研究目的
Investigating the therapeutic effects of human neural progenitor cells (hNPCs) on retinal degenerative diseases by identifying the retinal proteomic changes that occur following hNPC treatment.
研究成果
hNPCs aid in retinal survival by activating the NRF2-mediated oxidative stress response pathway, both in vitro and in vivo. This study suggests that similar hNPC-induced survival mechanisms occur in both environments, offering potential for therapeutic applications in retinal degenerative diseases.
研究不足
Technical sensitivity may limit the detection of low abundance proteins. The study provides a global view of retinal proteins, which may not accurately identify cell-specific mechanisms. Further proteomic analysis with specific cell types is needed.
1：Experimental Design and Method Selection:
A co-culture system of retinas and hNPCs was developed to mimic the retinal environment following hNPC injection. Proteomic analysis was performed to identify changes in protein expression.
2：Sample Selection and Data Sources:
Retinal tissue from RCS rats at different stages of degeneration (P30 and P60) was used. Media from hNPCs cultured alone and co-cultured with retinas were collected for proteomic analysis.
3：List of Experimental Equipment and Materials:
hNPCs were cultured in Stemline Neural Stem Cell Expansion Medium supplemented with EGF and LIF. Retinal samples were processed using various kits and reagents for protein digestion and analysis.
4：Experimental Procedures and Operational Workflow:
Retinas were co-cultured with hNPCs or alone for up to 7 days. Proteins were extracted, digested, and analyzed by liquid chromatography mass spectrometry (LC MS/MS).
5：Data Analysis Methods:
Proteomic data were analyzed using bioinformatic tools including Ingenuity Pathway Analysis (IPA) to identify significantly affected pathways and upstream regulators.
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Stemline Neural Stem Cell Expansion Medium
Sigma-Aldrich
Culture medium for human neural progenitor cells (hNPCs)
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epidermal growth factor (EGF)
Sigma-Aldrich
Supplement for hNPC culture medium
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leukemia inhibitory factor (LIF)
Millipore
Supplement for hNPC culture medium
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Accutase
Sigma-Aldrich
Dissociation reagent for hNPC neurospheres
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24-well plate
Greiner Bio-One
Cell culture plate for hNPCs and retinal co-culture
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Thincert 0.4 μm pore translucent transwell chamber
Greiner Bio-One
Co-culture system for retinas and hNPCs
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Amicon Ultra-0.5 Centrifugal Filter Ultracel – 3K Device
Millipore
Protein concentration device
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NanoDrop One Analyzer
Thermo-Fisher
Protein quantification
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FASP Protein Digestion Kit
Expedeon
Protein digestion for mass spectrometry
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Oasis HLB 96-well plate 30 μm/5mg
Waters Corporation
Sample desalting for mass spectrometry
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Dionex Ultimate 3000 NanoLC
Thermo Fisher
Liquid chromatography system for mass spectrometry
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Orbitrap Elite
Thermo Fisher
Mass spectrometer for proteomic analysis
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EasySpray ion source
Thermo Fisher
Ion source for mass spectrometry
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PepMap RSLC C18 2μm, 100?, 50μm i.d. x 15cm
Thermo Fisher
Analytical column for liquid chromatography
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Leica CM1850 cryostat microtome
Leica Biosystems
Cryosectioning of retinal tissue
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Leica DM6000B microscope
Leica Microsystems
Imaging of retinal sections
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TUNEL Apoptosis Detection Kit
Millipore
Detection of apoptotic cells in retinal sections
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