Volume 10, Issue 1 (2019)
JMBS 2019, 10(1): 125-131 |
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Kavoosi S, Shirzad H, Jalili S, Sadeghizadeh M, Motahari P. Monitoring Lead Toxicity by Huh7-1x-ARE-luc Cell Line Lucifersae Biosensor. JMBS 2019; 10 (1) :125-131
URL: http://biot.modares.ac.ir/article-22-13931-en.html
URL: http://biot.modares.ac.ir/article-22-13931-en.html
1- Genetics Department, Biologic Sciences Faculty, Tarbiat Modares University, Tehran, Iran
2- Research Institute of Police Science and Social Studies, Tehran, Iran, Research Institute of Police Science and Social Studies, Tehran, Iran , hadi_shirzad@yahoo.com
3- Research Institute of Police Science and Social Studies, Tehran, Iran
2- Research Institute of Police Science and Social Studies, Tehran, Iran, Research Institute of Police Science and Social Studies, Tehran, Iran , hadi_shirzad@yahoo.com
3- Research Institute of Police Science and Social Studies, Tehran, Iran
Abstract: (6948 Views)
Aims: The accumulation of free radicals in the body leads to damages to cellular biopolymers through oxidative stress. Due to the increasing proliferation of heavy metals in soil and water environments, finding efficient methods for diagnostic detection and measurement of heavy metals in contaminated environments is very important. Cell-based biosensors can produce a measurable signal in response to specific chemical or physical agent in their environment. In this study, stable hepatoma Huh7 ARE-reporter cell line was developed containing luciferase gene with the aim of monitoring lead toxicity. This biosensor is reported to be able to detect lead by expressed signal which is measurable. The luciferase assay and Real time-PCR were performed.
Materials and Methods: In this experimental research, the Huh7-1x-ARE-luc was stably transferred in to the Huh7 cells and transfected cells were selected. After 5 passages, stable clones were isolated to confirm plasmid entrance. Luciferase activity of the Huh7-1x-ARE-luc cell line was performed with 0-80μM lead concentration to induce oxidative stress response. Cell viability was assessed by MTT. With Real time PCR, AREKEAP1 pathway gene expression were detected. Statistical analysis was performed by ΔCt method, using graphpad prism 6 software.
Findings: The gene expression of the reporter gene increased with increasing oxidative stress. Reducing the expression of the reporter gene was observed after 30 μM. 35 μM lead inhibited 50% cell metabolism. Expression of antioxidant pathway genes was significantly increased in 30 μM leaded cells compared to control gene.
Conclusion: The biosensor prepared from Huh7-1x-ARE-luc cell line of the reporter gene can be a convenient and efficient means for measuring oxidative compounds such as heavy metals such as lead.
Materials and Methods: In this experimental research, the Huh7-1x-ARE-luc was stably transferred in to the Huh7 cells and transfected cells were selected. After 5 passages, stable clones were isolated to confirm plasmid entrance. Luciferase activity of the Huh7-1x-ARE-luc cell line was performed with 0-80μM lead concentration to induce oxidative stress response. Cell viability was assessed by MTT. With Real time PCR, AREKEAP1 pathway gene expression were detected. Statistical analysis was performed by ΔCt method, using graphpad prism 6 software.
Findings: The gene expression of the reporter gene increased with increasing oxidative stress. Reducing the expression of the reporter gene was observed after 30 μM. 35 μM lead inhibited 50% cell metabolism. Expression of antioxidant pathway genes was significantly increased in 30 μM leaded cells compared to control gene.
Conclusion: The biosensor prepared from Huh7-1x-ARE-luc cell line of the reporter gene can be a convenient and efficient means for measuring oxidative compounds such as heavy metals such as lead.
Article Type: _ |
Subject:
Agricultural Biotechnology
Received: 2016/11/16 | Accepted: 2018/02/1 | Published: 2019/03/16
Received: 2016/11/16 | Accepted: 2018/02/1 | Published: 2019/03/16
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