Method for establishing chrysotile-induced BEAS-2B malignant transformation cell strain

Abstract

The invention discloses a method for establishing a chrysotile induced BEAS-2B malignant transformation cell strain, and relates to the technical field of cell engineering, and the method comprises the following steps: selecting and preparing an experimental subject; determining a proper concentration, and infecting BEAS-2B cells with chrysotile contamination liquid; and evaluating the cell malignant transformation. After 46 generations of continuous contamination, the BEAS-2B cells have different degrees of malignant transformation, the chrysotile malignant transformation cell strain is successfully constructed, by establishing a stable cell or animal model, the process that chrysotile induces the cells to cancerate can be more comprehensively elaborated, the ability and degree of chrysotile for inducing the cells to generate malignant transformation can be evaluated, and the clinical application prospect is broad. Furthermore, the carcinogenic molecular mechanism of chrysotile can be elaborated, and a new strategy and target are provided for early diagnosis and treatment of occupational diseases caused by chrysotile exposure.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to a method for establishing a chrysotile-induced BEAS-2B malignant transformation cell strain. Background Art

[0002] Chrysotile is a general term for serpentine asbestos, among which chrysotile is the most widely produced mineral in nature, and has a high degree of crystallization and good divisibility. It is a type of asbestos that is still being mined. The carcinogenicity of asbestos has long been known to the academic community, but it has not been banned in domestic industries, especially the construction industry. Although the government stipulates that protective equipment must be worn when mining and processing chrysotile asbestos, it has not given corresponding regulations and guidance for its application. Comprehensive epidemiological and in vitro and in vivo experiments have shown that chrysotile can induce lung-related diseases through oxidative stress, chronic inflammation, and DNA damage. If exposed for a long time, it can further lead to malignant tumors such as lung cancer and mesothelioma.

[0003] Cellular malignant transformation is the basis of tumorigenesis. Chrysotile asbestos may cause tumorigenesis at certain concentrations and exposure times, but there is still a lack of a complete mechanism for the gradual malignant transformation of normal cells to the final tumor formation. In order to more comprehensively explain the process of chrysotile asbestos inducing cell carcinogenesis, it is urgent to establish a stable cell or animal model to evaluate its ability and degree of inducing cell malignant transformation, and then explain the molecular mechanism of chrysotile carcinogenesis, so as to provide new strategies and targets for the early diagnosis and treatment of occupational diseases caused by chrysotile asbestos exposure. Summary of the invention

[0004] The present invention provides a method for establishing a chrysotile-induced BEAS-2B malignant transformation cell line, and aims to solve the technical problem of how to establish a stable cell or animal model, evaluate its ability and degree of inducing malignant transformation of cells, and further explain the molecular mechanism of chrysotile carcinogenesis.

[0005] In view of the above problems in the prior art, according to one aspect disclosed in the present invention, the present invention adopts the following technical solution:

[0006] A method for establishing a chrysotile asbestos-induced malignant transformation cell line of BEAS-2B, comprising:

[0007] Step 1: Selecting experimental objects: BEAS-2B cells and chrysotile asbestos; grinding the chrysotile asbestos into fiber dust with a particle size of less than 5 μm; and characterizing the crystal structure and main components of the chrysotile asbestos;

[0008] Step 2: Infection method

[0009] Prepare complete culture medium: prepare complete culture medium by mixing DMEM basal culture medium with fetal bovine serum and double antibody in a ratio of 9:1:0.1;

[0010] Preparation of chrysotile asbestos poisoning solutions: adding chrysotile fiber dust to prepare 0μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, and 400μg / mL poisoning solutions, respectively, as poisoning solutions for BEAS-2B cells; the normal control group was cultured with complete culture medium;

[0011] Determine the optimal concentration of chrysotile asbestos: Use cell activity experiments to determine the optimal concentration of chrysotile asbestos, based on the IC 80 The concentration of asbestos was taken as the optimum concentration of poisoning; according to the experiment, the optimum concentration of chrysotile asbestos poisoning solution was determined to be 200μg / mL;

