CN114854669A - Method for constructing esophageal epithelial cell precancerous lesion cell model - Google Patents
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Abstract
The invention relates to the technical field of cell model construction methods, in particular to a construction method of an esophageal epithelial cell precancerous lesion cell model, and the following scheme is provided, wherein the method comprises the steps of continuously intervening HEEC to the 30 th generation for a long time by using a 1 mu mol/LAFB1 solution, detecting the influence of HEEC activity of AFB1 infected cells of different generations by adopting a CCK8 method, detecting apoptosis and cell cycle of cells of different generations by adopting a flow cytometer, and detecting invasion and migration capacity of the cells of 30 generations infected cells by adopting a Transwell method. The invention further explores the related molecular mechanism of AFB1 in the esophageal cancer generating process, lays a foundation for the etiology research of esophageal cancer, and provides a certain theoretical basis for preventing the toxicity of AFB1 in food to esophagus.
Description
Technical Field
The invention relates to the field of cell model construction methods, in particular to a construction method of an esophageal epithelial cell precancerous lesion cell model.
Background
The international cancer research institution reports show that the incidence rate of esophageal cancer ranks seventh among all cancers, the total mortality rate ranks sixth among all cancers, and the environment, dietary habits and life styles play an important role in the incidence process of esophageal cancer, wherein mycotoxin infection is considered to be one of important risk factors for the incidence of esophageal cancer in residents in high incidence areas of esophageal cancer in China. Aflatoxin B1 is the most common and most toxic mycotoxin in food, and AF B1 was defined by IARC as a class I carcinogen in 1993. A case-control study shows that the high exposure of aflatoxin B1 can be an important risk factor of esophageal precancerous lesion of Huaian China (high incidence area of esophageal cancer). In recent years, the establishment method of the esophageal precancerous lesion thin cell model is rarely seen, and the methyl benzyl nitrosamine is mostly used for inducing establishment of the esophageal precancerous lesion variable model, so that the invention provides the establishment method of the esophageal epithelial cell precancerous lesion thin cell model.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for constructing a premalignant esophageal epithelial cell thin cell model.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for constructing an esophageal epithelial cell precancerous lesion cell model comprises the following steps:
step one, HEEC is cultured;
step two, contamination treatment: dissolving AFB1 powder in DMSO to obtain 10mmol/L mixed solution, and adding complete culture solution to dilute the mixed solution to obtain 1 μmol/LAFB1 venom;
seeding the HEEC at 40% density for 25cm 2 Culturing overnight in a culture bottle, treating with AFB1 after HEEC is attached to the wall and recovered in shape, carrying out passage, wherein the first generation of cells are treated with AFB1, and after the cells are recovered in shape and attached to the wall overnight, repeating the treatment with AFB1, and repeating the culture for 30 generations, wherein each treatment for 5 generations, the corresponding cells are frozen and stored in liquid nitrogen, and the AFB1 treatment is adding 1 mu mol/LAFB1 into HEEC to culture in a venom staining solution for 48-72 hours.
Further, the HEEC culturing step is as follows: HEEC were placed in complete medium and placed in 5% CO 2 And 37 ℃, wherein the complete culture solution comprises high-sugar DMEM culture solution, FBS and double antibody, and the high-sugar DMEM culture solution comprises: FBS: the volume ratio of the double antibody is 10: 1: 0.1, 100U/ml streptomycin and 100U/ml penicillin for double antibody.
Further, the construction method comprises the step of detecting HEEC activity after the infection treatment by adopting a CCK8 method.
Further, the step of detecting the activity of HEEC after the infection treatment comprises the following steps:HEEC at 6X 10 3 The cells were seeded in 96-well plates at a density of 100. mu.L/well and placed at 37 ℃ in 5% CO 2 Culturing in an incubator; after 24h, respectively adding 100 mu L of AFB1 culture solution containing 0, 0.01, 0.1, 1, 10, 100 and 1000 mu mol/L into the plate, each group having 5 multiple wells, and setting blank wells for 24h of contamination; then the culture solution is discarded, 100 mu L of complete culture solution is replaced, 10 mu L of CCK8 solution is added into each interference hole in an adherence manner, the incubation is carried out for 1.5h in an incubator, the OD value of each hole is measured under a CCK8 mode of an enzyme labeling instrument, and the recorded result is averaged.
