Malignant transformed cell strain induced by benzo [g, h, i] perylene and application thereof

Abstract

The invention discloses a malignant transformed cell strain induced by benzo [g, h, i] perylene and application thereof. The cell strain is preserved in Guangdong Microbial Culture Collection Center on June 27, 2024, and the preservation number is GDMCC No: 64810. The inventor adopts benzo [g, h, i] perylene with environmental exposure concentration to continuously act on human lung bronchial epithelial cells to construct an in-vitro malignant transformed cell strain, the in-vitro malignant transformed cell strain is successfully constructed through identification of a soft agar clone formation experiment and a mouse-loaded tumorigenesis experiment, and the model is subjected to multiplication capacity, apoptosis, period, invasion and migration capacity and DNA damage degree detection, so that the in-vitro malignant transformed cell strain is obtained. The general characteristics of the metabolite cells are met. The in-vitro malignant transformed cell strain / cell model provides more intuitive evidence for the carcinogenicity research of benzo [g, h, i] perylene and provides an experimental basis for the subsequent research of the carcinogenic mechanism of benzo [g, h, i] perylene.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and more specifically to a malignant transformation cell line induced by benzo[g,h,i]perylene and application thereof. Background Art

[0002] As a recognized carcinogen, the research on carcinogenicity of PAHs has received extensive attention from the scientific community. Currently, the U.S. Environmental Protection Agency and the European Commission have listed 16 PAHs as environmental priority pollutants, but the China Ambient Air Quality Standard (GB3095-2012) only stipulates annual average and 24-hour average limits for benzo[a]pyrene among PAHs. As early as 2012, the International Agency for Research on Cancer (IARC) listed benzo[a]pyrene as a Class 1 carcinogen. Therefore, most of the current research on the carcinogenicity and mechanism of PAHs is focused on benzo[a]pyrene, and it is unknown whether other PAHs are also carcinogenic.

[0003] Benzo(g,h,i)perylene, as a cyclized hexacyclic aromatic hydrocarbon, is also one of the 16 priority PAHs. In recent years, many studies have shown that the pollution level of benzo(g,h,i)perylene in the environment is high, and even in some cities, its environmental exposure concentration is higher than that of benzo(a)pyrene. my country's air quality monitoring data in 2019 also showed that benzo(g,h,i)perylene is one of the three most abundant types of PAHs in my country. Since a study by the International Agency for Research on Cancer (IARC) in 1983 showed that benzo(g,h,i)perylene is not carcinogenic to humans and animals, research on benzo(g,h,i)perylene has basically come to a standstill. However, in 1993, there was a document pointing out that benzo(g,h,i)perylene has significant DNA binding ability both in vivo and in vitro, speculating that it may be a weak tumor initiator. In 2001, a study also pointed out that benzo[g,h,i]perylene would enhance the carcinogenicity of benzo[g,h,i]perylene through a synergistic effect, but since then, research on the carcinogenicity of benzo[g,h,i]perylene has stagnated.

[0004] At present, the evaluation of the carcinogenicity of environmental pollutants mainly relies on population epidemiological surveys and animal experiments, but the credibility of epidemiological survey results depends on strict design and is limited and interfered by many factors; animal carcinogenicity tests are expensive and time-consuming, so it is difficult to meet actual needs. In recent years, the method of using exogenous compounds to induce cell malignant transformation in vitro to construct a cell malignant transformation model has become a faster and more efficient method for screening carcinogens and studying carcinogenic mechanisms. At present, there is still some controversy about whether benzo[g,h,i]perylene is carcinogenic, and the evidence that it is carcinogenic is not direct enough. Therefore, in order to clarify whether benzo[g,h,i]perylene is carcinogenic and further explore its carcinogenic mechanism, it is necessary to provide a cell malignant transformation model for research. Patent CN 105802915 A discloses a cell that undergoes malignant transformation. The cell has a preservation number of CCTCC NO: C2014243 in the China Center for Type Culture Collection. The cell is obtained by inducing malignant transformation of the cell in vitro using benzo[a]pyrene. However, the cell cannot be used for the study of benzo[g,h,i]perylene. Therefore, it is urgent to develop an in vitro cell malignant transformation model specifically for benzo[g,h,i]perylene. Summary of the invention

[0005] The purpose of the present invention is to overcome the above defects and deficiencies in the prior art and provide a malignant transformation cell line induced by benzo[g,h,i]perylene. An in vitro cell malignant transformation model is constructed by long-term exposure of benzo[g,h,i]perylene to human lung bronchial epithelial cells, aiming to lay an experimental foundation for clarifying whether benzo[g,h,i]perylene is carcinogenic and its carcinogenic mechanism, overcoming the problems of the existing technology that the population epidemiological survey is limited by many factors, the animal cancer induction experiment is expensive, and the in vitro cell malignant transformation model of benzo[g,h,i]perylene is lacking.

[0006] The second object of the present invention is to provide a method for constructing the above-mentioned malignant transformed cell line.

[0007] The third object of the present invention is to provide the application of the above-mentioned malignant transformed cell line.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a malignant transformed cell line induced by benzo[g,h,i]perylene, and the cell line was deposited in Guangdong Provincial Microbiological Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City on June 27, 2024, with a deposit number of GDMCC No: 64810.

[0010] The present invention selects benzo[g,h,i]perylene as a research object, uses environmental exposure concentration of benzo[g,h,i]perylene to continuously act on human lung bronchial epithelial cells, and constructs an in vitro malignant transformation model. This malignant transformation model has been successfully constructed through soft agar cloning experiment and mouse tumor formation experiment. At the same time, a series of in vitro experiments were used to detect the cell functions of the cell model, and it was found that the proliferation ability of cells induced by benzo[g,h,i]perylene to transform was significantly enhanced, the apoptosis rate was decreased, the cell cycle was disordered, and the invasion and migration ability were significantly enhanced, accompanied by the occurrence of DNA damage, indicating that the malignant transformation model has similar characteristics to the currently confirmed malignant cells and cancer cells, and can be used for the study of the biological effects and molecular mechanisms of benzo[g,h,i]perylene, the study of the carcinogenic mechanism of benzo[g,h,i]perylene, or the screening / evaluation of cancer treatment drugs caused by benzo[g,h,i]perylene.

