CN110423725B - A chrysotile-induced malignant transformation strain of human pleural mesothelial cells and its application - Google Patents

Abstract

Translated fromChinese

本发明公开了一种温石棉诱导的人胸膜间皮细胞恶性转化株及其应用，所述人胸膜间皮细胞恶性转化株分类命名为：人胸膜间皮细胞，株号为：AS‑T，保藏号为：CCTCC NO.C201923。本发明人胸膜间皮细胞恶性转化株由人胸膜间皮细胞(MeT‑5A)经温石棉多阶段多次染毒处理后，发生恶性转化获得，在作为致癌物致癌机制研究的细胞模型等多个方面具有良好的应用前景。

The invention discloses a chrysotile-induced malignant transformation of human pleural mesothelial cells and its application. The deposit number is: CCTCC NO.C201923. The malignant transformation strain of human pleural mesothelial cells of the present invention is obtained from human pleural mesothelial cells (MeT-5A) after being treated with chrysotile in multiple stages and multiple times, and then undergoes malignant transformation. This aspect has a good application prospect.

Description

Chrysotile-induced human pleural mesothelial cell malignant transformation strain and application thereof

Technical Field

The invention relates to the technical field of biology and oncology, in particular to a chrysotile-induced malignant transformation strain of human pleural mesothelial cells and application thereof.

Background

Asbestos is a generic term for natural fibrous silicate-like minerals, having high tensile strength, high flexibility, resistance to chemical and thermal attack, electrical insulation and spinnability. The asbestos mainly comprises asbestos, chrysotile, iron asbestos and the like. IARC (international agency for research on cancer) has identified all types of asbestos as a class i carcinogen, i.e., all types of asbestos can lead to the development of malignant tumors. Asbestos exposure results in the development of more than 80% of malignant mesotheliomas. Malignant Mesothelioma (MM) has hidden diseases, the clinical manifestations lack specificity, the misdiagnosis rate is high, the diagnosis is mostly in the middle and late stages, and most patients die within 1 year of confirmed diagnosis. The developing countries of asia such as china, india and thailand are major countries of asbestos imports and exports, and it is presumed that the incidence of malignant mesothelioma will tend to increase year by year for decades in the future. The pathogenesis of malignant mesothelioma is unknown, so that an effective early diagnosis and treatment method is lacking clinically.

The cell malignant transformation model is a material for excellently researching carcinogenic mechanism of carcinogen and can also be used for basic research of diagnosis and treatment of malignant mesothelioma. However, since the genome of human cells is stable and has an effective DNA repair mechanism, and has strong resistance to foreign damage, especially damage to carcinogens is not as sensitive as some animal cells, it is difficult to transform animal cells, so that the research on the carcinogenic activity of cells has been generally performed by using animal fibroblasts, such as NIH/3T3 cells. The human normal mesothelial cells are adopted, and the obtained cell model can directly reflect the actual condition of the effect of the chrysotile on the human target cells. Cell transformation is a change in the genetic alteration of a cell, involving a change in DNA or gene, and is a multifactorial, multistage, multi-pathway process. Thus, it is difficult to cause malignant transformation by 1 treatment regardless of the treatment of normal human cells with chemical, physical, viral, oncogene, etc.

Therefore, the chrysotile-induced malignant transformation of mesothelial cells is very difficult and is rarely reported at present. Yang H et al (Yang H, Bocchetta M, Kroczynska B, et al. TNF-alpha inhibitors for-induced cytotoxicity via NF-kappaB-dependent pathway, a porous mechanism for inhibitors-induced oncogenesis. Proc Natl Acad Sci U S A.2006.103(27):10397 10402.) induce malignant transformation of mesothelial cells with chrysotile, which was specifically: co-culturing mesothelial cell HM and macrophage, pretreating with 10ng/ml TNF-alpha, and adding 5 μ g/cm²After 48 hours of treatment with chrysotile, the culture was continued for 4 weeks with a culture medium containing TNF-. alpha.and cell foci (foci) were formed as a result. In the conversion process, the method adopts a co-culture technology with macrophage and adds TNF-alpha to reduce the toxicity of chrysotile, and the method only adopts chrysotile to treat alone and does not adopt other methods or reagents; this method only detects foci formation of cells cultured in a culture plate and does not verify anchorage-independent growth of transformed cells by soft agar colony formation experiments, which are important criteria for verifying success of malignant transformation of cells, and thus it does not prove successful transformation.