[0012] Continuous exposure: According to the determined optimal concentration of 200 μg / mL, when BEAS-2B cells are in the logarithmic growth phase, the chrysotile asbestos solution is exposed to BEAS-2B cells for 24 hours, then washed twice with PBS, digested with trypsin at a ratio of 1:4, and the first generation of exposure is obtained; then complete medium is used to continue culturing, and when the cells are in the logarithmic growth phase, the complete medium is removed, washed twice with PBS, and then 200 μg / mL of the above-mentioned solution is added for exposure for 24 hours, washed twice with PBS, digested with trypsin at a ratio of 1:4, and the second generation of exposure is obtained; the above operations are repeated in sequence until the cells undergo malignant transformation;

[0013] Step 3: Evaluation of cell malignant transformation

[0014] After several generations of infection in step 2, the following experiment was used to evaluate the degree of malignant transformation of BEAS-2B cells;

[0015] The morphology of normal BEAS-2B cells and cells exposed to chrysotile asbestos for several generations were observed by Wright-Giemsa staining.

[0016] The concentration changes of supernatant components of normal BEAS-2B cells and cells exposed to chrysotile asbestos for several generations were detected, including glucose (Glu), lactate dehydrogenase (LDH), interleukin-6 (IL-6), and neuron-specific enolase (NSE). Compared with the supernatant of normal BEAS-2B cells, the consumption of Glu in the supernatant of cells exposed to chrysotile asbestos for several generations increased, the amount of LDH produced by cells increased, and the inflammatory factors IL-6 and NSE increased, indicating that the metabolic capacity of cells was enhanced, and there was inflammation and the formation of tumor malignant transformation.

[0017] The colony formation experiment was used to observe and compare the proliferation ability of BEAS-2B cells after several generations of exposure to chrysotile asbestos and the original generation. Compared with the original generation, the number of colonies formed by BEAS-2B cells exposed to chrysotile asbestos was more, showing a stronger proliferation ability.

[0018] The scratch test was used to observe the migration ability of cells. When cells underwent malignant transformation, the migration and growth ability of cells was compared after the cells were scratched under the same culture conditions. After comparison, the cells exposed to chrysotile asbestos had stronger migration and growth ability, showing stronger malignant transformation ability.

[0019] The Transwell assay was used to evaluate the migration and invasion abilities of cells. The experiments showed that cells exposed to chrysotile asbestos had stronger migration and invasion abilities.

[0020] The primary BEAS-2B cells and cells exposed to chrysotile asbestos were tested for mesenchymal genes and proteins. The results showed that the cells exposed to chrysotile had a tendency to become tumorous.

[0021] Tumor gene and protein detection was performed on primary BEAS-2B cells and cells exposed to chrysotile asbestos. The experiment showed that compared with normal BEAS-2B cells, mesenchymal and tumor indicators were reduced.

[0022] Nude mouse tumorigenesis experiments were performed on primary BEAS-2B cells and cells exposed to chrysotile asbestos, and tumor morphology, tissue pathology, tumor gene detection and immunohistochemistry analysis were performed to evaluate the expression of malignantly transformed cells in vivo.

[0023] After several generations of continuous exposure from step 1 to step 3, BEAS-2B cells have undergone malignant transformation, and a malignant transformation cell line exposed to chrysotile asbestos solution has been constructed.

[0024] In order to better realize the present invention, a further technical solution is:

[0025] Furthermore, XRD and XRF were used to characterize the crystal structure and composition of the samples, respectively.

[0026] Furthermore, after 46 generations of infection in step 2, an experiment was performed to evaluate the degree of malignant transformation of BEAS-2B cells.

[0027] Furthermore, the chrysotile asbestos is Qinghai Mangya chrysotile asbestos.

