Establishment method of esophageal squamous epithelial cell precancerous lesion and cancerous cell model
Through NMBzA infection treatment and microscopy, morphological changes were observed, combined with cell proliferation, invasion, migration ability detection and nude mouse tumor formation experiment, a precancerous lesions and cancer model of esophageal squamous epithelial cells was constructed, solving the shortcomings in the model construction in the existing technology, and achieving accurate lesion distinction and verification.
Patent Information
- Application Number
- CN202510536009.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-27
- Publication Date
- 2025-07-29
AI Technical Summary
There is a lack of effective methods for constructing precancerous lesions and cancerous models in the prior art, making it difficult to accurately distinguish and verify precancerous lesions and cancerous processes.
NMBzA staining venom was used to treat esophageal squamous epithelial cells, and morphological changes were observed by microscopy. By detecting cell proliferation, invasion and migration capabilities, and nude mice tumorigenesis experiments were carried out to verify the model successfully.
A precancerous lesions and cancer model of esophageal squamous epithelial cells was constructed, providing the judgment efficiency and accuracy, verifying the method of distinguishing precancerous lesions and cancer, and laying the foundation for the etiology of esophageal squamous cell carcinoma.
Smart Images
Figure CN120384051A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of cell model construction, and specifically relates to a method for constructing a precancerous lesion and canceration cell model of esophageal squamous epithelial cells. Background Art
[0002] The early symptoms of esophageal cancer are not obvious, and it is mostly in the advanced stage when discovered, with a poor prognosis. Therefore, the mortality rate has been remaining high in the global cancer spectrum. The report of the International Agency for Research on Cancer shows that the global incidence rate of esophageal cancer ranks 14th among all cancers, while the total mortality rate ranks 8th. Therefore, paying attention to the occurrence and development process of esophageal cancer is of great significance for the precise prevention and treatment of esophageal cancer.
[0003] Esophageal cancer is mainly divided into esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) according to histological types. In recent years, there are not few methods for constructing a model of esophageal squamous epithelial cell canceration, but there are rarely methods for constructing a model that pays attention to the process of esophageal squamous epithelial cell canceration, and it is difficult to find references for differentiating between a precancerous lesion model and a canceration model of esophageal cells. For this reason, the present invention proposes a method for constructing a precancerous lesion and canceration cell model of esophageal squamous epithelial cells. Summary of the Invention
[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for constructing a precancerous lesion and canceration cell model of esophageal squamous epithelial cells, and solve the problems in the prior art.
[0005] The purpose of the present invention can be achieved by the following technical solutions:
[0006] A method for constructing a precancerous lesion and canceration cell model of esophageal squamous epithelial cells, comprising the following steps:
[0007] Cultivate esophageal squamous epithelial cells;
[0008] Perform a poisoning treatment on the esophageal squamous epithelial cells with an NMBzA poisoning solution;
[0009] Record the change in the passage time of the esophageal squamous epithelial cells after poisoning, observe the morphological changes of the esophageal squamous epithelial cells under a microscope, and determine the precancerous lesion and canceration generations;
[0010] Determine whether the precancerous lesion and canceration cell model is successfully constructed by detecting the cell proliferation, invasion, and migration abilities;
[0011] Verify whether the precancerous lesion and canceration cell model is successfully constructed by detecting the tumorigenic ability of the cells through a nude mouse tumorigenesis experiment.
[0012] Furthermore, the process of cultivating esophageal squamous epithelial cells is: place the esophageal squamous epithelial cells in a complete culture medium and culture them in an incubator.
[0013] Furthermore, the complete culture medium includes: DMEM culture medium, FBS and double antibody, and the volume ratio is 10:1:0.1.
[0014] Furthermore, the double antibody is streptomycin at 100 U / ml and penicillin at 100 U / ml.
[0015] Furthermore, the concentration of the NMBzA poisoning solution is 2 μmol / L.
[0016] Furthermore, the preparation process of the NMBzA poisoning solution is as follows: dissolve NMBzA in DMSO to obtain a 10 mmol / L mixed solution, and dilute it to 2 μmol / L with the complete culture medium, then it is ready.
[0017] Furthermore, the process of poisoning esophageal squamous epithelial cells is as follows:
[0018] 1) Inoculate esophageal squamous epithelial cells into a culture flask and perform poisoning treatment with the NMBzA poisoning solution;
[0019] 2) After the cells proliferate to show contact inhibition, passage them and repeat the poisoning treatment with the NMBzA poisoning solution.
