CN114854669A - A method for constructing a cell model of precancerous lesions of esophageal epithelial cells - Google Patents

Abstract

The invention relates to the technical field of cell model construction methods, in particular to a construction method for a cell model of precancerous lesions of esophageal epithelial cells. The following scheme is now proposed, which includes using 1 μmol/LAFB1 solution to continuously intervene HEEC to the 30th generation for a long time, and adopting the CCK8 method. To detect the effect of AFB1 exposure on HEEC viability of different passages, flow cytometry was used to detect cell apoptosis and cell cycle of different passages, and Transwell method was used to detect the invasion and migration ability of 30 passages of exposed cells. The present invention further explores the relevant molecular mechanism of AFB1 in the occurrence process of esophageal cancer, 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 the esophagus.

Description

A method for constructing a cell model of precancerous lesions of esophageal epithelial cells

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 technique

According to the report of the International Agency for Research on Cancer, the global incidence of esophageal cancer ranks seventh among all cancers, and the total mortality ranks sixth among all cancers. Environment, dietary habits, and lifestyle play an important role in the occurrence of esophageal cancer. Among them, mycotoxin infection is considered to be one of the important risk factors for the occurrence of esophageal cancer in residents of high-incidence areas of esophageal cancer in China. Aflatoxin B1 is the most common and the most toxic mycotoxin in food, and AF B1 was defined as a class I carcinogen by IARC in 1993. A case-control study suggests that high exposure to aflatoxin B1 may be an important risk factor for esophageal precancerous lesions in Huai'an, China, an area with a high incidence of esophageal cancer. In recent years, the establishment method of esophageal precancerous lesion cell model is rarely seen, and most of them use methylbenzylnitrosamine to induce the establishment of esophageal precancerous lesion animal model. Therefore, the present invention proposes a precancerous esophageal epithelial cell Methods of constructing diseased cell models.

SUMMARY OF THE INVENTION

In order to solve the problems in the prior art, the present invention provides a method for constructing an esophageal epithelial cell precancerous lesion cell model.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for constructing an esophageal epithelial cell precancerous lesion cell model, comprising the following steps:

Step 1, culturing HEEC;

Step 2, poisoning treatment: dissolving the AFB1 powder in DMSO to obtain a 10 mmol/L mixed solution, and then adding the complete culture solution to dilute the mixed solution to obtain a 1 μmol/LAFB1 poisoning solution;

The HEECs were inoculated into a 25cm ² culture flask at a density of 40% and cultured overnight. After the HEECs adhered to the walls and recovered their shape, they were treated with AFB1 and passaged. This was the first generation of cells treated with AFB1. AFB1 treatment was performed, and the culture was repeated for 30 generations, wherein the corresponding cells were cryopreserved in liquid nitrogen for each treatment for 5 generations. The AFB1 treatment was performed by adding 1 μmol/LAFB1 venom solution to HEECs and cultured for 48-72 hours.

Further, the step of culturing HEECs is: placing HEECs in a complete culture medium and in an incubator with 5% CO ₂ and 37° C. The complete culture medium includes high-glucose DMEM medium, FBS and double antibody , the volume ratio of high glucose DMEM medium: FBS: double antibody is 10: 1: 0.1, and the double antibody is 100U/ml streptomycin and 100U/ml penicillin.

Further, the construction method includes using the CCK8 method to detect the HEEC activity after the exposure treatment.

Further, the steps of detecting the HEEC viability after exposure treatment include the following: inoculating HEECs at a density of 6×10 ³ /well in a 96-well plate, with a volume of 100 μL per well, and placing them in a 37°C, 5% CO ₂ incubator After 24 hours, 100 μL of AFB1 medium containing 0, 0.01, 0.1, 1, 10, 100, and 1000 μmol/L were added to the plate, 5 replicate wells in each group, and blank wells were set to infect them for 24 hours; then discarded The culture medium was replaced with 100 μL of complete culture medium, and then 10 μL of CCK8 solution was added to the wall of each intervention well, incubated in the incubator for 1.5 h, placed in the CCK8 mode of the microplate reader to measure the OD value of each well, and the recorded results were averaged.

