMV-EGFP-AKTs-PGK-puro plasmid;

[0060] (2) Plasmids pSLenti-CMV-EGFP-AKTs-PGK-puro, pMD2G and pSPAX2 were simultaneously transfected into HEK293T cells to form lentivirus;

[0061] (3) The new HEK293T cells were transfected with lentivirus, and finally HEK293T cell lines that stably expressed GFP-AKT1, GFP-AKT2, and GFP-AKT3 were selected by puromycin (2 μg / mL).

[0062] Example 1

[0063] This embodiment provides a method for screening cell-embedded cell death agonists and a series of cell-embedded cell death agonists obtained through screening. The operation method is as follows:

[0064] (1) Mix ordinary HEK293T cells with HEK293T cells overexpressing GFP and AKT1 prepared in Preparation Example 1 at a ratio of 1:1, and culture them in a glass-bottomed culture dish for 24 h until they adhere (37℃, 5% CO2). The culture medium is DMEM medium containing 10% bovine serum and 1% penicillin antibiotics.

[0065] (2) The candidate substances (nocodazole, mebendazole, oligomycin, FCCP, CCCP, cysteine, glutathione, acetylcysteine, paclitaxel, KX2-391, GNE-2861, ENMD-2076, PF-3758309 hydrochloride, tetrandrine, 3-norcolchicine, octendaazole, PF-04554878, PF-06409577, compressine phosphate, compressine disodium phosphate) were dissolved in the culture medium and adjusted to the corresponding concentrations. They were then mixed with the adherent cells obtained in step (1) and co-incubated for 1 h (37℃, 5% CO2).

[0066] (3) After co-incubation, the cells were fixed with 4% formalin, stained with HOECHST 33342, and then observed under a fluorescence microscope. The reaction process is shown in the schematic diagram below. Figure 1 As shown. The cell embedding mortality rate was calculated as (a+b) / (a+b+c)%, where a = number of phagocytes, b = number of phagocytes, and c = number of all other cells. If a+b+c > 200, cell embedding death agonists were screened, and the results are shown in Table 1 (DMSO was used as the control group, and each group was analyzed in triplicate). Fluorescence microscopy images of the DMSO group and the CCCP (50 μM) group are shown below. Figure 2 As shown in the figure (scale bar is 50 μm), it can be seen that under the stimulation of the agonist CCCP, a certain proportion of cells undergo in-line cell death, and the triangle marks the cells that undergo in-line cell death. Figure 3 yes Figure 2 A magnified view of a portion of the image (scale bar is 10 μm) clearly shows the hollowed-out structures appearing in the cells where embedded cell death has occurred.

[0067] Table 1

[0068]

[0069]

[0070] As shown in Table 1, nocodazole, mebendazole, oligomycin, FCCP, CCCP, KX2-391, GNE-2861, ENMD-2076, PF-3758309 hydrochloride, tetrandrine, 3-norcolchicine, octendaazole, PF-04554878, PF-06409577, compressoritine phosphate, and compressoritine disodium phosphate all significantly promoted the in-cell death of HEK293T cells and can be used as a new class of in-cell death agonists.

[0071] Example 2

[0072] This embodiment provides a method for screening cell-embedded cell death antagonists and a series of cell-embedded cell death antagonists obtained through screening. The operation method is as follows:

[0073] (1) Mix ordinary HEK293T cells with HEK293T cells overexpressing GFP and AKT1 prepared in Preparation Example 1 at a ratio of 1:1, and culture them in a glass-bottomed culture dish for 24 h until they adhere (37℃, 5% CO2). The culture medium is DMEM medium containing 10% bovine serum and 1% penicillin antibiotics.