[0020] Furthermore, the proliferation ability of esophageal squamous epithelial cells is detected by CCK8; the proliferation ability and invasion ability of esophageal squamous epithelial cells are detected by plate cloning; the invasion ability of esophageal squamous epithelial cells is detected by Transwell experiment; the migration ability of esophageal squamous epithelial cells is detected by scratch experiment.
[0021] Furthermore, the esophageal squamous epithelial cells are: HEEC or HET-1A.
[0022] Furthermore, the process of the nude mouse tumorigenesis experiment is as follows:
[0023] Digest and centrifuge esophageal squamous epithelial cells at different passage numbers, suspend them in PBS and Matrigel glue prepared in a 1:1 ratio, and inoculate them subcutaneously under the right axilla of nude mice; record the body weight and tumor volume of nude mice every 3 days, take out the tumor after 21 days of inoculation, take pictures and record, and perform histopathological detection.
[0024] Advantages of the present invention:
[0025] 1. The present invention discloses a method for long-term intervention of esophageal squamous epithelial cells (HEEC and HET-1A) by NMBzA to establish a model of esophageal squamous precancerous lesions and cancer cells, explores the development of esophageal squamous epithelial cells during precancerous lesions and carcinogenesis, lays a foundation for the etiological study of esophageal squamous cell carcinoma, and provides a certain theoretical basis for the subsequent research on the related mechanisms of the occurrence and development of esophageal squamous cell carcinoma.
[0026] 2. The present invention discloses a method for judging precancerous lesions and carcinogenic generations of cells by using cell morphological changes and passage time changes, which enables subsequent cell biology experiments to play a role in determination and verification, increases the determination efficiency, and improves the determination accuracy.
[0027] 3. The present invention discloses a method for verifying and differentiating precancerous lesions and carcinogenic models by using nude mouse subcutaneous tumorigenesis; the precancerous lesion cells with proliferation, invasion, and migration variations fail to form tumors, while the carcinogenic cells can successfully form tumors, effectively differentiating and verifying the two-stage model. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0029] Figure 1 is the cell morphology diagram of the present invention;
[0030] Figure 2 is the CCK8 result diagram of the present invention;
[0031] Figure 3 is the flat plate cloning result diagram of the present invention;
[0032] Figure 4 is the Transwell result diagram of the present invention;
[0033] Figure 5 is the migration result diagram of the present invention;
[0034] Figure 6 is the nude mouse tumorigenesis result diagram of the present invention;
[0035] Figure 7 is the cell model construction flow chart of the invention. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0037] Example 1
[0038] As Figure 7 shown, the construction method of the esophageal squamous epithelial cell precancerous lesion and carcinogenic cell model includes the following steps:
[0039] S1, Culture esophageal squamous epithelial cells (HEEC and HET-1A);
[0040] Place the esophageal squamous epithelial cells in complete culture medium and place them in an incubator with 5% CO2 and 37 °C until they grow to the pre-contact inhibition stage; the complete culture medium includes: DMEM culture medium, fetal bovine serum (FBS) and double antibody; among them, the volume ratio of DMEM culture medium, FBS and double antibody is 10:1:0.1, and the double antibody is 100 U / ml streptomycin and 100 U / ml penicillin.
[0041] S2, Use N-methylbenzylnitrosamine (NMBzA) to treat esophageal squamous epithelial cells with toxicants;
[0042] The process of toxicant treatment includes:
[0043] 1) Dissolve N-methylbenzylnitrosamine (NMBzA) in DMSO to obtain a 10 mmol / L mixed solution, and then add complete culture medium to dilute the mixed solution to obtain a 2 μmol / L NMBzA toxicant solution.
[0044] 2) Inoculate the HEEC and HET-1A at a density of 40% into 25 cm 2 culture flasks and perform NMBzA toxicant treatment. This is the first generation of cells treated with NMBzA. After the cells proliferate to show contact inhibition, subculture and repeat the NMBzA toxicant treatment; HEEC is repeatedly cultured to the 40th generation, and HET-1A is repeatedly cultured to the 50th generation. Among them, every 5 generations of treatment, the corresponding cells are cryopreserved in liquid nitrogen;
[0045] The NMBzA toxicant treatment is: add 2 μmol / L NMBzA toxicant solution to HEEC and HET-1A and culture for 48 - 72 hours.
[0046] S3, Record the change of cell passage time of HEEC and HET-1A, observe the morphological changes of HEEC and HET-1A cells under a microscope, and determine the precancerous lesions and canceration generations;
[0047] During the toxicant treatment of HEEC and HET-1A cells, observe the morphology of each generation of cells under an inverted microscope. When the cell passage time is significantly shortened and obvious morphological changes occur in the cells, mark the generation for subsequent experiments. The microscope found that the 15th generation of HEEC after toxicant treatment had precancerous lesion morphology, and the 40th generation had canceration morphology; the 20th generation of HET-1A after toxicant treatment had precancerous lesion morphology, and the 50th generation had canceration morphology.
