研究目的
To investigate the role of TEMs and autophagy in the development of AMD.
研究成果
Our results showed that the knockdown of TEMs in laser-induced CNV mice can reduce autophagy and suppress experimental CNV in vivo and in vitro. The TEMs regulation of autophagy and the VEGF mediation of angiogenesis may occur through an AMPK pathway. Our data suggest that TEMs play an important role in CNV, and they may be a novel preventive target for nAMD.
研究不足
The intra-choroidal infiltration of TEMs during laser-induced CNV needs to be detected, and the mechanism of TEMs infiltrating into the CNV lesions is still unclear and needs to be further evaluated. Also, the specific association of Ang2-TEMs pathway with VEGF-A/VEGFR2 signal is still unknown, and the different effects of TEMs and other types of macrophages on CNV still require further evaluation.
1：Experimental Design and Method Selection:
Employed macrophage-specific Tie2 knockout mice and used a laser-induced choroidal neovascularization (CNV) model.
2：Sample Selection and Data Sources:
Used Tie2flox/+ mice and LyzCre+ mice to generate Tie2flox/flox; LyzCre+ mice (TKO mice), and Tie2flox/+; LyzCre+ mice (WT mice, as control).
3：List of Experimental Equipment and Materials:
Included a 532-nm laser photocoagulator, 3-Methyladenine (3-MA), phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium, fetal bovine serum, and various antibodies for western blotting.
4：Experimental Procedures and Operational Workflow:
Included laser-induced CNV, intravitreal injection of 3-MA, fundus fluorescein angiography, cell isolation and culture, quantitative real-time PCR, western blotting analysis, cell migration assay, transwell chamber assay, cell tube formation assay, and immunofluorescence histochemistry.
5：Data Analysis Methods:
Statistical data were analyzed using SPSS 19.0 software, and results were expressed as mean ± SD. A one-way analysis of variance (ANOVA) was performed to evaluate differences between groups.
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532-nm laser photocoagulator
Used to induce choroidal neovascularization (CNV) in mice.
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3-Methyladenine (3-MA)
MCE
Used to inhibit autophagy.
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phosphate-buffered saline (PBS)
Used to dissolve 3-MA for intravitreal injection.
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Dulbecco’s modified Eagle’s medium
Sigma
Used for cell culture.
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fetal bovine serum
Sigma
Used as a supplement in cell culture medium.
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TRC-50IA retinal camera
TRC-50IA
Topcon
Used for fundus fluorescein angiography.
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TRIzol reagent
Invitrogen
Used for total RNA extraction.
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Prime Script RT Master Mix
TaKaRa
Used for reverse transcription.
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SYBR Green PCR Master Mix
TaKaRa
Used for quantitative Real-time PCR.
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Enhanced Chemiluminescence fluorescence detection system
Used to visualize protein levels in western blotting.
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Matrigel matrix
cat. no. 356234
BD Biosciences
Used for cell tube formation assay.
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SPSS 19.0 software
19.0
SPSS Inc.
Used for statistical data analysis.
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