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Chronic low-dose arsenite’s metabolites exposure induced human keratinocytes transformation.
China (IFMSA-China) - Soochow University, Suzhou
Department of Toxicology, School of Public Health
Type of Research Project
- Basic science
What is the background of the project?
Inorganic arsenic (arsenate/arsenite) has long been recognized as human carcinogens by the International Agency for Research on Cancer (IARC). However, the exact molecular and cellular mechanisms involved in arsenic carcinogenesis remain obscure due to the difficulty in establishment of animal models over the years. In recent research of arsenic carcinogenesis, many researchers have directed their attention to methylated metabolites of inorganic arsenics. Recently, study of arsenic species toxicity made rapid progress. Ultimate chemical form and mechanism of arsenic carcinogenesis have been more obvious. The project may establish transformed the immortalized human keratinocytes (Hacat) via low level and long term arsenite’s metabolites treatment. It will offer a new insight for further understanding the molecular mechanism of sodium arsenite exposure to humans.
What is the aim of the project?
This project aims to understand the molecular mechanism of arsenite exposure to keratinocyte transformation.
What techniques and methods are used?
1. Growth kinetics: The population doubling time was obtained using a hemocytometer under a microscope. 2. Measurement of matrix metalloproteinase-9 (MMP-9) levels: The levels of MMP-9 were measured using a commercial Human MMP-9 (Matrix Metalloproteinase 9) ELISA (enzyme linked immunosorbent assay) Kit according to the manufacturer's instructions. 3. Cell proliferation assay: Cell proliferation assay was performed using a CCK-8 (Cell Counting Kit-8) kit according to the manufacturer's directions. 4. Colony formation assay. Soft agar plates were prepared in 35-mm (diameter) dishes with an underlayer of 0.70% agarose in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum). To test for soft-agar colony growth capacity, cells were plated in triplicate at a density of 5×103 in 2 ml 0.35% agarose overlaid on 0.70% agarose in the same medium. The plates were incubated at 37°C in a humidified 5% CO2 atmosphere and the cultures were fed every 3 days. After 3 weeks, colonies with >30 cells were examined microscopically and photographed with a dissection microscope。 Hemocytometer, Microscope, Microplate reader must be used.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
1. He will learn the relevant steps of the cell culture and be able to conduct cell culture independently. 2. The student will establish transformed the immortalized human keratinocytes (Hacat) via chronic low-dose arsenite’s metabolites treatment; 3. He will expose the cells continuously to low doses of arsenic metabolites, and finally detect the changes in related indicators, using cell proliferation assay, colony formation assay and ELISA; He will conduct literature retrieval and reading, and learn to use software including Endnote, ImageJ, SPSS, and GraphPad Prism.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Preliminary readings will be provided. In addition, the tutor will discuss and explain in detail about the project that students will be conducting. Students are encouraged to take part in the group meeting and seminars. Doctoral or master students will help them once a week to study the theory related to this study, and the tutor will organize a discussion every two weeks in the form of a PPT report. Meetings and seminars are in English.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a poster - The student will prepare a scientific report - The student’s name will be mentioned in a future publication - The student will have the opportunity to present the results together with the supervisor at a conference
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
The student should be able to: 1. Read scientific literatures; 2. Conduct General cell culture; 3. Conduct Cell Lysis and Total Protein Extraction; Use instruments including: Hemocytometer, Microscope, Microplate reader.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- 1. Xu Yang; et al. Continuous Activation of Nrf2 and Its Target Antioxidant Enzymes Leads to Arsenite-Induced Malignant Transformation of Human Bronchial Epithelial Cells. Toxicology and Applied Pharmacology. 2015. 289：231-239.
- Dapeng Wang; et al. Hypermethylation of Keap1 Gene Inactivates Its Function and Promotes Nrf2 Nuclear Accumulation Is Involved in Arsenite-Induced Human Keratinocyte Transformation. Free Radical Biology and Medicine. 2015. 89：209-219.
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