[0048] S4, Detect the cell proliferation ability by CCK8;
[0049] 1) For the 0th, 15th, and 40th generations of HEEC cells at 2×103 Inoculate at a density of [number] cells / well into a 96-well plate, with a volume of 100 μL per well. Incubate in an incubator at 37 °C and 5% CO₂. Each group has 6 - 8 replicate wells, and a blank well is set. After the cell growth density of the group with the fastest proliferation reaches 90%, discard the culture medium. Dilute the CCK8 solution with DMEM culture medium at a dilution ratio of 9:1 to prepare the detection reagent. After incubating in the incubator for 15 min, measure the OD value of each well in the CCK8 mode of the microplate reader every 5 min until the OD value stabilizes. Record the results and take the average value.
[0050] 2) Repeat the above operations for HET-1A cells at passage 0, 20, and 50.
[0051] S5. Detect the cell proliferation ability and invasion ability by plate cloning.
[0052] 1) Inoculate HEEC cells at passage 0, 15, and 40 at a density of 1×10 3 cells / well into a six-well plate, with a volume of 2 mL per well. Incubate in an incubator at 37 °C and 5% CO₂. Each group has 3 replicate wells. When single cells grow into colonies without contacting each other, discard the culture medium. Fix the cells with 1 mL of 4% paraformaldehyde per well for 10 min, and then stain with crystal violet staining solution for 15 min. Count the number of colonies and take the average value.
[0053] 2) Repeat the above operations for HET-1A cells at passage 0, 20, and 50.
[0054] S6. Detect the cell invasion ability by Transwell experiment.
[0055] 1) Dilute Matrigel with DMEM culture medium without FBS at a dilution ratio of 8:1. Add 50 μL of the diluted solution to each well of the chamber, place it in a 24-well plate, with 3 replicate wells in each group. Let it stand in the incubator for 2 h, and then add 100 μL of PBS to each well to soften the matrix gel for 30 min. Discard the reagents in the chamber and reserve for use. Inoculate HEEC cells at passage 0, 15, and 40 at a density of 1×10 4 cells / well into the upper chamber with the gel coated. Add 600 μL of culture medium containing 20% FBS to the lower chamber. Incubate for 24 h, discard the culture medium, wipe off the inner layer of cells in the chamber with a sterile cotton swab. Fix the cells with 600 μL of 4% paraformaldehyde per well for 10 min, and then stain with crystal violet staining solution for 15 min. Count the number of colonies and take the average value.
[0056] 2) Repeat the above operations for HET-1A cells at passage 0, 20, and 50.
[0057] S7. Detect the cell migration ability by scratch assay.
[0058] 1) Seed HEEC cells at passages 0, 15, and 40 at a density of 40% in six-well plates, with a volume of 2 mL per well. Incubate them in an incubator at 37 °C and 5% CO2, with 3 replicates per group. When the cell density reaches 90%, gently draw a straight line on the cell layer with a 200 μL pipette tip to form a "scratch". Discard the culture medium and floating cells, add 2 mL of DMEM medium without FBS to each well, and incubate in the incubator for 24 h. Record the results and take the average value.
[0059] 2) Repeat the above operation for HET-1A cells at passages 0, 20, and 50.
[0060] S8. Detect the tumorigenic ability of cells through nude mouse tumorigenesis experiments to verify whether the model is successfully constructed;
[0061] 1) Digest and centrifuge HEEC cells at passages 15 and 40, and suspend them in a 1:1 mixture of PBS and Matrigel. Inoculate 1×10 7 / 150 μL per mouse subcutaneously into the right axilla of 5-week-old male BALB / c nude mice, with 6 - 8 mice per group. Record the body weight and tumor volume of nude mice every 3 days. After 21 days of inoculation, remove the tumors, take pictures and record, and perform histopathological detection.
[0062] 2) Repeat the above operation for HET-1A cells at passages 0, 20, and 50.
[0063] Example 2
[0064] In this example, the results involved in the cell model construction process in Example 1 are analyzed.