[0011] The present invention also provides a method for constructing the above-mentioned malignant transformation cell line, which comprises using 48-52 ng / mL of benzo[g,h,i]perylene to infect and culture normal human lung bronchial epithelial cells, monitoring the cell proliferation ability every 23-27 generations of infection, and identifying the malignant transformation of the cells after 198-202 generations of infection, thereby obtaining an in vitro cell line induced by benzo[g,h,i]perylene.

[0012] Furthermore, the concentration of benzo[g,h,i]perylene used was 50 ng / mL.

[0013] Furthermore, the human normal lung bronchial epithelial cells are human lung bronchial epithelial cells BEAS-2B.

[0014] Furthermore, the culture medium used for the poisoning culture is DMEM / F12 complete culture medium containing 5% fetal bovine serum and a benzo[g,h,i]perylene concentration of 50 ng / mL.

[0015] Furthermore, the monitoring of cell proliferation ability is performed using a cell activity detection test and a plate clone formation experiment.

[0016] Furthermore, the malignant transformation identification is carried out using soft agar colony formation experiment and mouse tumor formation experiment.

[0017] Furthermore, the proliferation capacity of cells was monitored every 25 generations of exposure, and the malignant transformation of cells was identified after 200 generations of exposure.

[0018] As a preferred embodiment, the method for constructing an in vitro cell line induced by benzo[g,h,i]perylene comprises the following steps:

[0019] (1) Determination of exposure concentration: The average daily exposure concentration of benzo[g,h,i]perylene in the air of Guangzhou from 2016 to 2021 ranged from 0.045 to 4.223 ng / m 3 The volume of air inhaled into the human body every day is 20m 3 , as well as the airway and lung fluid volume of 12.4mL and the effective deposition rate on the human lung surface of 10%, the annual intake concentration of benzo[g,h,i]perylene was obtained to be 2.649-248.612ng / mL; 5 gradient concentrations (0, 5, 50, 500, 5000ng / mL) of benzo[g,h,i]perylene were set up to act on human lung bronchial epithelial cells (BEAS-2B cells);

[0020] (2) After 24 hours of exposure, the relative activity of cells in each dose group was detected using a cell activity detection test (CCK-8 test), and 50 ng / mL was determined as the final exposure concentration;

[0021] (3) Construction of in vitro chronic malignant transformation model: The original culture medium of BEAS-2B cells in the logarithmic growth phase was discarded, and the infected culture medium was added and continued to be cultured for 24 hours. This was the first generation of infection, recorded as P1; 24 hours after infection, the cells were passaged, and when the cell confluence reached 80% again, the infection operation was performed again, recorded as P2, and so on;

[0022] (4) Monitoring of cell proliferation ability: In the process of constructing the in vitro malignant transformation model, the cell proliferation ability was monitored by using a cell activity detection test (CCK-8 test) and a plate clone formation test every 25 generations of exposure;

[0023] (5) Identification of in vitro malignant transformation model: After 200 generations of infection, the malignant transformation of the cell model was identified using soft agar colony formation assay and mouse tumor formation assay.

[0024] Preferably, the step (3) comprises diluting a 50 μg / mL benzo[g,h,i]perylene application solution with a DMEM / F12 complete medium containing 5% fetal bovine serum to obtain a benzo[g,h,i]perylene poisoning medium with a final concentration of 50 ng / mL, while ensuring that the volume fraction of dimethyl sulfoxide (DMSO) is 0.1% to control the effect of the solvent on the cells.

[0025] The present invention also provides the use of the above-mentioned malignant transformed cell strain as a cell model for studying the biological effects and molecular mechanisms of benzo[g,h,i]perylene, a cell model for studying the carcinogenic mechanism of benzo[g,h,i]perylene, or a cell model for screening / evaluating cancer therapeutic drugs caused by benzo[g,h,i]perylene.

[0026] The present invention also provides the use of the above-mentioned malignant transformed cell strain in the preparation of a drug for treating cancer caused by benzo[g,h,i]perylene.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention provides a malignant transformed cell line induced by benzo[g,h,i]perylene, which was deposited in Guangdong Microbiological Culture Collection Center on June 27, 2024, with a deposit number of GDMCC No: 64810. The malignant transformed cell line has significantly enhanced proliferation ability, decreased apoptosis rate, disordered cell cycle, and significantly enhanced invasion and migration ability, accompanied by the occurrence of DNA damage, indicating that the malignant transformed cell line has similar characteristics to the currently confirmed malignant transformed cells and cancer cells, and can be used for the study of the biological effects and molecular mechanisms of benzo[g,h,i]perylene, the study of the carcinogenic mechanism of benzo[g,h,i]perylene, or the screening / evaluation of cancer treatment drugs caused by benzo[g,h,i]perylene.

[0029] (2) The present invention uses exogenous compounds to induce cell malignant transformation in vitro to construct a cell malignant transformation model. Compared with animal carcinogenicity tests, this method can screen carcinogens and conduct carcinogenic mechanism research more quickly and efficiently. At the same time, compared with epidemiological studies, the present invention can also provide more direct experimental evidence, and can artificially control exposure factors to avoid the interference of many confounding factors. The establishment of this in vitro malignant transformation model provides more intuitive evidence for the carcinogenicity research of benzo[g,h,i]perylene and provides an experimental basis for subsequent research on its carcinogenic mechanism. At the same time, it reveals the carcinogenicity of other polycyclic aromatic hydrocarbons except for the recognized carcinogen benzo[a]pyrene, providing a more comprehensive insight into the evaluation of the carcinogenicity of polycyclic aromatic hydrocarbons, and laying a theoretical foundation for the control of polycyclic aromatic hydrocarbons and the formulation of relevant environmental air standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The changes in cell viability of BEAS-2B cells after treatment with 0, 5, 50, 500, and 5000 ng / mL benzo[g,h,i]perylene for 24 hours.