Further, the step of detecting the viability of HEECs after the exposure treatment comprises detecting the viability of cells every 5 passages after the intervention of AFB 1: will contain AFB 1 5, 10, 15, 20, 25, 30 th generation cells infected with culture medium were 6X 10 3 The cells were seeded in 96-well plates at a density of 100. mu.L/well and placed at 37 ℃ in 5% CO 2 Culturing for 48h in an incubator, discarding the culture solution, changing to 100 mu L of complete culture solution, immediately adding 10 mu L of CCK8 solution to each interference hole for adherence, carefully avoiding generating air bubbles, incubating for 1.5h in the incubator, measuring the OD value of each hole in a CCK8 mode of an enzyme labeling instrument, and recording the result and taking an average value.
Further, the construction method comprises calculating the survival rate of the cells: cell viability was 100% (test OD-blank OD)/(control OD-blank OD).
Further, the construction method comprises detecting the cell cycle by adopting flow cytometry.
Further, the construction method comprises the steps of detecting apoptosis by using a flow cytometer, detecting migration capacity of cells by using a Transwell method, and detecting invasion capacity of cells by using the Transwell method.
Further, the detection of the invasion capacity of the cells by using the Transwell method comprises the following steps: diluting Matrigel gel with a FBS-free culture solution in a dilution ratio of 1: 8; adding 50 mu L of diluent into each hole of the small chamber, refrigerating and standing at 4 ℃ until the experiment is used the next day; digesting the cells infected by non-infected HEEC and AFB1 for 30 generations by pancreatin, centrifuging, and adjusting the cell density of each group to 3 × 10 with FBS-free culture solution 5 One well, 200. mu.L of cell suspension was added to the upper chamber after the gel was applied, and 600. mu.L of 50% F was added to the lower chamberA culture solution of BS.
The invention has the beneficial effects that:
the invention discloses a method for long-term intervention of normal epithelial cells (HEEC) of esophagus into a cell model of esophageal precancerous lesion through aflatoxin B1(AFB1), which comprises the following steps: continuously intervening HEEC to 30 th generation for a long time by using a 1 mu mol/LAFB1 solution, detecting the influence of HEEC activity infected by AFB1 in different generations by adopting a CCK8 method, detecting apoptosis and cell cycle of cells in different generations by adopting a flow cytometer, detecting invasion and migration capacity of the cells infected by 30 generations by adopting a Transwell method, and detecting the expression of EMT related proteins (E-cadherin and N-cadherin) and cell cycle related proteins (cyclin A2, cyclin D1 and CDK2) by adopting a Western blot method. The invention further explores the related molecular mechanism of AFB1 in the esophageal cancer generating process, lays a foundation for the research on the etiology of esophageal cancer, and provides a certain theoretical basis for preventing AFB1 in food from toxicity to esophagus.
Drawings
FIG. 1 is a diagram showing the relationship between the activity of HEEC detected by CCK8 method according to the present invention;
FIG. 2 is a schematic diagram showing the relationship between the proliferation of HEEC detected by CCK8 method in the invention;
FIG. 3 is a schematic diagram showing the relationship between HEEC cell cycle status and HEEC cell cycle status in flow cytometry;
FIG. 4 is a schematic diagram showing the relationship between HEEC apoptosis in flow cytometry;
FIG. 5 is a schematic diagram showing the relationship of HEEC migration ability detection by Transwell method according to the present invention;
FIG. 6 is a schematic diagram showing the relationship of HEEC invasion ability detected by Transwell method according to the present invention;
fig. 7 is a schematic overall flow chart of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The meaning of each abbreviation in the present invention is: HEEC: normal esophageal epithelial cells, AFB 1: aflatoxin B1, PBS: phosphate buffer solution, DMSO: dimethyl sulfoxide, DMEM: dulbecco's modified eagle medium;
a method for constructing an esophageal epithelial cell precancerous lesion cell model is shown in figure 7, and HEEC is continuously intervened by AFB1 for a long time, and the specific method comprises the following steps:
(1) cell culture
Preparing a cell culture solution by using a high-glucose DMEM culture solution, FBS (fetal bovine serum) and a double antibody (100U/ml streptomycin and 100U/ml penicillin) according to a proportion of 10: 1: 0.1, in 5% CO 2 And culturing HEEC with the prepared complete culture solution in an incubator at 37 ℃ until the cell density reaches 80-90%, and performing operations such as passage, experiment or cryopreservation.
(2) Contamination treatment
Preparing AFB1 venom staining mother liquor: AFB1 powder was dissolved in DMSO at a concentration of 10mmol/L and placed in a refrigerator at 4 ℃ if used for a long period of time.
