Multiwalled carbon nanotube 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 problems such as difficult malignant transformation and stimulation, and achieve good application prospects

Active Publication Date: 2019-04-26
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, regardless of the treatment of normal human cells with chemical, physical, virus and oncogenes, malig

Method used

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  • Multiwalled carbon nanotube induced human pleural mesothelial cell malignant transformation strain and application thereof
  • Multiwalled carbon nanotube induced human pleural mesothelial cell malignant transformation strain and application thereof
  • Multiwalled carbon nanotube 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: MWCNT was purchased from sigma company, No. Aldrich 659258. When in use, weigh 5 mg of MWCNT powder and dissolve it in 1 ml of M199 culture solution to make a 5 mg / ml mother solution, and ultrasonically treat it in ice water to form a uniform suspension. Ultrasonic conditions: 180W, 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 4-5 days, and inoculated at a ratio of 1:4. Cells were divided into treatment group / transformation group and blank control group, and each group had 3 wells for parallel control.

[0025] Transformation experiment: cells in the logarithmic growth phase were digested with 0.25% trypsin, counted after being dispersed by pipetting, and diluted to 1-2×10 5 Cells / well were seeded on 6-...

Embodiment 2

[0030] Cell apoptosis was detected by flow cytometry.

[0031] The MeT-5A and MCN-MeT-5A cells were subcultured until the monolayer was confluent, digested with trypsin, collected in a centrifuge tube, centrifuged at 1000g for 5min, discarded the culture medium; then suspended and washed the cells with PBS, centrifuged at 1000g for 5min After the cells were resuspended with 500 μl of binding buffer, 15 μl of Annexin V-FITC / PI was added and left at room temperature for 5 minutes; finally, flow cytometry analysis was performed. The results showed that, compared with MeT-5A cells, MCN-MeT-5A cells showed resistance to apoptosis after MWCNT exposure for 12 months. However, when MWCNT was exposed to poison for 6 months, the early apoptosis rate and the total apoptosis rate of the cells were significantly increased ( Figure 5 ).

Embodiment 3

[0033] Cell cycle changes were detected by flow cytometry.

[0034] MeT-5A and MCN-MeT-5A cells were subcultured to monolayer confluence, digested with trypsin, collected in centrifuge tubes, centrifuged at 1000g for 5min; suspended and washed in PBS twice, centrifuged at 1000g for 5min and collected; Re-suspend cells in 0.3ml PBS to avoid cell clumping; add pre-cooled pure ethanol at a ratio of 70-75% of the final concentration, and fix for at least 2 hours; centrifuge at 1000g for 5 minutes, discard ethanol; suspend and wash the cell pellet in 1ml PBS, and place Centrifuge at 1 000 g for 5 min for 1 min; suspend the cell pellet with 500 μl PI / Triton X-100 staining solution, place at 37°C for 15 min, and then use flow cytometry for detection.

[0035] Table 1 Changes of MCN-MeT-5A cell cycle

[0036] cell name

[0037] The results are shown in Table 1. Compared with MeT-5A cells, MCN-MeT-5A cells increased in G1 phase and decreased in S phase.

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Abstract

The invention discloses a multiwalled carbon nanotube induced human pleural mesothelial cell malignant transformation strain and application thereof. The human pleural mesothelial cell malignant transformation strain is classified and named as a human pleural mesothelial cell, with the stain number of MCN-MeT-5A and the preservation number of C2018124. The human pleural mesothelial cell malignanttransformation strain is obtained by carrying out MWCNT multi-stage and multi-time exposure treatment on the human pleural mesothelial cells (MeT-5A) and performing malignant transformation. The multiwalled carbon nanotube induced human pleural mesothelial cell malignant transformation strain has a good application prospect in aspects such as cell models for studying a carcinogenic mechanism of carcinogens.

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 multi-walled carbon nanotubes and an application thereof. Background technique [0002] Multi-walled carbon nanotubes (MWCNTs) are currently one of the most commonly used nanomaterials and are widely used in various fields such as industrial manufacturing, nanotechnology and biomedicine. However, with the promotion and use of MWCNT in many fields, more and more MWCNT enter the environment and organisms and accumulate in various ways, such as occupational exposure in the production of MWCNT and its products; changes in physical and chemical factors in the environment such as pH value, ultraviolet radiation, etc. change the properties of MWCNT in the original product, so that the MWCNT that is not easy to escape will fall off, increasing the chance of entering the organism. MWCNT has the ...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693C12N2503/02G01N33/5011G01N2500/10
Inventor 鞠莉朱丽瑾应士波吴昊尹先宏楼建林贾振宇余珉张淑芝
Owner ZHEJIANG ACAD OF MEDICAL SCI
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