[0074] (2) Dissolve CCCP in culture medium and adjust to 50 μM, mix with adherent cells obtained in step (1), and co-incubate for 30 min (37℃, 5% CO2); then dissolve candidate substances (cysteine, glutathione, acetylcysteine) in culture medium and adjust to the corresponding concentration, mix with the above adherent cells, and co-incubate for 1 h (37℃, 5% CO2);

[0075] (3) After co-incubation, the cells were fixed with 4% formalin, stained with HOECHST 33342, and then observed under a fluorescence microscope. The ratio of the number of cells that underwent intercalation death to the total number of cells was calculated, i.e., the cell intercalation death rate, and cell intercalation death agonists were screened. The results are shown in Table 2 (CCCP was used as the control group, and each group of data was performed in parallel 3 times).

[0076] Table 2

[0077] Candidate material Cellular embedded mortality rate CCCP (50μM) 15.83 CCCP (50 μM) + Cysteine ​​(5 mM) 5.82 CCCP (50 μM) + Glutathione (1.4 mM) 4.2 CCCP (50 μM) + Acetylcysteine ​​(1 mM) 3.3

[0078] As shown in Table 2, cysteine, glutathione, and acetylcysteine ​​all significantly inhibited the in-cell death induced by CCCP in HEK293T cells, and have the potential to be used as a new class of in-cell death antagonists.

[0079] Example 3

[0080] This embodiment provides a method for screening cell-embedded cell death antagonists and the screened cell-embedded cell death antagonists. The operation method is as follows:

[0081] (1) Mix ordinary HEK293T cells with HEK293T cells overexpressing GFP and AKT1 prepared in Preparation Example 1 at a ratio of 1:1, and culture them in a glass-bottomed culture dish for 24 h until they adhere (37℃, 5% CO2). The culture medium is DMEM medium containing 10% bovine serum and 1% penicillin antibiotics.

[0082] (2) Dissolve nocodazole in culture medium and adjust to 0.5 μM, mix with adherent cells obtained in step (1), and co-incubate for 30 min (37℃, 5% CO2); then dissolve candidate substances (cysteine, glutathione, acetylcysteine ​​and paclitaxel) in culture medium and adjust to the corresponding concentration, mix with the above adherent cells, and co-incubate for 1 h (37℃, 5% CO2);

[0083] (3) After co-incubation, the cells were fixed with 4% formalin, stained with HOECHST 33342, and then observed under a fluorescence microscope. The ratio of the number of cells that underwent intercalation death to the total number of cells was calculated, i.e., the cell intercalation death rate, to screen for cell intercalation death agonists. The results are shown in Table 3 (with nocodazole as the control group, and each group of data was performed in parallel 3 times).

[0084] Table 3

[0085] Candidate material Cellular embedded mortality rate Nocodazole (0.5 μM) 22.39 Nocodazole (0.5 μM) + Paclitaxel (0.5 μM) 5.28 Nocodazole (0.5 μM) + Paclitaxel (5 μM) 2.2

[0086] As shown in Table 3, paclitaxel has a significant inhibitory effect on in-cell death induced by nocodazole in HEK293T cells, and has the potential to be used as a new in-cell death antagonist.

[0087] Example 4

[0088] This embodiment provides a method for screening cell-embedded cell death agonists, the operation method of which is as follows:

[0089] (1) The ordinary HEK293T cells were mixed with the following cells prepared in Preparation Example 1: ① HEK293T cells overexpressing GFP, ② HEK293T cells overexpressing both GFP and AKT1, ③ HEK293T cells overexpressing both GFP and AKT2, and ④ HEK293T cells overexpressing both GFP and AKT3, at a ratio of 1:1. The cells were cultured in glass-bottomed culture dishes for 24 hours until they adhered (37°C, 5% CO2). The culture medium was DMEM medium containing 10% bovine serum and 1% penicillin antibiotics.

[0090] (2) Dissolve CCCP in culture medium and adjust to 50 μM, mix with adherent cells obtained in step (1), and co-incubate for 1 h (37℃, 5% CO2);

[0091] (3) After co-incubation, the cells were fixed with 4% formalin, stained with HOECHST 33342, and then observed with a fluorescence microscope. The ratio of the number of cells that underwent intercalation death to the total number of cells was calculated, i.e., the cell intercalation death rate. The results are shown in Table 4 (DMSO was used as a control, dissolved in the culture medium at a volume ratio of 1:500, and each set of data was performed in triplicate).