② long-term AFB1 contamination treatment: according to the result of the previous CCK8 cell proliferation experiment, the infection concentration of AFB1 is determined to be 1 mu mol/L, and the cell malignant transformation is induced. The whole culture solution is diluted to 1 mu mol/L and is prepared for use. Cells were seeded at approximately 40% density for 25cm 2 Culturing overnight in a culture flask, culturing for 48-72 hours in DMEM culture solution containing 1 mu mol/L AFB1 after the cells are attached to the recovered morphology, treating the first generation of cells with AFB1, repeating the same AFB1 treatment after the cells are attached to the recovered morphology overnight, and repeating the culture for 30 generations. Wherein, the corresponding cells are frozen in liquid nitrogen for each 5 generations of treatment.
The activity and proliferation of the cells can be detected by the CCK8 method.
(1) HEEC viability assay after AFB1 intervention
HEEC at 6X 10 3 The cells were seeded in 96-well plates at a density of 100. mu.L/well and placed at 37 ℃ in 5% CO 2 Culturing in an incubator. After 24h, 100. mu.L of AFB1 culture medium containing 0, 0.01, 0.1, 1, 10, 100, 1000. mu. mol/L was added to each plate, each set of 5 duplicate wells, and blank wells (containing no cells, only culture medium, to remove the backScenic value) is infected for 24 hours; then the culture solution is discarded, 100 mu L of complete culture solution is replaced, 10 mu L of CCK8 solution is added into each interference hole in an adherence manner, care is taken to avoid generating air bubbles, the culture box is incubated for 1.5h, the wells are placed in a microplate reader CCK8 mode to measure the OD value of each hole, and the recorded results are averaged.
The experiment was repeated 3 times according to the above procedure. Calculating the survival rate of the cells: cell viability was 100% (test OD-blank OD)/(control OD-blank OD).
(2) Cell viability was measured every 5 passages after AFB1 intervention
The 5 th, 10 th, 15 th, 20 th, 25 th and 30 th generation cells infected with the culture solution containing AFB1 are treated at 6 x 10 th 3 The cells were seeded in 96-well plates at a density of 100. mu.L/well and placed at 37 ℃ in 5% CO 2 Culturing for 48h in an incubator, discarding the culture solution, changing to 100 mu L of complete culture solution, immediately adding 10 mu L of CCK8 solution to each interference hole for adherence, carefully avoiding generating air bubbles, incubating for 1.5h in the incubator, measuring the OD value of each hole in a CCK8 mode of an enzyme labeling instrument, and recording the result and taking an average value.
The experiment was repeated 3 times according to the above procedure. Calculating the proliferation rate of the cells: the cell proliferation rate was defined as (test OD-blank OD) - (control OD-blank OD)/(control OD-blank OD) × 100%.
Detecting the cell cycle by flow cytometry;
three parallel samples are set for each group of cells, and cells containing AFB1 culture solution infected with 0, 5, 10, 15, 20, 25 and 30 generations are planted in 25cm 2 The culture flask contains 3mL of culture medium, and is placed at 37 deg.C and 5% CO 2 Culturing in a saturated humidity incubator. And collecting each group of cells for flow detection after the cells grow adherent for 48 hours. Washing with PBS for 2 times, adding 1mL pancreatin cell containing 0.25% EDTA, stopping digestion until cell is completely shrunk and rounded and has small amount of floating, transferring to 15mL centrifuge tube, centrifuging at 1000rpm for 5min, washing with PBS twice, centrifuging at 1000rpm for 5min, adjusting cell density to 1 × 10 6 Then, transferred to a 1.5mL EP tube, and 500. mu.L of 70% glacial ethanol (prepared in advance and placed in a refrigerator for pre-cooling at 4 ℃) was added dropwise. Each set of EP tubes was placed in a refrigerator at 4 ℃ overnight, centrifuged the next day to discard the iced ethanol, and washed with PBSCentrifuging for 1 time, discarding, adding 500 μ L prepared stain solution (RNase PI ═ 1: 9), gently blowing, stirring, standing in dark for 30min, sucking single cell suspension, sieving with 300 mesh sieve, and detecting with flow cytometer.
Detecting apoptosis by using a flow cytometer;
five replicates of each group of cells were plated and 0, 5, 10, 15, 20, 25, 30 passages of cells infected with AFB1 culture medium were seeded at 25cm 2 The culture flask contains 3ml of culture medium, and is placed at 37 deg.C and 5% CO 2 Culturing in a saturated humidity incubator. And collecting each group of cells for flow detection after the cells adhere to the wall and grow for 48 hours. Collecting each group of culture solution, washing cells with PBS and collecting, adding 1ml of pancreatin digestive cells without EDTA, stopping digestion after the cells are all shrunk and rounded and have a small amount of floating, centrifuging at 1200rpm for 5min, washing twice with PBS and centrifuging, then discarding supernatant, adding Binding buffer with appropriate volume to ensure that the cell density is 5 multiplied by 10 5 And (4) lightly blowing and beating. Then 100 and 200 mul of single cell suspension is added with 5 mul LannexinV-FITC and 5 mul LPI, and after incubation for 20min in the dark, detection is carried out by an upper flow cytometer.