Disclosure of Invention

The invention provides a chrysotile-induced human pleural mesothelial cell malignant transformation strain and application thereof, aiming at the problem that a chrysotile-induced mesothelial cell malignant transformation cell model is lacked in the prior art.

A human pleural mesothelial cell malignant transformant is classified and named as: human pleural mesothelial cells, strain number: AS-T, deposited in the China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan, China in 2019, 1 month and 23 days, with the deposition number: CCTCC NO. C201923. The pleural mesothelial cell AS-T is subjected to multistage multiple toxication treatment by chrysotile on human pleural mesothelial cells (MeT-5A) to generate malignant transformation, and a cell is obtained and named AS human pleural mesothelial cell AS-T, and the cell has higher growth speed than the original MeT-5A cell and obtains the laminated growth characteristic.

The invention also provides a progeny cell of the human pleural mesothelial cell malignant transformant.

The invention also provides application of the human pleural mesothelial cell malignant transformant in a cell model used as a carcinogenic mechanism research. Wherein the carcinogen is chrysotile.

The invention also provides application of the human pleural mesothelial cell malignant transformant in a cell model for researching malignant mesothelioma occurrence mechanism.

The invention also provides application of the human pleural mesothelial cell malignant transformant in extracting a malignant mesothelioma specific tumor marker.

The invention also provides application of the human pleural mesothelial cell malignant transformant in screening or evaluating drugs for treating malignant mesothelioma.

The invention also provides application of the human pleural mesothelial cell malignant transformant in developing a malignant mesothelioma detection kit.

Adopting international standard chrysotile dust to perform intermittent toxicant exposure on normal human mesothelial cells, then removing chrysotile to continue culturing, and adopting an anchorage independence experiment to verify. As a result, after 28 times of contamination and further 15W of culture, the cells obtained malignant characteristics such as stacking growth and anchorage independence. Detecting the change of apoptosis, cell cycle, mitochondrial membrane potential and active oxygen level of chrysotile transformed cells. The invention provides an experimental cell model for the mechanism research, early diagnosis and treatment basic research of the malignant mesothelioma caused by chrysotile.

Drawings

Fig. 1 is a graph showing the results of cell activity assays, where p <0.05, p <0.01, and p <0.001 (the same applies below).

FIG. 2 is a view of microscopic observation of cell stack growth, in which A is MeT-5A cells and B is AS-T MeT-5A cells.

FIG. 3 is a graph of the results of cell anchorage independent growth assay, in which graph A is MeT-5A cells, graph B is AS-T MeT-5A cells, and graph C is the statistical results of the two cells.

FIG. 4 is a diagram of the results of cell scanning electron microscopy, wherein, the diagram A is MeT-5A cells, and the diagram B is AS-T MeT-5A cells.

FIG. 5 is a graph showing the result of detecting apoptosis change by flow cytometry, wherein, the graph A is MeT-5A cell, the graph B is AS-T MeT-5A cell, and the graph C is statistical result.

FIG. 6 is a graph of the statistics of cell cycle changes detected by flow cytometry.

FIG. 7 is a graph showing the results of mitochondrial membrane potential flow assay, in which A is MeT-5A cells, B is AS-T MeT-5A cells, and C is a statistical result graph.

FIG. 8 is a graph showing the results of ROS level measurement, in which graph A is a MeT-5A cell, graph B is an AS-T MeT-5A cell, and graph C is a statistical result graph.

Detailed Description

Example 1

Preparing fibers: chrysotile is from the japan mineral fibre association. When in use, 5mg of chrysotile fiber is weighed and suspended in 1ml of PBS solution to prepare 5mg/ml mother solution, and the mother solution is subjected to ultrasonic treatment in ice water to form uniform suspension. Ultrasonic conditions are as follows: 180W (Watt), 10s on, 5s off, for a total of 30 cycles. And (5) performing high-pressure sterilization after ultrasonic treatment.