[0028] Compared with the prior art, one of the beneficial effects of the present invention is:

[0029] According to the method for establishing a chrysotile-induced BEAS-2B malignant transformation cell line, after 46 generations of continuous exposure, BEAS-2B cells have undergone malignant transformation to varying degrees, and a chrysotile malignant transformation cell line has been successfully constructed. By establishing a stable cell or animal model, the process of chrysotile-induced cell carcinogenesis can be more comprehensively described, and the ability and degree of inducing cell malignant transformation can be evaluated, and the molecular mechanism of chrysotile carcinogenesis can be described, thereby providing new strategies and targets for the early diagnosis and treatment of occupational diseases caused by chrysotile exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application document or the technical solutions in the prior art, the drawings required for use in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only references to some embodiments in the present application document. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 The present invention is a flow chart of a method for establishing a chrysotile-induced BEAS-2B malignant transformation cell line according to an embodiment of the present invention.

[0032] Figure 2A Schematic diagram of normal BEAS-2B cell morphology (Wright-Giemsa staining) according to one embodiment of the present invention.

[0033] Figure 2B Schematic diagram of the cell morphology (Wright-Giemsa staining) after CA infection for 46 generations according to one embodiment of the present invention.

[0034] Figure 3A Schematic diagram of LDH concentration in the supernatant of cells infected for 46 generations according to an embodiment of the present invention.

[0035] Figure 3B Schematic diagram of Glu concentration in the supernatant of cells infected for 46 generations according to an embodiment of the present invention.

[0036] Figure 3C Schematic diagram of NSE concentration in the supernatant of cells infected for 46 generations according to an embodiment of the present invention.

[0037] Figure 3D Schematic diagram of IL-6 concentration in the supernatant of cells infected with 46 generations according to an embodiment of the present invention.

[0038] Figure 4A Schematic diagram of the colony number of primary BEAS-2B cells in a colony formation experiment according to one embodiment of the present invention.

[0039] Figure 4BSchematic diagram of the colony count of cells in a colony formation experiment after 46 generations of CA poisoning according to an embodiment of the present invention.

[0040] Figure 4C This is a graph showing the relative number of colonies of primary BEAS-2B cells after being infected with CA for 46 generations according to an embodiment of the present invention.

[0041] Figure 5A The original cell morphology of normal BEAS-2B cells at 0h and 48h after scratching according to one embodiment of the present invention is shown.

[0042] Figure 5B The original morphology of cells after normal CA infection at 0h and 48h after scratching according to one embodiment of the present invention.

[0043] Figure 5C Graph showing the percentage of scratch healing rate after primary BEAS-2B cells were intoxicated with CA for 46 generations according to one embodiment of the present invention.

[0044] Fig. 6A Schematic diagram of the results of a Transwell cell migration experiment of primary BEAS-2B cells (crystal violet staining) according to one embodiment of the present invention.

[0045] Figure 6B Schematic diagram of the Transwell cell migration experiment results (crystal violet staining) of the CA group according to an embodiment of the present invention.

[0046] Fig. 7A Schematic diagram of the results of Transwell cell invasion experiment (crystal violet staining) of primary BEAS-2B cells according to one embodiment of the present invention.

[0047] Figure 7B Schematic diagram of the Transwell cell invasion experiment results (crystal violet staining) of the CA group according to an embodiment of the present invention.

[0048] Fig. 8A The Western blot diagram of each protein in the cells of the NC and CA poisoning groups according to one embodiment of the present invention.

[0049] Figure 8B Schematic diagram of the relative expression levels of various proteins in cells of NC and CA poisoning groups according to an embodiment of the present invention.

[0050] Fig. 9 Schematic diagram of the relative expression levels of mRNA of each gene in NC and infected group cells according to one embodiment of the present invention. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0052] See also Figure 1 As shown, a method for establishing a cell line of BEAS-2B malignant transformation caused by chrysotile and artificial glass fiber is preferably used as the research object, and a cell model of malignant transformation is constructed by continuously infecting bronchial epithelial cells (BEAS-2B). The specific construction and evaluation methods are as follows:

[0053] Step 1. Experimental object: BEAS-2B cells, chrysotile asbestos (CA). Grind the chrysotile asbestos into fiber dust with a particle size of less than 5um using a planetary ball mill. Then use XRD and XRF to characterize the crystal structure and main components of the sample respectively.

