B101099139 李佳駺introduction
B101099047 黃冠閔results
More harmful to body comparing with LDL Damage to:
◦ Endothelial Cells ◦ Cardiac myocytes ◦ Smooth muscle cells
◦ Platelet endothelium interaction ◦ Macrophages
◦ Kidney
◦ Chondrocytes
OxLDL ECs apoptosis Homeostatic
Life extension *:
◦ Worm, fruit fly, short-lived fish Cancer prevention:
◦ Skin and GI tumor
Cardioprotective effects: ◦ eNOS activity ↑
◦ Platelet aggregation↓ ◦ LDL peroxidation↓ Antidiabetic effects
Neuroprotective effects (RIMA) ◦ Reversible inhibition of MAO-A Anti-inflammatory effects
binds, internalizes and degrades oxidized
low-density lipoprotein
regulation of Fas-induced apoptosis
atherosclerosis, risk of myocardial infarction,
Isolation of mouse Cerebrovascular Ecs (CECs):
◦ Trypsin, collagenase B, centrifuge
LDL and OxLDL isolation
◦ Human blood LDL , + Cu2SO4 (48hrs)
ROS quantification
◦ ROS sensitive Dye and flow cytometry
DNA Ladder Analysis
Quantification of DNA Fragmentation
◦ Anthos 2010 microplate photometer, 450 nm wavelength
Analysis of apoptotic Cells
◦ Flow cytometry
Knockdown and over expression of Lox-1
◦ RNAi and siRNA
Immunoblot and RT-PCR Statistical Analysis
No adverse effect under the concentrations of 0.5,
1, 5, and 10 μmol/L resveratrol for 24, 48, and 72 hours
After exposure for 72 hours, resveratrol at 25
μmol/L caused 30% decrease in cell viability
When concentration reached 50 μmol/L,
administration of resveratrol for 48 and 72 hours decreased the viability of mouse CECs by 44%
and 59% , respectively
No adverse effect observed below the concentration of 10 μmol/L
LDL was reacted with copper sulfate for 1, 6, 12, 18, and 24 hours Formation of MDA (which implies the increase of oxLDL)
Level of oxLDL drop as the concentration of resveratrol increase
Concentration of resveratrol is 10μmol/L
CECs in this group is pretreated with N-acetylcystein, a strong anti-oxidant
No significant difference oxLDL group and pretreatment with Res+oxLDL Pretreatment is not protective
Cell viability in post treatment with Res+oxLDL group is significantly less than control, yet higher than pretreatment with Res+oxLDL group
These two proteins are essential for the formation of tight junction
Apoptotic changes (shrinkage of cell) induced by oxLDL
Fragmentation of DNA (formation of DNA ladder) induced by oxLDL
Fragmentation of DNA increase OD values at 450nm
More effect if co-treated with Resveratrol and
Lox-1 cDNA can increase the Produciton of Lox-1 receptor
Resveratrol alone doesn’t decrease Lox-1 mRNA production
Lox-1 mRNA production decreased Significantly comparing these to group
Resveratrol alone
doesn’t decrease Lox-1 protein production
Lox-1 protein production decreased Significantly comparing these to group
Most Bax locate inside of cytoplasm Bax translocate to mitochondria Decreased translocation of Bax Decreased translocation of Bax Decreased translocation of Bax Decreased translocation of Bax
According to the previous research, when
resveratrol is about 4μmol/L, it has antitumor activity.
Thus, the concentration of 1~10 μmol/L may
be higher than it can be in vivo applied.
However, the first experiment of this study has showed that it is within the safe range.
Resveratrol protects CECs from
oxLDL-induced damage of CECs via an anti-apoptotic mechanism.
Resveratrol decreases alterations of the
fragmentation of genomic DNA, reduces sub-G1 phase arrested CECs, reduces the
translocation of Bax proteins, has synergistic effects with Z-VEID-FMK, and suppresses the overexpression of LOX-1.
This study shows resveratrol can protect
CECs from oxLDL-induced apoptotic insults via the way mentioned previously.
Q:紅酒要喝多少才可以達到研究中的效果？ A:根據北醫另外一位老師的研究，大約要十公升。 Q:把受傷的細胞就回來，會不會反而癌化？ A:Resveratrol是在源頭就吸收了自由基，進而拯 救細胞，而不是在細胞受損後才讓它不死。 Resveratrol中的苯環可以吸收電子，吸收自由基
Q:Z-VEID FMK的作用？ A:它是一種caspase-6的抑制劑。 Q:為什麼是這個而不是用其他的藥呢？ A:根據前人的研究，這個是最常用的，因為它是細 胞凋亡前的最後一個caspase，有caspase-6就一 定會有細胞凋亡，有3或9則不一定，所以抑制 caspase-6可以抑制凋亡，所以選這個藥。
曾士剛：為什麼老師不把定量後的數據做計算，得 到更多的推論？(例如把oxLDL的量和resveratrol 的量相除，得到最適比例等等) 連老師：這個實驗的假設比較是要實驗定性的，之 所以定量是可以協助我們進行差異比較，這樣就可 以說明我們的假設是否成立，這麼一點數據的數據 計算，並沒辦法多說明什麼，不如留在下次實驗， 一次做的更完整些。