Further, the step of detecting the viability of HEECs after exposure treatment includes detecting the viability of cells at every 5th passage after AFB1 intervention: cells at passages 5, 10, 15, 20, 25, and 30 infected with AFB1 _- containing culture medium were 6×10 ³ cells/well were inoculated into 96-well plates, with a volume of 100 μL per well, and placed in a 37°C, 5% CO ₂ incubator for 48 h, then discarded the culture medium and replaced it with 100 μL of complete culture medium. Add 10 μL of CCK8 solution to the wall of the intervention well, be careful to avoid bubbles, incubate in the incubator for 1.5 h, place the microplate reader in CCK8 mode to measure the OD value of each well, record the results and take the average value.

Further, the construction method includes calculating the cell survival rate: cell survival rate=(OD of test group-OD of blank group)/(OD of control group-OD of blank group)×100%.

Further, the construction method includes using flow cytometry to detect cell cycle.

Further, the construction method includes detecting cell apoptosis by flow cytometry, detecting cell migration ability by Transwell method, and detecting cell invasion ability by Transwell method.

Further, using the Transwell method to detect the invasive ability of cells includes: diluting Matrigel gel with FBS-free culture medium at a dilution ratio of 1:8; adding 50 μL of diluent to each well of the chamber, refrigerating at 4°C and standing until the next day for experimental use ; Uninfected HEEC and AFB1-infected cells for 30 passages were digested with trypsin and centrifuged, and the cell density of each group was adjusted to 3×10 ⁵ cells/well with culture medium without FBS. 200 μL of cell suspension was added, and 600 μL of culture medium containing 50% FBS was added to the lower chamber.

Beneficial effects of the present invention:

The invention discloses a method for long-term intervention of normal esophageal epithelial cells (HEEC) by aflatoxin B1 (AFB1) into an esophageal precancerous lesion cell model. , CCK8 method was used to detect the effect of AFB1 exposure on HEEC viability of different passages, flow cytometry was used to detect cell apoptosis and cell cycle of different passages, and Transwell method was used to detect the invasion and migration ability of 30 passages of exposed cells. Western blot was used to detect the expressions of EMT-related proteins (E-cadherin, N-cadherin) and cell cycle-related proteins (cyclin A2, cyclin D1 and CDK2). The present invention further explores the relevant molecular mechanism of AFB1 in the occurrence process of esophageal cancer, 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 the esophagus.

Description of drawings

Fig. 1 is that the present invention adopts CCK8 method to detect the relationship schematic diagram of the vitality situation of HEEC;

Fig. 2 is the relational schematic diagram that the present invention adopts CCK8 method to detect the proliferation situation of HEEC;

3 is a schematic diagram of the relationship between the present invention using flow cytometry to detect the cell cycle of HEEC;

4 is a schematic diagram of the relationship between the present invention using flow cytometry to detect the apoptosis of HEEC;

Fig. 5 is the relational schematic diagram that the present invention adopts Transwell method to detect the migration ability of HEEC;

Fig. 6 is the relational schematic diagram that the present invention adopts Transwell method to detect HEEC invasion ability;

FIG. 7 is a schematic diagram of the overall flow of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments.

The meanings of the abbreviations in the present invention are: HEEC: normal esophageal epithelial cells, AFB1: aflatoxin B1, PBS: phosphate buffered solution, DMSO: dimethyl sulfoxide, DMEM: dulbecco's modified eagle medium;

A method for constructing an esophageal epithelial cell precancerous lesion cell model, as shown in Figure 7, uses AFB1 to continuously intervene HEEC for a long time. The specific method is as follows:

(1) Cell culture

Prepare cell culture medium with high glucose DMEM medium, FBS (fetal bovine serum), and double antibody (100U/ml streptomycin, 100U/ml penicillin) in a ratio of 10:1:0.1, placed in 5% _CO2 , in a 37°C incubator, culture HEEC with the prepared complete culture medium, and when the cell density reaches 80-90%, operations such as passage, experiment or cryopreservation can be performed.