[0054] Step 2. Infection method:

[0055] 1) Prepare complete culture medium: prepare complete culture medium by mixing DMEM basal culture medium with fetal bovine serum and double antibody in a ratio of 9:1:0.1.

[0056] 2) Preparation of CA poisoning solution: Add CA fiber dust to the prepared complete culture medium to prepare 0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL poisoning solutions, which are used as poisoning solutions for BEAS-2B cells. The normal control group is cultured with complete culture medium.

[0057] 3) Determine the optimal concentration of CA poisoning solution: Use cell activity test (CCK-8) to determine the optimal concentration of poisoning, based on the IC 80 The optimal concentration of CA was determined to be 200 μg / mL based on previous experiments.

[0058] 4) Continuous infection: According to the optimal concentration (200 μg / mL) determined previously, when BEAS-2B cells are in the logarithmic growth phase, the CA infection solution is used to infect BEAS-2B cells for 24 h, and then the cells are washed twice with PBS and digested with trypsin at a ratio of 1:4 for passage, which is the first infection generation (T1); then complete culture medium is used to continue culturing, and when the cells are in the logarithmic growth phase, the complete culture medium is removed, the cells are washed twice with PBS, and 200 μg / mL of the infection solution is added again for infection for 24 h, and then the cells are washed twice with PBS and digested with trypsin at a ratio of 1:4 for passage, which is the first infection generation (T2); the above operations are repeated in sequence until the cells undergo malignant transformation.

[0059] Step 3. Evaluation of cell malignant transformation

[0060] After 46 generations of infection using the above method, the following experiments were used to evaluate the degree of malignant transformation of BEAS-2B cells, including cell proliferation, migration, invasion ability, cell mesenchymalization, expression of tumor genes and proteins, and in vivo experiments.

[0061] 1) Wright-Giemsa staining: observe changes in cell morphology.

[0062] Normal BEAS-2B cells are tightly connected and have regular shapes, such as polygonal and long spindles. After 46 generations of CA infection, the cells become loosely connected, irregular in shape, and have different sizes, such as round and oval mesenchymal changes. Compared with the primary cells, the morphology has changed. Figure 2A-2B shown.

[0063] 2) Changes in the concentration of supernatant components: The detection indicators include glucose (Glu), lactate dehydrogenase (LDH), interleukin-6 (IL-6), and neuron-specific enolase (NSE). Compared with the normal BEAS-2B cell supernatant, the consumption of Glu in the cell supernatant after 46 generations of CA poisoning increased, the amount of LDH produced by the cells increased, and the inflammatory factor IL-6 and tumor marker NSE both increased, indicating that the metabolic capacity of the cells was enhanced, and there was inflammation and tumor malignant transformation. The results are as follows Figure 3A-3D As shown, the concentrations of the components in the supernatant of cells infected with 46 generations: a. LDH, b. Glu, c. NSE, d. IL-6; compared with NC, the differences were statistically significant.

[0064] 3) Colony formation experiment: observe the proliferation ability of cells. When cells undergo malignant transformation, the number of colonies formed will increase, and the cells may show stronger proliferation ability. Figure 4A-4B As shown, the normal control group (NC, Figure 4A ) and the poisoned group ( Figure 4B ) Cell colony formation experiment, crystal violet staining, compared with the primary BEAS-2B cells, the number of colonies formed after 46 generations of CA infection was more, the difference was statistically significant, and it showed a stronger proliferation ability.

[0065] 4) Scratch test to observe the migration ability of cells. When cells undergo malignant transformation, under the same culture conditions, the area of ​​the blank area will become smaller after the cells are scratched, and the cells will show stronger migration and growth ability. Figure 5A-Figure 5C After 46 generations of infection, the fusion rate and scratch area of ​​CA at 48h after scratching ( Figure 5B , Figure 5C ) was 28.8%, while the normal BEAS-2B cell scratch area ( Figure 5A , Figure 5C) was 44.9%, and the difference was statistically significant. This showed that cells infected with CA had stronger migration and growth abilities and showed stronger malignant transformation abilities.