Cell culture: human pleural mesothelial cells (MeT-5A) were cultured in M199 medium containing 10% fetal bovine serum at 37 ℃ in 5% CO₂Culturing in saturated humidity environment, subculturing thecells 1 time every 3-4 days, and inoculating according to a ratio of 1: 3. The cells were divided into a transformation group and a negative control group, each group was provided with 3 wells for parallel control.

Transformation experiments: digesting the cells in logarithmic growth phase by 0.25% of pancreatin, blowing, dispersing, counting, diluting with the whole culture solution to 1-2 × 10⁵The density of each cell/well was plated on 6-well plates and placed in a 37 ℃ incubator. 24h after cell inoculation, old culture medium was discarded, and 5. mu.g/cm diluted with PBS was added to the transformed group²Chrysotile, PBS of equal volume was added to the control group, and three replicates were set for each group. After 24h, the medium and chrysotile fibers were aspirated, washed 3 times with PBS and replaced with conventional medium. Human pleura mesothelial cells were treated with intermittent exposure to chrysotile for 24 h/time, once a week, 28 times total, after which exposure to chrysotile was discontinued and culture was continued for 15W (weeks) with M199 containing 10% fetal bovine serum. The growth and morphological changes of the cells were observed and recorded during the experiment using a microscope.

After the MeT-5A cells are subjected to malignant transformation by a multi-stage multi-time contamination experimental method, a cell is harvested, and the cell is classified and named as: human pleural mesothelial cells, strain number: AS-T, deposited in the China Center for Type Culture Collection (CCTCC) at the university of Wuhan, China in 2019, 1 month and 23 days, with the preservation number: c201923, the growth rate of the cell is increased compared with that of the original MeT-5A cell (figure 1), and the stacked growth characteristic is obtained (figure 2).

Anchorage independent growth experiments: after the cells are treated for 72 times and continuously cultured for 2 months, 3ml of 0.7% bottom agar-whole culture medium is added into each hole of a 6-hole plate, 3ml of 0.35% agar-whole culture medium containing 1000 cells to be detected is added into the top layer after solidification, 3 parallel samples are arranged in each group of cells, the cells are cultured for 3 weeks, the colony forming rate of each group of cells is counted, and more than 50 cells are counted into 1 colony. Synchronously cultured blank control cells were cultured in soft agar as described above and observed for colony formation. After multiple exposure treatment, the cells showed the characteristics of laminated growth and anchorage-independent growth (figure 3), and a malignant transformation cell model was successfully obtained.

And the change generated inside the malignant transformation cell AS-T cell is observed by a scanning electron microscope, compared with the MeT-5A cell and the cell which is not transformed by chrysotile, the edge of the cell is observed to have protrusion and the organelle is clear under the scanning electron microscope (figure 4A); cells transformed with chrysotile showed smooth edges, rounded cells, enlarged nuclei, significant decrease in organelles, and abnormal mitochondria and fleshy reticulum (FIG. 4B).

Comparative example 1

The cells were cultured in the same manner as in example 1, but the transformation experiment was carried out in the following manner:

1. the incubation was discontinued at 28W (weeks) and continued after withdrawal without exposure to chrysotile (i.e.no further 15 weeks of incubation were performed as compared to the incubation method of example 1).

2. While the chrysotile was intermittently treated, 800ng/ml phorbol ester (TPA), which is a cancer-promoting agent, was added thereto, and after 28W (weeks) of treatment, culture was continued for 15W (weeks) with or without removal of the treatment product.

3. While the chrysotile was intermittently treated, TNF-. alpha.was added at a concentration of 0.5. mu.g/ml, and after 28W treatment, culture was continued for 15W (weeks) with or without the removal of the treatment product.

In the experiment process, the growth and morphological change of the cells are observed by using a microscope, and a soft agar colony forming experiment is carried out, so that the groups do not form clones on soft agar, namely, cell transformation is not obtained.