Detecting the migration capacity of the cells by adopting a Transwell method;
digesting, centrifuging and collecting the cells infected by 30 generations of non-infected HEEC and AFB 1. Resuspending the cells with PBS, centrifuging again, adding FBS-free culture medium to obtain single cell suspension, and adjusting cell density of each group to 5 × 10 4 One well, 200. mu.L of cell suspension was added to the upper chamber, and 600. mu.L of culture medium containing 20% FBS was added to the lower chamber, and the cells were cultured for 24 hours. After the culture is finished, the residual cells in the chamber are lightly wiped by a cotton swab, 600 mu L of methanol is added in the lower chamber for fixing the chamber, the fixing solution is absorbed after 15min, 600 mu L of crystal violet staining solution is added for staining for 15min, then PBS is washed for 1-2 times, the chamber is air-dried in the dark, the chamber is observed under an inverted microscope, 5 visual fields are randomly selected for each sample to take a picture, and the number of migration is counted.
Detecting the invasion capacity of the cells by adopting a Transwell method;
matrigel gel was diluted with FBS-free medium at a dilution ratio of 1: 8. Adding 50 μ L of diluent into each well of the chamber, refrigerating at 4 deg.C, standing until the next dayAnd (4) testing and using. Digesting the cells infected by non-infected HEEC and AFB1 for 30 generations by pancreatin, centrifuging, and adjusting the cell density of each group to 3 × 10 with FBS-free culture solution 5 One well, 200. mu.L of cell suspension was added to the upper chamber, and 600. mu.L of 50% FBS-containing medium was added to the lower chamber. After 24h of incubation, the subsequent steps were as in 5.
Example 1: the HEEC is continuously intervened to the 20 th generation for a long time by using a 1mol/LAFB1 solution, the proliferation rate of the HEEC is 14.31 +/-6.20 percent by adopting a CCK8 method, the G0/G1 phase ratio in the cell cycle of the HEEC is 56.78 +/-0.89 percent by adopting a flow cytometer, the S phase ratio is 33.60 +/-0.93 percent by adopting the flow cytometer, and the apoptosis rate of the HEEC is 18.63 +/-0.36 percent by adopting the flow cytometer.
Example 2: the HEEC is continuously intervened to the 30 th generation for a long time by using a 1mol/LAFB1 solution, the proliferation rate of the HEEC is 45.79 +/-1.12 percent by adopting a CCK8 method, the G0/G1 phase ratio in the cell cycle of the HEEC is 47.44 +/-0.92 percent by adopting a flow cytometer, the S phase ratio is 43.69 +/-0.58 percent by adopting the flow cytometer, and the apoptosis rate of the HEEC is 9.17 +/-0.60 percent by adopting the flow cytometer.
As a result, the long-term continuous intervention of HEEC to passage 30 by using 1mol/LAFB1 solution increases the proliferation capacity of cells, the cell cycle is converted from G0/G1 phase to S phase, and the apoptosis is reduced.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (9)
1. A method for constructing an esophageal epithelial cell precancerous lesion cell model is characterized by comprising the following steps:
step one, HEEC is cultured;
step two, contamination treatment: dissolving AFB1 powder in DMSO to obtain 10mmol/L mixed solution, and adding complete culture solution to dilute the mixed solution to obtain 1 μmol/L AFB1 venom;
seeding the HEEC at 40% density for 25cm 2 Culturing overnight in a culture bottle, treating with AFB1 after HEEC is attached to the wall and recovers the shape, carrying out passage, namely treating the first generation cells with AFB1, repeatedly treating with AFB1 after the cells recover the shape and are attached to the wall overnight, and repeatedly culturing for 30 generations, wherein each treatment for 5 generations, carrying out liquid nitrogen cryopreservation on the corresponding cells, and the treating with AFB1 is to add 1 mu mol/L AFB1 staining solution into HEEC to culture for 48-72 hours.