(2) Poisoning treatment

①Configure AFB1 poisoning liquid mother solution: Dissolve AFB1 powder in DMSO, the concentration is 10mmol/L, if it is used for a long time, it can be placed in a 4°C refrigerator.

②Long-term AFB1 exposure treatment: According to the results of the previous CCK8 cell proliferation experiments, it was determined that the AFB1 exposure concentration was 1 μmol/L to induce malignant transformation of cells. It was diluted to 1 μmol/L with complete culture medium, and it was prepared and used now. The cells were inoculated into a 25cm ² culture flask at a density of about 40%, and cultured overnight. After the cells adhered to the wall and recovered their shape, they were cultured in DMEM medium containing 1 μmol/L AFB1 for 48-72 hours and then passaged. This is the first generation of cells treated with AFB1. After the cells recovered their form and adhered overnight, the same AFB1 treatment was repeated, and the culture was repeated for 30 generations. The corresponding cells were cryopreserved in liquid nitrogen for each treatment for 5 passages.

The cell viability and proliferation can be detected by CCK8 method.

(1) Measure HEEC activity after AFB1 intervention

HEECs were seeded in a 96-well plate at a density of 6×10 ³ cells/well, with a volume of 100 μL per well, and cultured in a 37° C., 5% CO ₂ incubator. After 24 hours, 100 μL of AFB1 medium containing 0, 0.01, 0.1, 1, 10, 100, and 1000 μmol/L were added to the plate, 5 replicate wells in each group, and blank wells (without cells, only with culture medium, In order to remove the background value) exposure for 24 hours; then discard the culture medium and replace it with 100 μL of complete culture medium, then add 10 μL of CCK8 solution to the wall of each intervention well, be careful to avoid bubbles, incubate in the incubator for 1.5 hours, and place it on a microplate reader The OD value of each well was measured in CCK8 mode, and the results were recorded as the average value.

Repeat the above steps for 3 times. Calculate the cell viability: cell viability=(OD of test group-OD of blank group)/(OD of control group-OD of blank group)×100%.

(2) Measure cell viability every 5 passages after AFB1 intervention

Cells at passages 5, 10, 15, 20, 25, and 30 infected with AFB1-containing culture medium were seeded in a 96-well plate at a density of 6×10 ³ cells/well, with a volume of 100 μL per well, at 37°C, 5 After culturing in a %CO ₂ incubator for 48 hours, discard the culture medium and replace it with 100 μL of complete culture medium, then add 10 μL of CCK8 solution to each intervention well to adhere to the wall, be careful to avoid air bubbles, incubate in the incubator for 1.5 hours, and place it on a microplate reader. The OD value of each well was measured in CCK8 mode, and the results were recorded as the average value.

Repeat the above steps for 3 times. Calculate the cell proliferation rate: cell proliferation rate=(OD of test group-OD of blank group)-(OD of control group-OD of blank group)/(OD of control group-OD of blank group)×100%.

Cell cycle detection by flow cytometry;

Three parallel samples were set for each group of cells, and the cells containing AFB1 culture medium were infected at passages 0, 5, 10, 15, 20, 25, and 30 in 25cm ² culture flasks, containing 3 mL of culture medium, and placed at 37 °C , 5% CO ₂ saturated humidity incubator culture. After the cells adhered and grown for 48 h, the cells in each group were collected for flow cytometry. Wash twice with PBS, then add 1 ml of EDTA containing 0.25% EDTA to trypsinize the cells, stop the digestion after the cells are all shrunken and rounded with a small amount of floating, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, and wash with PBS for two more times. After the pass, centrifuge at 1000rpm for 5min, adjust the cell density to 1×10 ⁶ cells, transfer to a 1.5mL EP tube, and add 500μL of 70% ice ethanol (prepared in advance and pre-cooled at 4°C) dropwise. The EP tubes of each group were placed in the refrigerator at 4°C overnight, the ice ethanol was discarded by centrifugation the next day, washed once with PBS, and then put into 500 μL of the pre-prepared dye solution (Rnase enzyme: PI=1:9) , Gently blow and beat to mix evenly, place in the dark for 30 min, suck up the single cell suspension, pass through a 300-mesh sieve, and detect by flow cytometry.