Example 2

Flow cytometry detects apoptotic changes.

Subculturing the cells until the cells are fully paved in a single layer, collecting the cells in a centrifuge tube after trypsinization, centrifuging for 5min at 1000g, and removing the culture solution; washing the cells by PBS suspension, and centrifuging the cells at 1000g for 5 min; suspending cells in 500. mu.l of binding buffer, adding Annexin V-FITC/PI 15. mu.l, and standing at room temperature for 5 min; flow cytometry analysis was performed. The results show that chrysotile transformed AS-T MeT-5A cells exhibit apoptosis-resistant properties and apoptosis is significantly reduced compared to MeT-5A cells (FIG. 5).

Example 3

Flow cytometry detects cell cycle changes.

Subculturing the cells until the cells are fully paved in a single layer, digesting the cells by pancreatin, collecting the cells in a centrifuge tube, and centrifuging the cells for 5min at 1000 g; washing cells with PBS suspension for 2 times, centrifuging at 1000g for 5min, and collecting; then 0.3ml PBS is used for fully suspending the cells again, so that the cell agglomeration phenomenon is avoided; adding pre-cooled pure ethanol with a final concentration of 70-75%, and fixing for at least 2 h; centrifuging 1000g for 5min, and discarding ethanol; 1ml PBS suspension washing cell precipitation, standing for 1min, centrifuging for 5min at 1000 g; the cell pellet was suspended in 500. mu.l of PI/Triton X-100 stain, left at 37 ℃ for 15min and then examined by flow cytometry. As shown in FIG. 6, the proportion of the chrysotile transformed cells in the G1 phase was decreased and the proportion of the G2+ M phase was increased as compared with the control group MeT-5A cells, indicating that the proportion of the chrysotile transformed cells arrested in the G1 phase was decreased, indicating that the proliferation of the chrysotile transformed cells was accelerated.

Example 4

Mitochondrial Membrane Potential (MMP) flow assay.

Subculturing the cells until the cells are fully paved in a single layer, and digesting each group of cells by using pancreatin; adding 1mL of culture solution into each group of 1 × 105 cells, washing for 2 times, centrifuging, and removing supernatant; 0.5mL of cell culture medium was added and resuspended. 0.5mL JC-1 staining working solution is added, inverted for a plurality of times and mixed evenly. Incubating in a cell incubator at 37 ℃ for 20 min; the cells were collected by centrifugation (1000rpm/min, 5min) and the supernatant was discarded. Washing with 1mL JC-1 staining buffer for 2 times; adding 0.5mL JC-1 staining buffer, re-suspending, detecting by a flow cytometer according to a standard program, exciting by mercury with the wavelength of 488nm,counting 10⁴And (4) cells.

As shown in FIG. 7, the mitochondrial membrane potential of AS-T MeT-5A cells was slightly higher than that of the control MeT-5A cells, and was significantly different.

Example 5

And (5) detecting the ROS level of the active oxygen.

Subculturing the cells until the cells are fully paved in a single layer, removing the culture solution, and adding 1mL (1: 1000 dilution) of DCFH-DA diluted by a serum-free culture solution; incubate at 37 ℃ for 20min in a cell culture box. Washing the cells three times with serum-free cell culture medium; pancreatin digestion ofgroup 2 cells; centrifuging to collect cells (1000rpm/min, 5min), and discarding the supernatant; flow cytometry detection, 488nm excitation, counting 10⁴And (4) cells. The results are shown in FIG. 8, and compared with control group MeT-5A cells, AS-T MeT-5A cells have reduced ROS levels, and show significant differences.

Claims (1)

Translated fromChinese

1. 一种人胸膜间皮细胞恶性转化株，其特征在于，命名为人胸膜间皮细胞，株号AS-T，保藏号为CCTCC NO. C201923。1. a human pleural mesothelial cell malignant transformation strain, is characterized in that, named as human pleural mesothelial cell, strain number AS-T, deposit number is CCTCC NO.C201923.

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