Download The Effect of Pathway-Interconnectors in SEB Induced Apoptosis

The Effect of Pathway-Interconnectors in SEB Induced Apoptosis Related Events in Human PBMCs
By: James D Waldschmidt, Advisor- Dr. Chanaka Mendis
Summary
Within the human immune system, cells such as peripheral blood mononuclear cells (PBMCs) exist as the
first line of defense against toxins such as Staphylococcal Enterotoxin B (SEB). The RNA and proteins that
are created by the PBMC caused by SEB can be analyzed through processes such as PCR and ELISA
assays. Through these processes, gene regulation levels and expression patterns can be analyzed to further
gene therapy treatment through a mechanistic and signal flow understanding.
Introduction
Human peripheral blood mononuclear cells (PBMCs) are considered the first line of defense in the
human immune system. The PBMC consists of a single, round nucleus and is a non-isolated circulatory
cell. Some examples of these include white blood cell (phagocyte), monocyte, or macrophages. In order to
begin the signal to trigger an immune response, some of the cells die and release signals to start chemical
lines of defense. The death of these cells is known as apoptosis. The apoptosis cycle begins with the toxin,
in this case Staphylococcal Enterotoxin B (SEB), binding to the outside cell membrane. The trigger begins
a cascade of cellular chemical signaling arriving at the nucleus. Genes in the DNA of the nucleus are
expressed and a cascade of responses begins. Eventually, chemicals are produced that cause the final
apoptosis and disruption of the cell.
The research of pathway definition and analysis began with an experiment I designed based upon
parameters outlined by my advisor, Chanaka Mendis. In vitro (Outside of body) simulations of these
effects were performed at a partnering lab in Maryland. The samples were then sent back to UWPlatteville to be analyzed by our research group. The cells initially had an RNA extraction performed on
them to gather the product of the DNA (transcription). The RNA was then placed into a more stable form
of cDNA (complementary DNA) through reverse-transcription polymerase chain reaction (RT-PCR). The
cDNA was then taken and had regular polymerase chain reaction (PCR) to create more copies of the
selected gene for analysis relative to a normal of background genomic DNA. The PCR was then analyzed
using Syber Green gel electrophoresis, fluorescence, and intensity comparisons using a program known
as ImageJ. Based upon controls, conditions, and inhibition, gene expression levels could be quantified.
Materials & Methods
Figure 1. Pathway Identification.
RNA Extraction: Trizol Reagent Method
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Reagents: Sample, Trizol Reagent, Chloroform, Isopropyl Alcohol, 75% Ethanol, RNase-free water
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Instrumentation: Centrifuge (12,000g maximum), Heat block (55-60°C)
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Consumables: 1.5 mL polypropylene microcentrifuge tubes, 10-1000μL pipettes & tips
RT-PCR: iScript cDNA synthesis kit (25 x 20uL reactions)
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Reagents: Sample, 5x iScript reaction mix, Nuclease-free water, iScript reverse transcriptase, RNA
template
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Instrumentation: Eppendorf PCR Thermocycler
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Consumables: 100 μL polypropylene microcentrifuge tubes, 10-100 μL pipettes & tips
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Reaction Protocol:
o 5 min. @ 25°C
o 30 min. @ 42°C
o 5 min. @ 85°C
o ∞ @ 4°C
PCR: Roche Diagnostics PCR Master kit
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Reagents: Sample, 1x PCR Mastermix, Left Primer, Right Primer, Sterile water
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Instrumentation: Eppendorf PCR Thermocycler
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Consumables: 100 μL polypropylene microcentrifuge tubes, 10-100 μL pipettes & tips
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Reaction Protocol:
o 2 min @ 94°C (1 cycle)
o 30 sec. @ 94°C, 60 sec. @ 58°C, 3 min. @ 72°C (30 cycles)
o 7 min. @ 72°C (1 cycle)
Gel Electrophoresis:
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Reagents: Agarose, Run Buffer, Deionized water, Syber Green Dye, Sample, Loading Dye
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Instrumentation: Gel Electrophoresis Tank, UV lamp @ 265nm, Camera with Syber Green lens
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Consumables: Gloves, 10-100 μL pipettes & tips
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Run: 30 min. @ 115V
Results & Discussion
Figure 2. Syber Green Fluorescence Gel Electrophoresis Image
Table 1. Human PBMC Gene Expression Time-point Inhibition Analysis.
According to the relative gene expression levels, every gene within the pathway was upregulated by
SEB. The inhibitor dropped certain levels and made others higher.
Conclusion
To conclude, the human PBMCs were indeed affected by the SEB and the inhibitors did have an effect
of up or down-regulating the gene expression. Future work on this experiment will entail analysis of protein
expression levels to verify RNA expression.