[0092] Table 4

[0093] Group CCCP DMSO HEK293T overexpressing GFP 2.66±0.6147 0.00±0.0000 HEK293T overexpressing GFP and AKT1 15.04±1.9500 0.56±0.2138 HEK293T overexpressing GFP and AKT2 7.22±0.9943 0.14±0.2521 HEK293T overexpressing GFP and AKT3 11.66±1.4500 0.72±0.4484

[0094] As shown in Table 4, HEK293T cells overexpressing AKT1 are more suitable as the cell model for the screening method of this invention compared with HEK293T cells overexpressing AKT2 or AKT3.

[0095] The applicant declares that this invention illustrates a method for screening cell-embedded cell death agonists or antagonists and the resulting products through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for screening cell-embedded cell death agonists or antagonists, characterized in that, The screening method includes: using adherent HEK293T cells as a screening model, co-culturing them with candidate substances, calculating the ratio of the number of cells undergoing embedded cell death to the total number of cells, and screening out cell embedded cell death agonists or antagonists. The screening method includes the following steps: (1) Mix ordinary HEK293T cells with HEK293T cells overexpressing protein kinase B and culture until they adhere to the culture vessel; the protein kinase B includes AKT1, AKT2 or AKT3; (2) The candidate material is then mixed with the adherent cells obtained in step (1) and co-incubated. (3) After co-incubation, observe with a fluorescence microscope, calculate the ratio of the number of cells that undergo embedded cell death to the total number of cells, and screen for cell embedded cell death agonists or antagonists. The agonists include nocodazole, mebendazole, oligomycin, FCCP, CCCP, KX2-391, GNE-2861, ENMD-2076, PF-3758309 hydrochloride, tetrandrine, 3-norcolchicine, octendaazole, PF-04554878, PF-06409577, compressorium phosphate, or compressorium disodium phosphate; the antagonists include cysteine, glutathione, or acetylcysteine.

2. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, The ratio of ordinary HEK293T cells to HEK293T cells overexpressing protein kinase B was (1:10)-(10:1).

3. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, The HEK293T cells overexpressing protein kinase B were labeled with a fluorescent protein.

4. The method for screening cell-embedded cell death agonists or antagonists as described in claim 3, characterized in that, The fluorescent protein includes GFP.

5. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, The protein kinase B is AKT1.

6. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, The mixed culture time is 12-48 h.

7. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, The screening concentration of the candidate substances is 1 nM-5 mM.

8. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, The co-incubation time is 20-90 min.

9. The method for screening cell-embedded cell death agonists or antagonists as described in claim 1, characterized in that, After the co-incubation is completed, the cells will be fixed and stained.

10. The method for screening cell-embedded cell death agonists or antagonists as described in claim 9, characterized in that, The fixation is performed using 4-10% formalin.

11. The method for screening cell-embedded cell death agonists or antagonists as described in claim 9, characterized in that, The dyeing agent used includes HOECHST 33342.

12. The use of cysteine ​​or its salts, glutathione or its salts, acetylcysteine ​​or its salts in the preparation of a CCCP-induced HEK293T cell embedded cell death antagonist, characterized in that, The HEK293T cell-embedded cell death antagonist was obtained by screening using the screening method described in any one of claims 1-11.

13. A method for inhibiting CCCP-induced embedded cell death in HEK293T cells for non-disease treatment purposes, characterized in that, The method comprises administering to HEK293T cells any one or a combination of at least two of the following drugs: cysteine ​​or a salt thereof, glutathione or a salt thereof, acetylcysteine ​​or a salt thereof; wherein the drugs are obtained by screening using the screening method according to any one of claims 1-11.

Images