[0031] Figure 2 It is used to monitor the cell proliferation ability during the construction of in vitro chronic malignant transformation model. Figure 2 A in the figure is a cell activity detection test (CCK-8 test) to monitor the changes in relative cell activity; B is a plate clone formation experiment to monitor the changes in cell proliferation ability.

[0032] Figure 3 Soft agar colony formation assay to characterize malignant transformation in a cell model induced by benzo[g,h,i]perylene poisoning.

[0033] Figure 4 The present invention is to identify the malignant transformation of the cell model of benzo[g,h,i]perylene poisoning. Figure 4 A is the tumor isolated from the mouse after tumor formation; B is the growth curve of the tumor; C is the weight of the tumor; D is HE staining of tumor tissue; EF is Ki-67 immunohistochemistry.

[0034] Figure 5 This is to detect the cell proliferation ability of BEAS-2B cells after benzo[g,h,i]perylene-induced malignant transformation. Figure 5 A in the middle is a CCK-8 experiment to detect the proliferation ability of cells after malignant transformation; BC is an EdU experiment to detect the proliferation ability of cells after malignant transformation.

[0035] Figure 6 This is the detection of apoptosis and cell cycle of BEAS-2B cells after benzo[g,h,i]perylene induced malignant transformation. Figure 6 AB in the middle is the apoptosis of cells after malignant transformation detected by flow cytometry; CD is the cycle of cells after malignant transformation detected by flow cytometry.

[0036] Figure 7 To detect the cell invasion and migration ability of BEAS-2B cells after benzo[g,h,i]perylene induced malignant transformation. Figure 7 AB in the middle represents the changes in the invasion ability of cells after malignant transformation; CD represents the changes in the migration ability of cells after malignant transformation.

[0037] Figure 8 This is to detect the DNA damage of BEAS-2B cells after benzo[g,h,i]perylene induced malignant transformation. Figure 8 AD is the comet assay to detect DNA damage after cell transformation; EF is the Western blot experiment to detect the changes in the expression level of γ-H2AX after cell transformation; GH is the immunofluorescence experiment to detect the changes in the number of γ-H2AX foci. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] 1. Experimental Materials

[0041] 1.1 Cell lines

[0042] Human lung bronchial epithelial cells BEAS-2B were purchased from the American Type Culture Collection (ATCC).

[0043] 1.2 Test substances

[0044] The analytical standard of benzo[g,h,i]perylene was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China.

[0045] 1.3 Experimental animals

[0046] Twenty 3- to 4-week-old specific pathogen-free (SPF) NCG mice, half male and half female, were quarantined and allowed to acclimatize to the environment for one week. They were randomly divided into a control group and a benzo[g,h,i]perylene group, with 5 male and 5 female mice in each group. The NCG mice were purchased from Guangdong Medicilon Biotech Co., Ltd., and the experimental animal production license number is: SCXK(Yue) 2020-0054.

[0047] 2. Main experimental reagents

[0048] 2.1 Cell resuscitation, culture, medium change, subculture and cryopreservation: DMEM / F12 medium (Gibco, USA), fetal bovine serum (FBS, Shanghai Sijiqing Co., Ltd.), PBS buffer (Zhejiang Senrui Biotechnology Co., Ltd.), trypsin (Guangzhou Jingxin Biotechnology Co., Ltd.), dimethyl sulfoxide (DMSO, MP Biomedicals, USA).

[0049] 2.2 Cell viability detection test (CCK-8 assay): Cell Counting kit-8 (CCK-8 kit, Shanghai Beyotime Co., Ltd.).

[0050] 2.3 Colony formation assay in vitro: 4% paraformaldehyde fixative (Guangzhou Jingxin Biotechnology Co., Ltd.), crystal violet solution (Shanghai Beyotime Co., Ltd.).

[0051] 2.4 Soft agar colony formation assay: Low melting point agarose (Guangzhou Yisheng Technology Co., Ltd., China).

[0052] 2.5 Tumorigenicity assay in nude mice: Matrigel (Corming, USA), 4% paraformaldehyde fixative (Guangzhou Jingxin Biotechnology Co., Ltd.).

[0053] 2.6 HE staining: Xylene and absolute ethanol (Sinopharm Chemical Reagent Co., Ltd.), HE staining kit (Wuhan Servier Biotechnology Co., Ltd.), neutral gum (Sinopharm Chemical Reagent Co., Ltd.).

[0054] 2.7 Immunohistochemistry experiment: anhydrous ethanol, xylene and n-butanol (Sinopharm Chemical Reagent Co., Ltd.), environmentally friendly dewaxing transparent liquid, 20× citric acid antigen retrieval solution, 20× Tris-EDTA antigen retrieval solution, bovine serum albumin (BSA), hematoxylin staining solution, differentiation solution and blue night, mounting glue, histochemistry kit DAB color developer (Wuhan Savier Biotechnology Co., Ltd.).

[0055] 2.8 EdU experiment: 4% paraformaldehyde fixative (Guangzhou Jingxin Biological Company), EdU cell proliferation detection kit (Guangzhou Ruibo Biotechnology Co., Ltd.).

[0056] 2.9 Cell apoptosis: Annexin V-PE / 7-ADD double staining cell apoptosis detection kit (Jiangsu KeyGen Biotechnology Co., Ltd.).

[0057] 2.10 Cell cycle: Cell cycle detection kit (Jiangsu KeyGen Biotech Co., Ltd.).

[0058] 2.11 Transwell invasion assay: Matrigel (Corning, USA), BSA (MPbio, USA).