[0066] 5) Transwell assay: evaluate the migration and invasion ability of cells. When cells undergo malignant transformation, their ability to pass through the Transwell chamber will be enhanced, and there will be more cells in the lower layer of the chamber, indicating that the cells have stronger migration ability. If matrix gel is added to the chamber, the invasion ability of cells can be evaluated. Figure 6A-6B , Figure 7A-7B As shown, compared with the normal control group (NC), the number of cells in the treated group increased significantly, and the difference was statistically significant, indicating that cells treated with CA for 46 generations had stronger migration and invasion abilities.

[0067] 6) Changes in tumor-related proteins. After cells are poisoned by CA, the tumor suppressor genes in the cells tend to be under-expressed, increasing the risk of cell proliferation and thus promoting the occurrence of tumors. Figure 8A-B As shown, compared with the NC group, the tumor-related proteins C-myc, P53, and Bcl-2 were down-expressed in the CA group, and the difference was statistically significant.

[0068] 7) Changes in tumor-related genes: including detection of c-Jun, c-myc, bcl-2, P53, and Akt genes. When cells undergo malignant transformation, the expression of tumor genes will change, such as downregulation of the expression of tumor suppressor genes c-myc, bcl-2, and P53. Fig. 9 As shown, compared with normal BEAS-2B cells (NC), tumor indicators showed different degrees of reduction, among which P53 and C-myc, both gene and protein were lowly expressed.

[0069] 8) Nude mouse tumor formation experiment: including tumor tissue size, pathological observation, tumor gene detection, and evaluation of the expression of malignantly transformed cells in vivo.

[0070] In summary, after 46 generations of continuous infection, BEAS-2B cells have undergone malignant transformation to varying degrees, and the CA malignant transformation cell line has been successfully constructed. The preservation information of the constructed cell line is as follows: the depositor is Mianyang 404 Hospital, with an address of No. 56, Yuejin Road, Fucheng District, Mianyang City, Sichuan Province, the deposit name is human bronchial epithelial malignant transformation cell line BEAS-2B-CA (Homosapiens), the preservation center is the China Center for Type Culture Collection, the preservation number is C2024354, and the preservation date is October 22, 2024.

[0071] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0072] References to "one embodiment", "another embodiment", "embodiment", etc. in this specification refer to specific features, structures or characteristics described in conjunction with the embodiment as included in at least one embodiment generally described in this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Further, when a specific feature, structure or characteristic is described in conjunction with any embodiment, it is claimed that the realization of such feature, structure or characteristic in conjunction with other embodiments also falls within the scope of the present invention.