[0065] Figure 1 It is a cell morphology diagram; where Figure 1 A and B in it respectively represent the cell morphologies of HEEC and HET-1A. During the poisoning process, it was found that the passage interval of HEEC P0 was about 72 h, HEEC P15 was about 48 h, and HEEC P40 was about 24 h; compared with HEEC P0, P15 and P40 gradually lost the polarity of the original epithelial cells significantly, showing a spindle-shaped irregular morphology, with an increase in the volume and number of nucleoli, and some cells showed fragmented nuclei. The passage interval of HET-1A P0 was about 96 h, HET-1A P20 was about 48 h, and HET-1A P50 was about 24 h; compared with HET-1A P0, P20 and P50 gradually lost the polarity of the original epithelial cells significantly, showing a spindle-shaped irregular morphology, with an increase in the volume and number of nucleoli, and some cells showed fragmented nuclei.
[0066] Figure 2 It is a CCK8 result diagram, Figure 2 A and B in it respectively correspond to the CCK8 results of HEEC cells and HET-1A cells;Figure 2 In A of [[]], when comparing HEEC-15 and HEEC-40 with the HEEC-0 group, *P < 0.05; when comparing HEEC-40 with the HEEC-15 group, # P < 0.05. Figure 2 In B of [[]], when comparing HET-1A-20 and HET-1A-50 with the HET-1A-0 group, *P < 0.05; when comparing HET-1A-50 with the HET-1A-20 group, # P < 0.05.
[0067] Figure 2 It can be seen from [[]] that: compared with HEEC P0, the OD values of P15 and P40 increased significantly (P < 0.05); compared with HEEC P15, the OD value of P40 increased significantly (P < 0.05). Compared with HET-1A P0, the OD values of P20 and P50 increased significantly (P < 0.05); compared with HET-1A P20, the OD value of P50 increased significantly (P < 0.05).
[0068] Figure 3 is the result graph of plate cloning, Figure 3 A and B in [[]] correspond to HEEC cells and HET-1A cells respectively; Figure 3 In A of [[]], when comparing HEEC-15 and HEEC-40 with the HEEC-0 group, *P < 0.05; when comparing HEEC-40 with the HEEC-15 group, # P < 0.05. Figure 3 In B of [[]], when comparing HET-1A-20 and HET-1A-50 with the HET-1A-0 group, *P < 0.05; when comparing HET-1A-50 with the HET-1A-20 group, # P < 0.05.
[0069] Figure 3 It can be seen from [[]] that: compared with HEEC P0, the colony numbers of P15 and P40 increased significantly (P < 0.05); compared with HEEC P15, the colony number of P40 increased significantly (P < 0.05). Compared with HET-1A P0, the colony numbers of P20 and P50 increased significantly (P < 0.05); compared with HET-1A P20, the colony number of P50 increased significantly (P < 0.05).
[0070] Figure 4 is the Transwell result graph, Figure 4 A and B in [[]] correspond to HEEC cells and HET-1A cells respectively; Figure 4 In A of [[]], when comparing HEEC-15 and HEEC-40 with the HEEC-0 group, *P < 0.05; when comparing HEEC-40 with the HEEC-15 group,# P < 0.05. Figure 4 In B of, compared with the HET-1A-0 group, *P < 0.05 for HET-1A-20 and HET-1A-50; compared with the HET-1A-20 group, # P < 0.05. Figure 4 It can be seen that: compared with HEEC P0, the migration rates of P15 and P40 were significantly increased (P < 0.05); compared with HEEC P15, the migration rate of P40 was significantly increased (P < 0.05). Compared with HET-1A P0, the migration rates of P20 and P50 were significantly increased (P < 0.05); compared with HET-1A P20, the migration rate of P50 was significantly increased (P < 0.05).
[0071] Figure 5 is the migration result graph, Figure 5 A and B in correspond to HEEC cells and HET-1A cells respectively; Figure 5 In A of, the red arrow points to the invasive cells; compared with the HEEC-0 group, *P < 0.05 for HEEC-40; compared with the HEEC-15 group, # P < 0.05. Figure 5 In B of, the red arrow points to the invasive cells; compared with the HET-1A-0 group, *P < 0.05 for HET-1A-50; compared with the HET-1A-20 group, # P < 0.05. Figure 5 It can be seen that: compared with HEEC P0, the number of cells of P15 increased; compared with HEEC P0 and P15, the number of cells of P40 was significantly increased (P < 0.05). Compared with HET-1A P0, the number of cells of P20 increased; compared with HET-1A P0 and P20, the migration rate of P50 was significantly increased (P < 0.05).