[0059] 2.12 Single-cell gel electrophoresis experiment (comet assay): sodium hydroxide (NaOH, Shanghai MacLean Biochemical Technology Co., Ltd.), PBS buffer (Zhejiang Senrui Biotechnology Co., Ltd.), sodium chloride (NaCl, Shanghai MacLean Biochemical Technology Co., Ltd.), low melting point agarose and normal melting point agarose (Guangzhou Yishen Technology Co., Ltd., China), analytical grade Tis base (Qingdao Shenggong Biotechnology Co., Ltd., China), disodium ethylenediaminetetraacetic acid (Na2EDTA, MP Biomedicals, USA), DMSO (MP Biomedicals, USA), Triton X-100 (MP Biomedicals, USA), nucleic acid red NA-Red (Guangzhou Biotech Co., Ltd., China).

[0060] 2.13 Protein immunoblotting experiment (Western blot): RIPA cell lysis buffer (strong), PMSF, Enhanced BCA Protein Assay Kit, SDS-PAGE protein loading buffer (5×), 1.5M PH=8.8 Tris-HCl solution, 1MPH=6.8 Tris-HCl solution, transfer buffer, secondary antibody diluent (Guangzhou Biyuntian Company), blocking buffer, Tween-20 (US MPBiomedicals Company), 30% Acr-Bis (29:1), primary antibody diluent, ammonium persulfate (AP), 10% SDS-PAGE electrophoresis buffer, TBS buffer (Wuhan Boster Company), rabbit monoclonal antibody anti-γ-H2AX (UK abcam Company), mouse monoclonal antibody anti-β-Actin, PageRulerTM Prestained Protein Ladder (US Thermo Scientific Company), TEMED, PVDF membrane (US Milipore Company), 800CW Goat anti-Rabbit IgG, 800CW Goat anti-Mouse IgG (LI-COR, USA).

[0061] 2.14 Cell immunofluorescence experiment: PBS buffer (Zhejiang Senrui Biotechnology Co., Ltd.), 4% paraformaldehyde fixative (Guangzhou Jingxin Biological Company), Tritonx-100 permeabilizer (US MP Biomedicals), blocking solution, primary antibody diluent, secondary antibody diluent, cell nucleus staining solution (DAPI), anti-fluorescence quenching sealing solution (Guangzhou Biyuntian Company), rabbit monoclonal antibody anti-γ-H2AX, goat anti-rabbit IgG H&L (Alexa 488)(abcam, UK).

[0062] The present invention selects benzo[g,h,i]perylene as a research object, uses environmental exposure concentration of benzo[g,h,i]perylene to continuously act on human lung bronchial epithelial cells (BEAS-2B), and constructs an in vitro malignant transformation model. This malignant transformation model has been successfully constructed through soft agar cloning experiment and mouse tumor formation experiment. At the same time, a series of in vitro experiments were used to detect the cell functions of the cell model, and it was found that the proliferation ability of the cells induced by benzo[g,h,i]perylene to transform was significantly enhanced, the apoptosis rate was decreased, the cell cycle was disordered, and the invasion and migration ability were significantly enhanced, accompanied by the occurrence of DNA damage, indicating that the malignant transformation model has similar characteristics to the currently confirmed malignant cells and cancer cells.

[0063] Example 1 Determination of the induced exposure concentration of benzo[g,h,i]perylene

[0064] 1. Methods

[0065] The average daily exposure concentration of benzo[g,h,i]perylene in the air of a certain city from 2016 to 2021 ranged from 0.045 to 4.223 ng / m 3 The volume of air inhaled into the human body every day is 20m 3 , as well as the airway and lung fluid volume of 12.4mL and the effective deposition rate on the human lung surface of 10%, the annual intake concentration of benzo[g,h,i]perylene is 2.649-248.612ng / mL. Therefore, 5 gradient concentrations (0, 5, 50, 500, 5000ng / mL) of benzo[g,h,i]perylene were set up to act on human lung bronchial epithelial cells (BEAS-2B cells). After 24 hours of exposure, the relative activity of cells in each dose group was detected using a cell activity detection test (CCK-8 test, the experimental process is as follows).

[0066] Cell activity detection test (CCK-8 experiment): Inoculate cells in a 96-well plate at a density of 2000 cells / well, set 5 replicate wells for each group, and inoculate cells in each group in a 96-well plate, place in a constant temperature incubator and continue to culture for 6-8 hours until the cells adhere to the wall, add 5 gradient concentrations (0, 5, 50, 500, 5000ng / mL) of benzo[g,h,i]perylene to each well, and continue to culture for 24 hours. After the culture is completed, discard the poisoned culture medium, add 110μL of a mixed solution of CCK-8 solution and complete culture medium to each well (CCK-8 solution: complete culture medium = 1:10), continue to place in a constant temperature incubator for 1 hour, and use a multifunctional microplate reader to detect the absorbance at 450nm.

[0067] 2. Results

[0068] The results are as follows Figure 1 As shown in the figure, when the concentration of benzo[g,h,i]perylene was 5ng / mL, the viability of BEAS-2B cells increased instead of decreasing, while the viability of BEAS-2B cells decreased significantly when the concentration reached 500 and 5000ng / mL, indicating that these two concentrations of benzo[g,h,i]perylene caused obvious acute toxic effects on BEAS-2B cells. Only when the concentration of benzo[g,h,i]perylene was 50ng / mL, the viability of BEAS-2B cells decreased to a certain extent, but no statistical difference was observed. Combining the toxicological design principles and relevant literature, 50ng / mL was finally selected as the final exposure concentration.