Cell apoptosis was detected by flow cytometry;

Five parallel samples were set for each group of cells, and the cells at passages 0, 5, 10, 15, 20, 25, and 30 containing AFB1 culture medium were seeded in 25cm ² culture flasks, containing 3ml of culture medium, and placed in 37 ℃, 5% CO ² saturated humidity incubator culture. After the cells adhered and grew for 48 h, the cells in each group were collected for flow cytometry. Collect the culture medium of each group, wash the cells with PBS and collect them, add 1 ml of EDTA-free trypsin to digest the cells, stop the digestion when the cells are all shrunken and round with a small amount of floating, centrifuge at 1200 rpm for 5 min, and then wash twice with PBS After centrifugation, the supernatant was discarded, and an appropriate volume of Binding buffer was added to ensure that the cell density was 5×10 ⁵ cells, and gently pipetted. Afterwards, 100-200 μL of single cell suspension was added with 5 μL AnnexinV-FITC and 5 μLPI, incubated in the dark for 20 min, and then detected by flow cytometer.

The migration ability of cells was detected by Transwell method;

Digest and centrifuge and collect uninfected HEEC, AFB1-infected cells for 30 passages. The cells were resuspended in PBS, centrifuged again, and FBS-free culture medium was added to make a single cell suspension. The cell density of each group was adjusted to 5×10 ⁴ cells/well. 600 μL of culture medium was incubated for 24 h. After the incubation, the residual cells in the chamber were gently wiped with a cotton swab, and 600 μL of methanol was added to the lower chamber to fix the chamber. After 15 minutes, the fixative was aspirated, and 600 μL of crystal violet staining solution was added for staining for 15 minutes. Air-dried, observed under an inverted microscope, and 5 fields of view were randomly selected for each sample to take pictures, and the number of migration was counted.

The invasive ability of cells was detected by Transwell method;

Dilute Matrigel gel with FBS-free medium at a dilution ratio of 1:8. Add 50 μL of diluent to each well of the chamber, refrigerate it at 4°C, and use it until the next day. The uninfected HEEC and AFB1-infected cells for 30 passages were digested with trypsin and centrifuged. The cell density of each group was adjusted to 3×10 ⁵ cells/well with culture medium without FBS, and added to the upper chamber where the gel was laid. 200 μL of cell suspension, 600 μL of culture medium containing 50% FBS was added to the lower chamber. After culturing for 24h, the following steps are the same as 5.

Example 1: Using 1mol/LAFB1 solution to continuously intervene HEECs for a long time to the 20th passage, using the CCK8 method to detect the proliferation rate of HEECs at 14.31±6.20%, and using flow cytometry to detect the proportion of G0/G1 phase in the cell cycle of HEECs 56.78±0.89%, S phase accounted for 33.60±0.93%, and the apoptosis rate of HEEC detected by flow cytometry was 18.63±0.36%.

Example 2: Using 1mol/LAFB1 solution to continuously intervene HEECs for a long time to the 30th passage, using the CCK8 method to detect the proliferation rate of HEECs of 45.79±1.12%, and using flow cytometry to detect the proportion of G0/G1 phase in the cell cycle of HEECs 47.44±0.92, the proportion of S phase was 43.69±0.58%, and the apoptosis rate of HEEC detected by flow cytometry was 9.17±0.60%.

The results showed that the long-term continuous intervention of HEEC with 1mol/LAFB1 solution to the 30th passage increased the proliferation ability of the cells, transformed the cell cycle from the G0/G1 phase to the S phase, and decreased cell apoptosis.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

Claims (9)

