Cellular Senescence Presentation
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Transcript of Cellular Senescence Presentation
Investigating Cellular Senescence Following Radiation Exposure
Stephen Liu, Mihai Dumbrava, Adrienne Wan and Laura Paterson UNRESTRICTED / ILLIMITÉ
Cellular Senescence
Cellular senescence refers to a process in which cell growth and development are irreversibly halted.
No known factor can induce senescent cells to re-enter the cell cycle.
Although they no longer divide, senescent cells remain metabolically active.
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Types of Senescence
Replicative Senescence Stress Induced
Premature Senescence (SIPS)
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Gamma Radiation
Gamma (γ) radiation is a form of indirectly ionizing radiation.
It can damage DNA through two different processes:1. Direct Action2. Indirect Action
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Lymphocytes
Lymphocytes are a type of white blood cell which recognize and respond to particular pathogens. They are one of the most radiosensitive and long-lived cell types in the human body.
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Gamma Radiation Doses
The Lethal Dose 50 (LD50) is between 4 to 7 Gy for humans (1 Gy= 1 J/kg).
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Absorbed Gamma Radiation Dose (Gy)
Irradation Time (s) Dose Rate (Gy/s) Blood Volume (mL)
0 - - 2.5
0.5 3.4 0.0607 2.5
0.75 7.5 0.0607 2.5
1 11.6 0.0607 2.5
2 28 0.0611 2.5
3 44.4 0.0611 2.5
4 60.8 0.0611 2.5
SIPS Pathway
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Metabolic processes
Gamma radiation exposure
DNA damage which can lead to senescence
Biomarkers of Senescent Cells
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Purpose
This project sets out to determine a relationship between the amount of absorbed gamma radiation and the occurrence of cellular senescence.
Absorbed Gamma Radiation
Perc
enta
ge o
f Se
nesc
ent c
ells
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Hypothesized Correlation
Procedure – Isolating Lymphocytes
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• Irradiate human blood samples
• Dilute blood using PBS
• Carefully layer diluted blood samples on Ficoll
• Centrifuge to separate blood into different layers
• Wash lymphocytes, transfer into RPMI Media
• Incubate for 48 hours at 37°C, 5% CO2
Procedure – Introducing p16 Antibody
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Ethanol Permeabilization
• Vortex and add ethanol to cell pellet
• Incubate at -20°C for at least 2 hours
• Add p16 antibody into cell suspension
• Incubate for 20-30 minutes in the dark
Flow Cytometry
1. Cell sample is inserted into flow cytometer through a small nozzle
2. Cells move in a single file past lasers
3. p16 probes are excited by these lasers to emit light in a specific channel
4. Detectors measure the light emitted
5. The amount of cells, as well as their morphological and photometric features, is determined
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p16 probes
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Phycoerythrin (PE) fluorochrome – a chemical that fluoresces to distinguish cellular properties.
p16 antibody
Normal Cell – Photometrically non-senescent
Fluorescent Cell – Photometrically and morphologically senescent
IDEAS® Software Analysis
Cell features are plotted on different graphs to distinguish senescence.
The percentage of senescent cells can be calculated using the equation:
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Experimental Results
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 50
0.5
1
1.5
2
2.5
3
3.5
4
f(x) = 0.142849977797818 x² − 0.0740297450563477 x
Effects of Radiation on Cellular Senescence γ
Radiation Dose (Gy)γ
Per
cen
tage
of S
enes
cen
t Ce
lls (
%)
Discussion – Acute Radiation SyndromeSe
nesc
ence
in %
Error Analysis
Laboratory Procedure
Software Analysis
Blood Donors
Limited Sample
Size
• Sample contamination
• Cell loss• Incorrect
morphology
• Different radiation exposure
• Lifestyle choices
• Insufficient data to draw conclusions
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Application
THE BRIGHT SIDE
Tumor Suppression
Cellular senescence can stop the uncontrolled division of cancerous
cells
Aging
Senescent cells can disrupt cell growth, differentiation, and initiate or promote age-related diseases
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THE DARK SIDE
Future Work
Radiation Therapy – determining the amount of radiation which will maximize the occurrence of cellular senescence.
Cell Repair
Cell Misrepair
Cell Death
Normal Cell
Division
Determining Accidental Radiation Dose
Conclusion
There is a positive quadratic correlation between the absorbed gamma radiation dose and the percentage of senescent lymphocytes.
Ranging from radiation therapy to age-related diseases, cellular senescence has a variety of applications and drawbacks.
This research has created a foundation for diagnosing an individual’s original radiation dose through the analysis of senescent cells.
Acknowledgements
Works Cited
Abcam. "Introduction to Flow Cytometry." Introduction to Flow Cytometry. Abcam, 2014. Web. 30 July 2014.
AbD Serotec. "Fluorochromes and Light." Fluorochromes and Light. Bio-Rad Laboratories, 2014. Web. 01 Aug. 2014.
Hall, Eric J., and Amato J. Giaccia. Radiobiology for the Radiologist. Philadelphia: Lippincott Williams & Wilkins, 2006. Print.
International Atomic Energy Agency. Radiation Biology: A Handbook for Teachers and Students. Vienna: International Atomic Energy Agency, 2010. IAEA. International Atomic Energy Agency, 2010. Web. 1 Aug. 2014.
Jefferson Lab. "Radiation Biological Effects." Radiation Biological Effects. Jefferson Lab, n.d. Web. 10 July 2014.
Rahman, Misha. "Introduction to Flow Cytometry." Introduction to Flow Cytometry. Bio-Rad Laboratories, 2014. Web. 24 July 2014.
Rodier, Francis, and Judith Campisi. "Four Faces of Cellular Senescence." Four Faces of Cellular Senescence. The Rockefeller University Press, 14 Feb. 2011. Web. 24 July 2014.
Tough, D. F., and J. Sprent. "Lymphocyte life-span and memory." National Center for Biotechnology Information. U.S. National Library of Medicine, May 1995. Web. 01 Aug. 2014.
The End
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