[0069] Example 2 Construction of in vitro chronic malignant transformation model and monitoring of cell proliferation ability

[0070] 1. Methods

[0071] Exposure method: 25 mg of benzo[g,h,i]perylene standard was dissolved in 5 mL of dimethyl sulfoxide (DMSO) to prepare a 5 mg / mL benzo[g,h,i]perylene stock solution, which was stored at 4°C in the dark. The 5 mg / mL benzo[g,h,i]perylene stock solution was diluted to a 50 μg / mL benzo[g,h,i]perylene application solution, which was diluted with DMEM / F12 complete medium containing 5% fetal bovine serum to a final concentration of 50 ng / mL benzo[g,h,i]perylene exposure medium, while the volume fraction of DMSO was kept at 0.1% to control the effect of the solvent on the cells. When BEAS-2B cells were in the logarithmic growth phase (growth confluence was 80%), the original culture medium was discarded, and the exposure medium was added to continue culturing for 24 hours. This was the first generation of exposure, recorded as P1. The cells were passaged 24 hours after exposure, and when the cells grew again and reached 80% confluence, they were exposed again, which was recorded as P2, and so on.

[0072] In the process of constructing the in vitro malignant transformation model, the cell proliferation ability was monitored using a cell activity detection test (CCK-8 test) and a plate clone formation test every 25 generations of exposure.

[0073] Cell activity detection test (CCK-8 experiment): Inoculate cells in a 96-well plate at a density of 2000 cells / well, set up 5 replicate wells for each group, and inoculate cells in each group in a 96-well plate and place it in a constant temperature incubator for 72 hours. After the culture is completed, discard the original culture medium in the 96-well plate, add 110 μL of a mixed solution of CCK-8 solution and complete culture medium to each well (CCK-8 solution: complete culture medium = 1:10), continue to place it in a constant temperature incubator for 1 hour, and use a multifunctional microplate reader to detect the absorbance at 450nm

[0074] Plate cloning experiment: Cells were digested, centrifuged and counted, and the cell concentration was adjusted to 1×10 4 / mL. Take 10μL of cell suspension (100 cells) and inoculate it in a 6-well plate containing 3mL of culture medium, with 3 replicates in each group. After inoculation, be careful to shake gently to make the cells evenly distributed. After culturing in a constant temperature incubator for 2 weeks, discard the old culture medium in the 6-well plate and wash it twice with PBS buffer, add 4% paraformaldehyde to fix the cells for 20 minutes. Discard the paraformaldehyde and add crystal violet stain to stain the cells for 30 minutes. Then wash it twice with PBS buffer, dry it at room temperature, take pictures and count it, and finally calculate the clone formation rate of the cells. Clone formation rate = (number of clones formed / number of inoculated cells) × 100%.

[0075] 2. Results

[0076] The results are as follows Figure 2As shown in A, the cell activity detection test (CCK-8 experiment) shows that the relative activity of cells exposed from the 50th generation has been differentially enhanced compared with normal BEAS-2B cells, and it continues to increase with the increase of exposure time. Figure 2 As shown in B, the plate cloning experiment also suggests that the cell proliferation ability has been significantly enhanced since the 100th generation, and it also continues to increase with the increase of the exposure time. The above results indicate that in the process of building a cell model, the cell proliferation ability continues to increase with the increase of the exposure time.

[0077] Example 3 Identification of in vitro malignant transformation model and detection of cell phenotype after malignant transformation

[0078] 1. Methods

[0079] Soft agar cloning experiment: Use ultrapure water to prepare 1.2% and 0.7% low-melting point agarose solutions, sterilize them in a high-temperature and high-pressure autoclave, and immediately mix the 1.2% low-melting point agarose solution and DMEM / F12 culture medium containing 10% fetal bovine serum in a volume ratio of 1:1. Inject the mixed solution into a 6-well plate, 2 mL per well, 3 replicates per group, place in a 4°C refrigerator to cool and solidify for 20 minutes, then place in an incubator to rewarm for 10 minutes as the bottom layer of gel. The 0.7% low-melting point agarose solution was placed in a 50°C water bath to prevent solidification, and was subsequently used to prepare the top layer of gel. Take cells in the logarithmic growth phase, digest, centrifuge and count, and adjust the cell density to 3×10 3 / mL. The cell suspension was mixed evenly with 0.7% low melting point agarose solution at a volume ratio of 1:1, and the mixture was injected into the 6-well plate with the bottom layer gel prepared above, 2mL per well, to form a double agar layer. After the upper layer gel solidified, it was placed in a constant temperature incubator and cultured for 4 weeks, with appropriate amount of liquid added 1-2 times a week. The clone formation visible to the naked eye was taken as a positive indicator, and the number of clones formed was taken and counted.

[0080] Tumor formation experiment in mice: 20 SPF-grade NCG mice aged 3-4 weeks, half male and half female, were quarantined and adapted to the environment for one week. They were randomly divided into a control group and a benzo[g, h, i]perylene group, with 5 males and 5 females in each group. They were fed SPF-grade feed and distilled water, with a 24-hour day and night cycle, and maintained at a constant temperature of 25℃±2℃ and a humidity of 50%-70%. Normal BEAS-2B cells (control group) and BEAS-2B cells treated with benzo[g, h, i]perylene for 200 generations (benzo[g, h, i]perylene group) were obtained, washed twice with PBS, resuspended in complete culture medium, counted the number of live cells and adjusted the cell number to 1.0×108 / mL. The matrix gel was placed in an ice bath at 4℃ overnight, and the complete culture medium containing cells and the matrix gel were mixed at a ratio of 1:1. A No. 6 needle syringe was used to subcutaneously inoculate the left armpit of the mouse, and the inoculation volume was 100μL (about 1×10 7The growth of mice was observed, the time of tumor appearance was recorded, the body weight and the long and short diameters of the tumor were recorded every three days, and the tumor volume was calculated (V = long diameter × short diameter). 2 / 2), and a growth curve was drawn. After 63 days, the mice were killed by cervical dislocation, and the tumor was removed and placed in 4% paraformaldehyde for subsequent section analysis. This experiment was approved by the Experimental Animal Ethics Committee of Guangzhou Medical University, number GY2023-311.