1. a construction method of esophageal epithelial cell precancerous lesion cell model, is characterized in that, comprises the steps: Step 1, culturing HEEC; Step 2, poisoning treatment: dissolving the AFB1 powder in DMSO to obtain a 10 mmol/L mixed solution, and then adding a complete culture solution to dilute the mixed solution to obtain a 1 μmol/L AFB1 poisoning solution; The HEECs were inoculated into a 25cm ² culture flask at a density of 40% and cultured overnight. After the HEECs adhered to the walls and recovered their shape, they were treated with AFB1 and passaged. This was the first generation of cells treated with AFB1. AFB1 treatment was carried out, and the culture was repeated for 30 generations, wherein the corresponding cells were cryopreserved in liquid nitrogen for each treatment for 5 generations. The AFB1 treatment was carried out by adding 1 μmol/L AFB1 poisoning solution to HEECs and cultured for 48-72 hours. 2. the construction method of a kind of esophageal epithelial cell precancerous lesion cell model according to claim 1, is characterized in that, the step of described cultivating HEEC is: HEEC is placed in complete culture liquid and placed in 5%CO ₂ . , in an incubator at 37°C, the complete culture medium includes high-glucose DMEM medium, FBS and double antibody, the volume ratio of high-glucose DMEM medium: FBS: double antibody is 10: 1: 0.1, and the double antibody is 100U/ ml of streptomycin and 100 U/ml of penicillin. 3 . The method for constructing a cell model of esophageal epithelial cell precancerous lesions according to claim 2 , wherein the method for constructing comprises adopting the CCK8 method to detect the HEEC viability after the exposure treatment. 4 . 4. The method for constructing a cell model of esophageal epithelial cell precancerous lesions according to claim 3, wherein the step of detecting the HEEC viability after the exposure treatment comprises the following steps: dividing the HEECs with 6×10 ³ cells/well Density inoculated in 96-well plates, each well volume of 100μL, placed in a 37 ℃, 5% CO ₂ incubator for culture; L of AFB1 culture medium, 5 duplicate wells in each group, and set blank wells for exposure to the virus for 24 hours; then discard the culture medium and replace it with 100 μL DMEM medium, then add 10 μL CCK8 solution to the wall of each intervention well, and incubate in the incubator for 1.5 h, place the microplate reader in CCK8 mode to measure the OD value of each well, and record the results to take the average value. 5. the construction method of a kind of esophageal epithelial cell precancerous lesion cell model according to claim 4, is characterized in that, the step of detecting the HEEC viability after exposure treatment comprises detecting AFB1 intervening cell viability every 5 generations: Cells at passages 5, 10, 15, 20, 25, and 30 infected with AFB ₁ culture medium were seeded in a 96-well plate at a density of 6×10 ³ cells/well, with a volume of 100 μL per well, and placed at 37°C, 5 After culturing in a %CO ₂ incubator for 48 hours, discard the culture medium and replace it with 100 μL of complete culture medium, then add 10 μL of CCK8 solution to the wall of each intervention well, incubate in the incubator for 1.5 hours, and place the microplate reader in CCK8 mode to measure each Hole OD value, record the results and take the average value. 6. the construction method of a kind of esophageal epithelial cell precancerous lesion cell model according to claim 5, is characterized in that, described construction method comprises calculating cell survival rate: cell survival rate=(test group OD-blank group OD) /(OD of control group-OD of blank group)×100%. 7 . The method for constructing an esophageal epithelial cell precancerous lesion cell model according to claim 6 , wherein the constructing method comprises using flow cytometry to detect cell cycle. 8 . 8. the construction method of a kind of esophageal epithelial cell precancerous lesion cell model according to claim 7, is characterized in that, described construction method comprises adopting flow cytometer to detect cell apoptosis, adopting Transwell method to detect the migration ability of cell . The invasive ability of cells was detected by Transwell method. 9. the construction method of a kind of esophageal epithelial cell precancerous lesion cell model according to claim 8, adopting the Transwell method to detect the invasive ability of the cell comprises: dilute Matrigel glue with the culture medium without FBS, and the dilution ratio is 1:8; Add 50 μL of diluent to each well of the chamber, and refrigerate at 4°C until the next day for experimental use; uninfected HEEC and AFB1-infected cells for 30 passages were digested with trypsin, centrifuged, and adjusted with culture medium without FBS. The cell density of each group was 3×10 ⁵ cells/well, 200 μL of cell suspension was added to the upper chamber covered with glue, and 600 μL of culture medium containing 50% FBS was added to the lower chamber.

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