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

A technology of skin cells and transformed strains, applied in the fields of biology and oncology, can solve the problem of lack of malignant transformation cell models of mesothelial cells

Active Publication Date: 2019-11-08
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the lack of chrysotile-induced malignant transformation cell models of mesothelial cells in the prior art, and provides a chrysotile-induced human pleural mesothelial malignant transformation strain and its application

Method used

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Fiber preparation: Chrysotile was obtained from the Japan Mineral Fiber Association. When in use, weigh 5 mg of chrysotile fiber and suspend it in 1 ml of PBS solution to make a 5 mg / ml mother solution, and ultrasonically treat it in ice water to form a uniform suspension. Ultrasonic conditions: 180W (watt), 10s on, 5s off, a total of 30 cycles. Autoclave after sonication.

[0024] Cell culture: Human pleural mesothelial cells (MeT-5A) were cultured in M199 medium containing 10% fetal bovine serum at 37°C, 5% CO 2 Cultured in a saturated humidity environment, the cells were subcultured once every 3-4 days, and inoculated at a ratio of 1:3. The cells were divided into a transformation group and a negative control group, and 3 wells were set up in each group as a parallel control.

[0025] Transformation experiment: the cells in the logarithmic growth phase were digested with 0.25% trypsin, dispersed by pipetting, counted, and diluted with the whole culture medium to 1...

Embodiment 2

[0036] Cell apoptosis was detected by flow cytometry.

[0037] The cells were subcultured until the monolayer was confluent, digested with trypsin, collected in a centrifuge tube, centrifuged at 1000g for 5min, and discarded the culture medium; the cells were suspended and washed in PBS, and centrifuged at 1000g for 5min; the cells were suspended in 500μl of binding buffer, and AnnexinV- 15 μl of FITC / PI, placed at room temperature for 5 minutes; analyzed by flow cytometry. The results showed that compared with MeT-5A cells, the AS-T MeT-5A cells transformed with chrysotile showed the characteristics of resistance to apoptosis, and the apoptosis was significantly reduced ( Figure 5 ).

Embodiment 3

[0039] Cell cycle changes were detected by flow cytometry.

[0040] The cells were subcultured until the monolayer was confluent, digested with trypsin, collected in a centrifuge tube, and centrifuged at 1000g for 5 minutes; the cells were suspended and washed twice in PBS, and collected after centrifuging at 1000g for 5 minutes; then the cells were fully resuspended with 0.3ml PBS to avoid There is agglomeration phenomenon; add pre-cooled pure ethanol at a final concentration of 70-75%, fix for at least 2 hours; centrifuge at 1000g for 5min, discard the ethanol; suspend and wash the cell pellet in 1ml PBS, place it for 1min, and centrifuge at 1000g for 5min; use 500μl PI / The cell pellet was suspended in Triton X-100 staining solution, placed at 37°C for 15 minutes, and then detected by flow cytometry. The result is as Figure 6 As shown, compared with the MeT-5A cells in the control group, the proportion of chrysotile-transformed cells in G1 phase was shortened, and the pro...

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Abstract

The invention discloses a chrysotile-induced human pleural mesothelial cell malignant transformation strain and an application thereof. The human pleural mesothelial cell malignant transformation strain is classified and named as human pleural mesothelial cells, the strain number is AS-T, and the preservation number is CCTCC NO.C201923. The human pleural mesothelial cell malignant transformation strain is obtained by carrying out malignant transformation on human pleural mesothelial cells (MeT-5A) after being subjected to chrysotile multi-stage multi-time contamination treatment, and has a good application prospect in multiple aspects such as a cell model for researching a carcinogenic mechanism of carcinogenic substances and the like.

Description

technical field [0001] The invention relates to the technical fields of biology and oncology, in particular to a malignant transformation strain of human pleural mesothelial cells induced by chrysotile and its application. Background technique [0002] Asbestos is a general term for natural fibrous silicate minerals with high tensile strength, high flexibility, chemical and thermal resistance, electrical insulation and spinnability. Asbestos mainly includes crocidolite, chrysotile, and amosite. IARC (International Agency for Research on Cancer) has classified all types of asbestos as Class I carcinogens, that is, all types of asbestos can lead to the occurrence of malignant tumors. Asbestos exposure is responsible for more than 80% of malignant mesotheliomas. Malignant mesothelioma (MM) has an insidious onset, a lack of specificity in clinical manifestations, and a high rate of misdiagnosis. Most of the patients are diagnosed in the middle and late stages, and most patient...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09G01N15/00G01N15/10G01N15/14
CPCC12N5/0693G01N15/10G01N15/00G01N15/14G01N15/1434G01N15/1031G01N2015/1006G01N2015/1486G01N2015/1477G01N15/01
Inventor 朱丽瑾尹先宏张敏鞠莉贾振宇肖芸楼建林陈钧强张幸
Owner ZHEJIANG ACAD OF MEDICAL SCI
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