[0081] HE staining: First, put the slices in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 75% alcohol for 5 minutes, and wash with tap water. Then put the slices in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, return to blue with blue solution, and rinse with running water. Then put the slices in 95% alcohol for dehydration for 1 minute, and put them in eosin staining solution for 15 seconds. Finally, put the slices in anhydrous ethanol I for 2 minutes, anhydrous ethanol II for 2 minutes, anhydrous ethanol III for 2 minutes, n-butanol I for 2 minutes, n-butanol II for 2 minutes, xylene I for 2 minutes, and xylene II for 2 minutes to make them transparent, and seal the slices with neutral gum. Microscope examination and image acquisition and analysis.

[0082] Immunohistochemistry experiment: Put the slices into environmentally friendly dewaxing solution I for 10 minutes, environmentally friendly dewaxing solution II for 10 minutes, environmentally friendly dewaxing solution III for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, and distilled water for washing. After antigen repair, put the slices into 3% hydrogen peroxide solution and incubate them at room temperature in the dark for 25 minutes to block endogenous peroxidase. Use 3% BSA to block for 30 minutes and add the primary antibody to incubate overnight at 4°C. After washing 3 times with PBS, add the secondary antibody (HRP labeled) of the corresponding species to the primary antibody to cover the tissue and incubate at room temperature for 50 minutes. Add freshly prepared DAB color development solution, control the color development time under a microscope, and rinse the slices with tap water to stop the color development. Hematoxylin counterstains the nucleus, and then put the slices into 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, n-butanol for 5 minutes, and xylene I for 5 minutes to dehydrate, seal the slices with sealing glue, and examine under a microscope.

[0083] Cell activity detection test (CCK-8 experiment): cells were inoculated in 96-well plates at a density of 2000 cells / well, 5 replicates were set for each group, and cells from each group were inoculated in 4 96-well plates to detect cell activity at 0h, 24h, 48h and 72h, and finally the 96-well plates inoculated with cells were placed in a constant temperature incubator and continued to be cultured until four detection time points. Among them, the time for cell attachment, i.e. 6-8h after seeding, was set to 0h, the original culture medium in the 96-well plate was discarded at the corresponding time, 110μL of a mixed solution of CCK-8 solution and complete culture medium was added to each well (CCK-8 solution: complete culture medium = 1:10), and the plate was placed in a constant temperature incubator for 1h, and the absorbance at 450nm was detected using a multifunctional microplate reader.

[0084] EdU experiment: cells in the logarithmic growth phase were seeded into 24-well plates at a cell density of 3×10 4 / mL. After culturing in a constant temperature incubator for 48 hours, the culture medium was aspirated and the cells were washed twice with PBS buffer. Complete culture medium and EdU solution were mixed evenly at a volume ratio of 1000:1, 300mL of the mixture was added to each well, and the cells were incubated for 2 hours and then washed twice with PBS buffer; 300μL of 4% paraformaldehyde was added to each well to fix it for 40 minutes, and the fixative was discarded; 300μL of glycine solution was added to each well, and the cells were incubated on a decolorizing shaker for 5 minutes, and the glycine solution was discarded; 300μL of PBS buffer was added to each well, and the cells were washed for 5 minutes, and the PBS buffer was discarded; 300μL of permeabilization agent (0.5% TritonX-100 in PBS) was added to each well, and the cells were incubated on a decolorizing shaker for 5 minutes. Incubate on a color shaker for 10 minutes, wash with PBS buffer for 5 minutes; add 300μL of Apollo staining reaction solution to each well, incubate on a decolorizing shaker for 40 minutes at room temperature and in the dark, and discard the staining reaction solution; add 300μL of permeabilization agent (0.5% TritonX-100 in PBS) to each well, wash on a decolorizing shaker 3 times, 10 minutes each time, and discard the permeabilization agent; mix deionized water and 100xHoechst33342 at a volume ratio of 100:1, add 300μL to each well, incubate on a decolorizing shaker for 40 minutes at room temperature and in the dark. Add 300μL PBS buffer to wash for 15 minutes, discard the PBS buffer, add 300μL PBS buffer, obtain pictures under a fluorescence microscope, and use ImageJ software to quantitatively analyze cell proliferation.

[0085] Apoptosis assay: 1×10 cells were seeded per well of a 6-well plate. 4Cells were cultured in a constant temperature incubator for 24 hours. Digested with EDTA-free trypsin for about 30 seconds, placed in a 1.5mL EP tube, centrifuged at 2000rpm for 5 minutes to collect the cells, and washed twice with pre-cooled PBS. 5μL 7-AAD dye solution and 50μL Binding Buffer were fully mixed and added to the cells. After mixing evenly, incubated in the dark for 15 minutes, 450μL Binding Buffer containing 1μL Annexin V-PE was added, mixed evenly, and continued to be in the dark for 15 minutes. Detected by flow cytometry, counted at medium speed 3×10 4 The results were analyzed using CytExpert software.

[0086] Cycle experiment: 1×10 cells were seeded in each well of a 6-well plate. 4 Cells were cultured in a constant temperature incubator for 24 hours. After digestion and collection of cells, they were washed once with pre-cooled PBS and centrifuged at 2000 rpm for 5 minutes. 1 mL of 70% ethanol was added to fix for at least 2 hours, washed once with PBS buffer, and centrifuged at 2000 rpm for 5 minutes. RnaseA:PI was prepared into a staining working solution at a ratio of 1:9. 500 μL of the working solution was added to each sample. After being protected from light for 30 minutes, 3×10 4 The results were analyzed using FlowJo software.

