研究目的
Investigating the potential role and regulatory mechanism of SIRT6 in hydrogen peroxide (H2O2)-induced oxidative damage of retinal ganglion cells (RGCs) in vitro.
研究成果
SIRT6 protects RGCs from H2O2-induced oxidative damage by promoting Nrf2/ARE signaling via inhibition of Bach1, suggesting it as a potential therapeutic target for retinal degenerative diseases.
研究不足
The study is conducted in vitro, which may not fully replicate in vivo conditions. The mechanisms may involve other pathways not explored, and further investigation is needed for clinical applications.
1：Experimental Design and Method Selection:
The study used in vitro experiments on primary RGCs isolated from rat retinas to investigate the role of SIRT6 in oxidative stress induced by H2O
2：Methods included cell culture, transfection with plasmids and siRNAs, RT-qPCR, western blot, MTT assay, LDH release measurement, caspase-3 activity assay, ROS detection, and luciferase reporter assay. Sample Selection and Data Sources:
Primary RGCs were isolated from neonatal SD rats.
3：List of Experimental Equipment and Materials:
Equipment includes microplate readers, real-time PCR systems, fluorometers, and centrifuges. Materials include H2O2, antibodies, kits for assays, and transfection reagents.
4：Experimental Procedures and Operational Workflow:
Cells were cultured, transfected, treated with H2O2, and subjected to various assays to measure viability, apoptosis, ROS, and signaling pathways.
5：Data Analysis Methods:
Statistical analysis was performed using SPSS with ANOVA and Bonferroni post hoc test.
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Applied Biosystems 7900HT Real-Time PCR System
7900HT
Applied Biosystems
Real-time quantitative PCR
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anti-Bach1 antibody
WH0000571M2
Sigma-Aldrich
Primary antibody for western blot
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OX-4
Abcam
Used to remove macrophages in cell culture
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Thy1.1 antibody
Abcam
Used to select retinal ganglion cells in cell culture
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DMEM
Gibco
Cell culture medium
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Lipofectamine RNAi Max
Invitrogen
Transfection reagent for siRNAs
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H2O2
Sigma-Aldrich
Inducing oxidative stress in cells
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TRIzol reagent
Invitrogen
RNA isolation
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High Capacity cDNA Reverse Transcription Kit
Thermo Fisher Scientific
Reverse transcription to cDNA
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PerfeCTa SYBR Green FastMix
Quanta Biosciences
PCR mix for real-time PCR
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RIPA lysis buffer
Protein extraction
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Nuclear Protein Extraction Kit
Beyotime Biotechnology
Extraction of nuclear proteins
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Bradford Protein Assay Kit
Beyotime Biotechnology
Protein concentration measurement
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PVDF membranes
Millipore
Membrane for western blot
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anti-SIRT6 antibody
ab191385
Abcam
Primary antibody for western blot
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anti-Nrf2 antibody
ab137550
Abcam
Primary antibody for western blot
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anti-GAPDH antibody
ab181602
Abcam
Primary antibody for western blot
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anti-Lamin B2 antibody
ab151735
Abcam
Primary antibody for western blot
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horseradish peroxidase-conjugated secondary antibodies
Sigma-Aldrich
Secondary antibody for western blot
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enhanced chemiluminescence detection reagents
GE Healthcare
Detection for western blot
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