[0072] Figure 6 is the tumor formation result graph in nude mice, Figure 6 A-1, A-2, and A-3 in are used to verify the HEEC model and respectively represent: the tumor body graph of nude mice, the body weight graph of nude mice, and the tumor body volume graph of nude mice; Figure 6 B-1, B-2, and B-3 in are used to verify the HET-1A model and respectively represent: the tumor body graph of nude mice, the body weight graph of nude mice, and the tumor body volume graph of nude mice. In Figure 6 In A-3 of, compared with the ESCC group, *P < 0.05 for EPL; compared with the 3rd day, & P < 0.05. In Figure 6 In B-2 of, compared with the ESCC group, *P < 0.05 for EPL. In Figure 6In B-3, when comparing the EPL group with the ESCC group, *P < 0.05. When comparing the other days of EPL with the 3rd day, & P < 0.05; when comparing the other days of ESCC with the 3rd day, # P < 0.05; when comparing ESCC with the 9th day which has the lowest volume value, a P < 0.05.
[0073] Figure 6 It can be seen that: After injecting P15 and P40 of HEEC for 12 days, the P15 cell mass in the nude mice of the EPL group gradually disappeared, while the volume of the P40 cell mass in the nude mice of the ESCC group gradually increased to form tumors; after 15 days, there was a significant difference in the tumor volume between the EPL and ESCC groups of nude mice (P < 0.05), and the difference gradually increased. The tumor formation effect of HET-1A cells was more obvious. After injecting P20 and P50 for 9 days, the P20 cell mass in the nude mice of the EPL group gradually disappeared, while the volume of the P50 cell mass in the nude mice of the ESCC group gradually increased to form tumors; after 12 days, there was a significant difference in the tumor volume between the EPL and ESCC groups of nude mice (P < 0.05), and the difference gradually increased. Compared with the 9th, 15th, 18th, and 21st days which have the smallest tumor volume in the ESCC group of nude mice, there was a significant increase (P < 0.05).
[0074] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.
[0075] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.
Claims
1. A method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells, characterized in that, It includes the following steps: Cultivate esophageal squamous epithelial cells; Use NMBzA contaminated solution to conduct contamination treatment on esophageal squamous epithelial cells; Record the change in the passage time of esophageal squamous epithelial cells after contamination, observe the morphological changes of esophageal squamous epithelial cells under a microscope, and determine the precancerous lesions and the generation number of canceration; By detecting the cell proliferation, invasion, and migration abilities, determine whether the precancerous lesion and canceration cell models are successfully constructed; Detect the tumorigenic ability of cells through nude mouse tumorigenesis experiments to verify whether the precancerous lesion and canceration cell models are successfully constructed.
2. The method for constructing a precancerous lesion and cancer cell model of esophageal squamous epithelial cells according to claim 1, wherein The process of cultivating esophageal squamous epithelial cells is as follows: Place esophageal squamous epithelial cells in complete culture medium and culture them in an incubator.
3. The method for constructing a precancerous and cancerous cell model of esophageal squamous epithelial cells according to claim 2, wherein The said complete culture medium includes: DMEM culture medium, FBS, and double antibodies, and the volume ratio is 10:1:0.
1.
4. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 3, wherein The said double antibodies are streptomycin at 100 U / ml and penicillin at 100 U / ml.
5. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 1, wherein, The concentration of the said NMBzA contaminated solution is 2 μmol / L.
6. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 5, wherein, The preparation process of the said NMBzA contaminated solution is as follows: Dissolve NMBzA in DMSO to obtain a 10 mmol / L mixed solution, and dilute it to 2 μmol / L with complete culture medium, then it is obtained.
7. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 1, characterized in that, The process of conducting contamination treatment on esophageal squamous epithelial cells is as follows: 1) Inoculate esophageal squamous epithelial cells in a culture flask and conduct contamination treatment with NMBzA contaminated solution; 2) After the cells proliferate to show contact inhibition, passage them and repeat the contamination treatment with NMBzA contaminated solution.
8. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 1, wherein Detect the proliferation ability of esophageal squamous epithelial cells through CCK8; detect the proliferation ability and invasion ability of esophageal squamous epithelial cells through plate cloning; detect the invasion ability of esophageal squamous epithelial cells through Transwell experiment; detect the migration ability of esophageal squamous epithelial cells through scratch experiment.
9. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 1, wherein The said esophageal squamous epithelial cells are: HEEC or HET-1A.
10. The method for constructing a precancerous lesion and cancerous cell model of esophageal squamous epithelial cells according to claim 1, characterized in that, The process of the said nude mouse tumorigenesis experiment is as follows: Digest and centrifuge esophageal squamous epithelial cells at different passage numbers, suspend them in PBS and Matrigel glue prepared in a ratio of 1:1, and inoculate them subcutaneously in the right axilla of nude mice; Record the body weight and tumor volume of nude mice every 3 days, take out the tumor after inoculation for 21 days, take pictures and record, and conduct histopathological detection.