[0087] Transwell invasion assay: Place the matrix gel in ice and place in a refrigerator at 4°C overnight. Dilute the matrix gel to 300 μg / mL using serum-free culture medium, add 100 μL / well to the 24-well plate transwell chamber, place in a constant temperature incubator for 1 hour, and remove the upper culture medium. Resuspend the cells in a pre-prepared BSA-containing culture medium and adjust the cell concentration to 2×10 5 / mL, and add 200μL to each well of the tanswell chamber of the 24-well plate, add 800μL of complete culture medium to each well of the lower chamber of the 24-well plate, and culture in a constant temperature incubator for 24h. Note that no bubbles should appear at the bottom of the chamber during the culture process; aspirate the culture medium in the chamber, gently wipe off the cells and glue in the upper chamber with a cotton swab, fix it with 4% paraformaldehyde for 30min, stain it with crystal violet stain for 30min, wash it with PBS buffer for 3 times, and dry it; observe and take pictures under a 10x microscope, and calculate the number of invasive cells.

[0088] Scratch experiment: Use a paintbrush to draw three horizontal lines on the bottom of a 6-well plate for positioning, that is, one well can see three positions, and the horizontal lines should just not appear in the field of view under the microscope to ensure that the same position is observed each time; cells in the logarithmic growth phase are inoculated in a 6-well plate, with 200,000 cells in each well; when the cell confluence is about 90%, use a 200μL gun tip to scratch, and pay attention to uniform strength when scratching; after scratching, wash away the debris with PBS buffer, add new culture medium, and take pictures under a 10x microscope, at this time 0h; after 24h, observe the cell migration again and take pictures, and calculate the cell migration rate.

[0089] Single cell gel electrophoresis experiment: Weigh 0.07g normal melting point agarose powder and dissolve it in 10mL PBS buffer solution, and heat it in a microwave to dissolve it, and prepare the first layer of gel. Take a clean and dry slide and preheat it in a 55℃ metal bath, draw 100μL normal melting point agarose gel and drop it on the slide, spread the agarose gel evenly through the cover glass, push the cover glass off after the gel solidifies, put it on the slide rack and store it in a 4℃ refrigerator for later use. Weigh 0.07g low melting point agarose powder and dissolve it in 10mL PBS buffer solution, and heat it in a microwave to prepare the second layer of gel. After cell digestion and centrifugation, resuspend it in PBS buffer solution. Take 70μL low melting point agarose gel and 30μL cell suspension and mix them evenly, draw 80μL of the mixture and drop it on the first layer of gel, cover it with a cover glass to spread the agarose gel evenly, and push the cover glass off after the gel solidifies. Place the slide in the cell lysis solution at 4℃ and avoid light for 1h. After gentle washing with pure water, place in the horizontal electrophoresis tank at 4℃ and avoid light for 30min. Turn on the power supply at a constant voltage of 25V and perform electrophoresis at 4℃ and avoid light for 30min. Take out the slide, aspirate Tris-HCl and drop it on the gel surface, wash for 5min, and neutralize the interference of the alkali solution. Add 100μL of staining solution (NA-Red: pure water = 1:500). Use a fluorescence microscope to take cell images at 10 times the microscope and analyze with CSAP analysis software.

[0090] Western blot: After the cell confluence reaches more than 70%, discard the original culture medium and add pre-cooled PBS buffer to wash twice. Evenly drip 200μL lysis solution (PMSF: IPA = 1: 100) and lyse on ice for 30min. Collect the cells and lysis solution at the bottom of the dish, centrifuge at 4℃, 14000rcf for 20min, and take the supernatant. Use the BCA kit to measure the cell protein concentration, adjust the protein concentration to the same concentration, add SDS-PAGE protein loading buffer, and denature the protein at 95℃ for 5min in a multi-functional gradient thermal cycler. Add 7.5mL10% SDS-PAGE separation gel and 5% SDS-PAGE concentration gel to the glass interlayer of the gel making device. After the gel solidifies, put the glass interlayer into the electrophoresis device and add the electrophoresis solution. Add 10μL PageRulerTM Prestained Protein Ladder and 20μL denatured protein sample to the sample wells, run electrophoresis at 80V for 30min, and then run electrophoresis at 100V for 70min. Cut the gel and activate the PVDF membrane in methanol for 30s. Prepare the transfer device and assemble it according to "positive electrode-sponge pad-filter paper-PVDF membrane-gel-filter paper-sponge pad-negative electrode", add the transfer solution, and run electrophoresis at a constant current of 280mA in an ice box for 50min. Place the PVDF membrane in an incubation box, add the blocking solution to block for 1h, then add the primary antibody and incubate overnight at 4℃. Wash with TBST solution 3 times, 10 minutes each time. Add the secondary antibody, shake in dark for 1h, and wash with TBST solution 3 times for 30min. Scan the image with the LI-COR Odyssey near-infrared imaging system and perform grayscale analysis.

[0091] Cell immunofluorescence experiment: Place the cell slides at the bottom of a 24-well plate and add 1 mL of 1×10 4 The suspension of cells was placed in a constant temperature incubator for 48 hours, and then the original culture medium was discarded. 300 μL PBS was added to wash twice, and the tissue fixative was added to fix for 20 minutes, and the fixative was discarded. 300 μL PBS was added to wash on a decolorizing shaker for 15 minutes. 0.5% Tritox-100 was added to permeate for 10 minutes, and 300 μL PBS was used to wash for 15 minutes. After blocking on a shaker for 1 hour, the primary antibody was added and incubated overnight at 4°C. After washing with PBS for 15 minutes, the fluorescent secondary antibody solution was added and incubated on a shaker in the dark for 1 hour. 10 μL DAPI staining solution was added, and the cells were incubated at room temperature in the dark for 10 minutes, and then washed with PBS for 15 minutes. The cells were picked up with a needle, dried in the dark, and then turned upside down on a slide, and 10 μL of anti-fluorescence attenuation sealing solution was added. The images were observed and collected under a 63x microscope using a Leica SP8 laser confocal microscope, and the number of foci of each cell was counted.

[0092] After 200 generations of infection, the above technical means were used to identify the malignant transformation of the cell model and detect the cell function after malignant transformation.