[0073] Although the present invention is described herein with reference to a plurality of illustrative embodiments of the present invention, it should be understood that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure and claims, a variety of variations and improvements may be made to the components and / or layout of the subject combination layout. In addition to variations and improvements made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A method for establishing a chrysotile-induced BEAS-2B malignant transformation cell line, characterized in that include: Step 1: Selecting experimental objects: BEAS-2B cells and chrysotile asbestos; grinding the chrysotile asbestos into fiber dust with a particle size of less than 5 μm; and characterizing the crystal structure and major components of said chrysotile; Step 2: Infection method Prepare complete culture medium: prepare complete culture medium by mixing DMEM basal culture medium with fetal bovine serum and double antibody in a ratio of 9:1:0.1; Preparation of chrysotile asbestos poisoning solution: adding chrysotile fiber dust to prepare 0μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, and 400μg / mL poisoning solutions, respectively, as poisoning solutions for BEAS-2B cells; the normal control group was cultured with complete culture medium; Determine the optimal concentration of chrysotile asbestos poisoning solution: Use cell activity experiment to determine the optimal concentration of poisoning, based on the IC 80 The concentration of asbestos was taken as the optimum concentration of poisoning; according to the experiment, the optimum concentration of chrysotile asbestos poisoning solution was determined to be 200μg / mL; Continuous exposure: According to the determined optimal concentration of 200 μg / mL, when BEAS-2B cells are in the logarithmic growth phase, the chrysotile asbestos solution is exposed to BEAS-2B cells for 24 hours, then washed twice with PBS, digested with trypsin at a ratio of 1:4, and the first generation of exposure is obtained; then complete medium is used to continue culturing, and when the cells are in the logarithmic growth phase, the complete medium is removed, washed twice with PBS, and then 200 μg / mL of the above-mentioned solution is added for exposure for 24 hours, washed twice with PBS, digested with trypsin at a ratio of 1:4, and the second generation of exposure is obtained; the above operations are repeated in sequence until the cells undergo malignant transformation; Step 3: Evaluation of cell malignant transformation After several generations of infection in step 2, the following experiment was used to evaluate the degree of malignant transformation of BEAS-2B cells; The morphology of normal BEAS-2B cells and cells exposed to chrysotile asbestos for several generations were observed by Wright-Giemsa staining. The concentration changes of supernatant components of normal BEAS-2B cells and cells exposed to chrysotile asbestos for several generations were detected, including glucose (Glu), lactate dehydrogenase (LDH), interleukin-6 (IL-6), and neuron-specific enolase (NSE). Compared with the supernatant of normal BEAS-2B cells, the consumption of GLU in the supernatant of cells exposed to chrysotile asbestos for several generations increased, the amount of LDH produced by cells increased, and the inflammatory factors interleukin-6 and neuron-specific enolase increased, indicating that the metabolic capacity of cells was enhanced, and inflammation and tumor malignant transformation were formed. The colony formation experiment was used to observe and compare the proliferation ability of BEAS-2B cells after several generations of exposure to chrysotile asbestos and the original generation. Compared with the original generation, the number of colonies formed by BEAS-2B cells exposed to chrysotile asbestos was more, showing a stronger proliferation ability. The scratch test was used to observe the migration ability of cells. When cells underwent malignant transformation, the migration and growth ability of cells was compared after the cells were scratched under the same culture conditions. After comparison, the cells exposed to chrysotile asbestos had stronger migration and growth ability, showing stronger malignant transformation ability. The Transwell assay was used to evaluate the migration and invasion abilities of cells. The experiments showed that cells exposed to chrysotile asbestos had stronger migration and invasion abilities. The primary BEAS-2B cells and cells exposed to chrysotile asbestos were tested for mesenchymal genes and proteins. The results showed that the cells exposed to chrysotile had a tendency to become tumorous. Tumor gene and protein detection was performed on primary BEAS-2B cells and cells exposed to chrysotile asbestos. The experiment showed that compared with normal BEAS-2B cells, mesenchymal and tumor indicators were reduced. Nude mouse tumorigenesis experiments were performed on primary BEAS-2B cells and cells exposed to chrysotile asbestos, and tumor morphology, tissue pathology, tumor gene detection and immunohistochemistry analysis were performed to evaluate the expression of malignantly transformed cells in vivo. After several generations of continuous exposure from step 1 to step 3, BEAS-2B cells have undergone malignant transformation, and a malignant transformation cell line exposed to chrysotile asbestos solution has been constructed.

2. The method for establishing a chrysotile asbestos-induced malignant transformation cell line of BEAS-2B according to claim 1, characterized in that XRD and XRF were used to characterize the crystal structure and composition of the samples.

3. The method for establishing a chrysotile asbestos-induced malignant transformation cell line of BEAS-2B according to claim 1, characterized in that After 46 generations of infection in step 2, the degree of malignant transformation of BEAS-2B cells was evaluated by experiment.

4. The method for establishing a chrysotile asbestos-induced malignant transformation cell line of BEAS-2B according to claim 1, characterized in that The chrysotile asbestos is the Mangya chrysotile from Qinghai.

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