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Heavy metal copper cytotoxicity detection method based on nitric oxide synthase NOS

A technology of nitric oxide and cytotoxicity, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as incompletely clear molecular mechanisms

Active Publication Date: 2015-11-04
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the specific toxicity mechanism of heavy metal copper, especially the molecular mechanism related to signal transduction, is still not fully understood.

Method used

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  • Heavy metal copper cytotoxicity detection method based on nitric oxide synthase NOS
  • Heavy metal copper cytotoxicity detection method based on nitric oxide synthase NOS
  • Heavy metal copper cytotoxicity detection method based on nitric oxide synthase NOS

Examples

Experimental program
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Embodiment 1

[0036] The test object of this embodiment: copper chloride.

[0037] Preparation method: Dissolve the standard product in sterile water to prepare a 0.1mol / L mother solution and store it at -20°C. Dilute it with culture medium to the required concentration before operation.

[0038] Model organism: human breast cancer MCF‐7 cells.

[0039] Reagents: RNA Rapid Extraction Kit (Spin-column was purchased from Bioteke; N-(6-methoxy-8-quinolyl) p-toluenesulfonamide (TSQ) was purchased from AAT Bioquest; N-acetyl-L-cysteine Amino acid (LNAC), L‐arginine (L‐Arg), dihydrorhodamine 123 (DHR), eNOS antibodies were purchased from Sigma; nNOS,, iNOS, β‐actin antibodies were purchased from Abcam; p‐elF2α Antibodies were purchased from Invitrogen; Western luminescence kits were purchased from Pierce; Fluo-3 / AM and nitric oxide detection kits were purchased from Beyond Biotechnology Research Institute; reverse transcription kits and RT-PCR kits were purchased from Bao Bioengineering Dalian Co...

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Abstract

The invention discloses a heavy metal copper cytotoxicity detection method based on nitric oxide synthase (NOS) and belongs to the technical field of heavy metal cytotoxicity. NOS dimers can be depolymerized by replacing zinc ions and calcium ions in the NOS dimers with heavy meter copper, the synthetic quantity of NO is reduced, and mRNA expression of NOS is regulated upwards in a feedback mode; on the other hand, the content of active oxygen in cells rises together with the monomer quantity of the NOS during depolymerization of the NOS dimmers, a cell translation initiation factor is phosphorylated, protein synthesis stops, finally, a toxicity mechanism of apoptosis is caused, the nitric oxide synthase (NOS) is used for heavy metal copper cytotoxicity detection, and a new solution is provided for performing biosafety assessment of heavy metal pollution and preventing and treating heavy metal poisoning and related diseases of heavy metal poisoning.

Description

technical field [0001] The invention relates to a method in the technical field of heavy metal toxicity, in particular to a method for detecting heavy metal copper cytotoxicity mediated by nitric oxide synthase NOS signaling pathway. Background technique [0002] Heavy metal copper is an essential trace element in living organisms, a cofactor of various enzymes, and participates in electron transfer in biological systems. At the same time, studies have shown that after exposure to copper ions, a large amount of reactive oxygen species are produced, leading to apoptosis of breast cancer epithelial cells. In neurons, copper exposure-induced apoptosis is associated with increased intracellular NO synthesis. Nitric oxide synthase (Nitric Oxide Synthase, NOS) is a kind of isoenzyme, which is the only kind of specificity in organisms that uses L‐Arginine (L‐Arginine) as a substrate and uses oxygen to generate NO Nitrogen (NO) enzymes. NO is a soluble gas. As a signal molecule, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/26C12Q1/02
Inventor 周培陆伟钟玲盈
Owner SHANGHAI JIAO TONG UNIV
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