[0093] 2. Results

[0094] The results are as follows Figure 3 As shown in B, cells poisoned with benzo[g,h,i]perylene formed obvious, dense, and large colonies in soft agar. The number of colonies was calculated and statistically analyzed, as shown in Figure 3 The clone formation rate of cells poisoned with benzo[g,h,i]perylene shown in A was significantly higher than that of normal BEAS-2B cells, and the difference was statistically significant.

[0095] like Figure 4 As shown in A, in the tumor formation experiment in mice, the tumors in the benzo[g,h,i]perylene-treated group were significantly larger than those in the control group; as time went on, the tumor volume in the benzo[g,h,i]perylene-treated group increased more significantly and with statistical significance ( Figure 4 B); Finally, the separated tumors were weighed and it was found that the tumor weight of the poisoned group increased significantly compared with the control group ( Figure 4 C) At the same time, if Figure 4 As shown in D, in the HE staining results, the tumor cells in the poisoning group were densely arranged, the cells were polygonal, the nuclear-cytoplasmic ratio was high, the nuclei were of different shapes and sizes, the nuclear chromatin was darker, and more nuclear division phases were observed (red arrows). In addition, immunohistochemistry was used to evaluate the expression level of Ki-67 (a human protein encoded by the MKI67 gene, which is highly correlated with the proliferation activity of cancer cells, making it a recognized marker of cell proliferation and tumorigenesis). Figure 4 As shown in E and 4F, the Ki-67 positive rate in the benzo[g,h,i]perylene-exposed group was significantly higher than that in the control group, and the difference was statistically significant. The above results showed that the benzo[g,h,i]perylene-induced malignant transformation cell line of BEAS-2B was successfully established.

[0096] like Figure 5 As shown in A, in the CCK-8 experiment, the proliferation ability of malignant cells induced by benzo[g,h,i]perylene was significantly enhanced compared with that of normal BEAS-2B cells. Figure 5 As shown in B and 5C, EdU experiments also showed that the proliferation ability of the constructed cell model was significantly enhanced.

[0097] like Figure 6 As shown in A and 6B, in the apoptosis experiment, the apoptosis rate of benzo[g,h,i]perylene-induced cells after malignant transformation was significantly reduced compared with normal BEAS-2B cells. Figure 6As shown in C and 6D, in the cycle experiment, compared with the control group, the proportion of malignantly transformed cells in the G0 / G1 phase increased significantly, while the proportion of S+G2 / M phase decreased significantly, indicating that the cell cycle was disrupted after malignant transformation.

[0098] like Figure 7 As shown in A and 7B, in the Transwell invasion assay, the invasion rate of cells after malignant transformation was significantly enhanced and statistically significant; Figure 7 As shown in C and 7D, in the scratch test, the migration rate of cells was significantly enhanced after malignant transformation and the difference was statistically significant.

[0099] like Figure 8 As shown in AD, in the comet assay, compared with the control group, BEAS-2B cells induced by benzo[g,h,i]perylene showed obvious "tailing" phenomenon, and the tail DNA%, tail length, and Olive tail distance were significantly increased. DNA damage is one of the causes of tumor formation. Figure 8 As shown in Figures E and 8F, in Western blot experiments, the DNA double-strand break marker protein γ-H2AX was significantly overexpressed in malignant cells. Figure 8 As shown in G and 8H, in the immunofluorescence experiment, the malignant cells generated more γ-H2AX foci than the normal BEAS-2B cells. The above results indicate that there is obvious DNA damage in the in vitro cell malignant transformation model we constructed.

[0100] The above experimental results all showed differentiated results, indicating that the BEAS-2B malignant transformation cell line induced by benzo[g,h,i]perylene was successfully established, and it was suggested that the cell model has general characteristics similar to cancer cells. The cell line was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with the deposit number GDMCC No: 64810. The cell line can be used for the study of the biological effects and molecular mechanisms of benzo[g,h,i]perylene, the study of the carcinogenic mechanism of benzo[g,h,i]perylene, or the screening / evaluation of cancer treatment drugs caused by benzo[g,h,i]perylene.

Claims

1. A malignant transformed cell line induced by benzo[g,h,i]perylene, characterized in that: The cell line was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 27, 2024, with the deposit number GDMCC No: 64810.

2. A method for constructing a malignant transformed cell line according to claim 1, characterized in that: Normal human lung bronchial epithelial cells were exposed to 48-52 ng / mL of benzo[g,h,i]perylene and cultured. The cell proliferation ability was monitored every 23-27 generations of exposure. The cells were identified for malignant transformation after 198-202 generations of exposure, and an in vitro cell line induced by benzo[g,h,i]perylene was obtained.

3. The construction method according to claim 2, characterized in that: The concentration of benzo[g,h,i]perylene used is 50 ng / mL.

4. The construction method according to claim 2, characterized in that: The human normal lung bronchial epithelial cells are human lung bronchial epithelial cells BEAS-2B.

5. The construction method according to claim 2, characterized in that: The culture medium used for the poisoning culture is a DMEM / F12 complete culture medium containing 5% fetal bovine serum and a benzo[g,h,i]perylene concentration of 50 ng / mL.

6. The construction method according to claim 2, characterized in that: The monitoring of cell proliferation ability is carried out by using cell activity detection test and plate clone formation experiment.

7. The construction method according to claim 2, characterized in that: The malignant transformation identification is carried out by using soft agar colony formation experiment and bone marrow mouse tumor formation experiment.

8. The construction method according to claim 2, characterized in that: The cell proliferation capacity was monitored every 25 generations of exposure, and the cells were identified for malignant transformation after 200 generations of exposure.

9. Use of the malignant transformed cell line of claim 1 as a cell model for studying the biological effects and molecular mechanisms of benzo[g,h,i]perylene, a cell model for studying the carcinogenic mechanism of benzo[g,h,i]perylene, or a cell model for screening / evaluation of cancer therapeutic drugs caused by benzo[g,h,i]perylene.

10. Use of the malignant transformed cell line according to claim 1 in the preparation of a drug for treating cancer caused by benzo[g,h,i]perylene.

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