2. The method for constructing the esophageal epithelial cell precancerous lesion thin cell model according to claim 1, wherein the HEEC culturing step is as follows: HEEC were placed in complete medium and placed in 5% CO 2 And 37 ℃, wherein the complete culture solution comprises high-sugar DMEM culture solution, FBS and double antibody, and the high-sugar DMEM culture solution comprises: FBS: the volume ratio of the double antibody is 10: 1: 0.1, 100U/ml streptomycin and 100U/ml penicillin for double antibody.
3. The method for constructing the esophageal epithelial cell precancerous lesion thin cell model according to claim 2, wherein the construction method comprises the step of detecting HEEC activity after infection treatment by using a CCK8 method.
4. The method for constructing the esophageal epithelial cell precancerous lesion thin cell model according to claim 3, wherein the step of detecting HEEC activity after infection treatment comprises the following steps: HEEC at 6X 10 3 The cells were seeded in 96-well plates at a density of 100. mu.L/well and placed at 37 ℃ in 5% CO 2 Culturing in an incubator; after 24h, respectively adding 100 mu L of AFB1 culture solution containing 0, 0.01, 0.1, 1, 10, 100 and 1000 mu mol/L into the plate, wherein each group comprises 5 multiple wells, and setting blank wells for 24h to infect; then the culture solution is discarded, 100 mu L of DMEM culture solution is replaced, 10 mu L of CCK8 solution is added into each interference hole in an adherence manner, the mixture is incubated in an incubator for 1.5h, the mixture is placed in a microplate reader CCK8 mode to measure the OD value of each hole, and the recorded result is averaged.
5. The method for constructing a pre-cancerous esophageal epithelial cell line model according to claim 4,the step of detecting the HEEC viability after the contamination treatment comprises detecting the cell viability every 5 passages after the intervention of AFB 1: will contain AFB 1 The 5 th, 10 th, 15 th, 20 th, 25 th and 30 th generation cells infected by the culture medium are 6 x 10 th 3 The cells were seeded in 96-well plates at a density of 100. mu.L/well and placed at 37 ℃ in 5% CO 2 Culturing for 48h in an incubator, discarding the culture solution, changing to 100 mu L of complete culture solution, immediately adding 10 mu L of CCK8 solution to each interference hole for adherence, incubating for 1.5h in the incubator, measuring the OD value of each hole in a CCK8 mode of an enzyme labeling instrument, recording the result, and taking an average value.
6. The method for constructing the esophageal epithelial cell precancerous thin cell model according to claim 5, wherein the construction method comprises the following steps of calculating the survival rate of cells: cell viability was 100% (test OD-blank OD)/(control OD-blank OD).
7. The method for constructing the esophageal epithelial cell precancerous thin cell model according to claim 6, wherein the construction method comprises detecting cell cycle by flow cytometry.
8. The method for constructing the esophageal epithelial cell precancerous lesion thin cell model as claimed in claim 7, wherein the method comprises detecting apoptosis by a flow cytometer, detecting migration ability of cells by a Transwell method, and detecting invasion ability of cells by the Transwell method.
9. The method for constructing the esophageal epithelial cell precancerous lesion cell model according to claim 8, wherein the step of detecting the invasion capacity of the cells by adopting a Transwell method comprises the following steps: diluting Matrigel gel with a FBS-free culture solution in a dilution ratio of 1: 8; adding 50 mu L of diluent into each hole of the small chamber, refrigerating and standing at 4 ℃ until the experiment is used the next day; digesting the cells infected by non-infected HEEC and AFB1 for 30 generations by pancreatin, centrifuging, and adjusting the cell density of each group to 3 × 10 with FBS-free culture solution 5 One/well, 200. mu.L of cell suspension was added to the upper chamber, and the lower chamber, after the gel was spread600 μ L of 50% FBS-containing medium.
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| CN106110336A (en) * | 2016-08-12 | 2016-11-16 | 郑州大学 | A kind of construction method of esophageal carcinoma chemoprophylaxis research mode |
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| CN106110336A (en) * | 2016-08-12 | 2016-11-16 | 郑州大学 | A kind of construction method of esophageal carcinoma chemoprophylaxis research mode |
| CN108018258A (en) * | 2016-11-04 | 2018-05-11 | 江苏齐氏生物科技有限公司 | A kind of isolation and culture method of primary people's esophageal epithelial cell |
| CN109295156A (en) * | 2018-09-17 | 2019-02-01 | 国家食品安全风险评估中心 | Chemicals induce the method for building up of the vitro detection model of human esophagus cancer |
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| 付凌萌等: "木犀草素与叶酸对黄曲霉毒素B_1致食管上皮细胞毒性及MTHFR基因高甲基化的影响", 《中国优秀硕士论文库医药卫生科技辑》 * |
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