ObjectiveTo analyze the early changes of gene expression levels and signaling pathways in 661W cell line under hypoxic conditions and to find potential functional target genes.MethodsThe cultured mouse 661W cells were divided into hypoxia treatment group and normoxia control group. Cells in the hypoxia treatment group were cultured in a three-gas incubator with volume fraction of 1% and 5% CO2 at 37 ℃. Cells in the normoxia control group were cultured in an incubator at 37 ℃ with volume fraction of 5% CO2. High-throughput sequencing technology was used to sequence the transcriptome of 661W cell treated with hypoxia and normoxia for 4 hours to screen for differentially expressed genes (DEG). Clustering heat map analysis, gene ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network (PPI) analysis were performed. The reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the accuracy of the sequencing results.ResultsA total of 506 differentially expressed genes were screened, including 459 up-regulated genes and 47 down-regulated genes. GO functional enrichment analysis showed that the main biological processes of DEG were the cell's response to hypoxia, glycolysis, negative regulation of cell proliferation and apoptosis. hypoxia inducible factor (HIF)-1α pathway, glycolysis, Forkhead box O (FoxO) pathway, Insulin signaling pathway and Adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway were involved in the above process. PPI analysis results showed that hub genes related to hypoxia were Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10 and Fbxo27. The RT-PCR results showed that the relative expression levels of 15 DEG mRNA in the hypoxic treatment group were higher than that of the normoxic control group, and the difference was statistically significant (P＜0.05). The mRNA expression levels of N-myc downstream-regulated gene-1 (Ndrg1), Mt1, and vascular endothelial growth factor A (VEGFA) were time-dependent on hypoxia.ConclusionsUnder hypoxia, DEG is mainly related to glucose metabolism, cell response to hypoxia, regulation of proliferation and apoptosis. HIF-1α pathway, glycolysis, FoxO pathway and AMPK pathway are involved in the early changes of 661W cells under hypoxia. Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10, Fbxo27 may play key roles in the response of 661W cells to hypoxia. Ndrg1, Mt1 and VEGFA could be potential functional target genes for the study of ischemia and hypoxia-related fundus diseases.
ObjectiveTo observe the interobserver agreement of classification of macular degeneration in severe pathological myopia (PM) by ophthalmologists with different clinical experience. MethodsA retrospective study. From January 2019 to December 2021, 171 eyes of 102 patients with severe PM macular degeneration who were examined at Eye Center of Beijing Tongren Hospital of Capital Medical University were included in the study. The clinical data such as age, gender, axial length, spherical equivalent power, fundus color photography, and optical coherence tomography (OCT) were collected in detail. Six independent ophthalmologists (A, B, C, D, E, F) classified each fundus photography based on META-PM and ATN classification of atrophy (A) system and interobserver agreement was assessed by Kappa statistics. According to the classification standard of traction (T) in the ATN classification, the OCT images were interpreted and classified, in which T0 was subdivided into retinal pigment epithelium (RPE) and choroidal thinning, choroidal neovascularization (CNV) with partial RPE and choroidal atrophy, RPE, and choroidal atrophy. Lamellar macular hole can't be classified by ATN system, which was defined as TX. Kappa (κ) test was used to analyze the consistency of classification results between physicians A, B, C, D, E and F. κ value ≤0.4 indicates low consistency, 0.4＜κ value ≤ 0.6 indicates moderate consistency, and κ value ＞0.6 indicates strong consistency. ResultsAmong the 171 eyes of 102 cases, there were 20 males with 37 eyes (19.6%, 20/102), and 82 females with 134 eyes (80.4%, 82/102); age was 61.97±8.78 years; axial length was (30.87±1.93) mm; equivalent spherical power was (－16.56±7.00) D. Atrophy (A) classification results in META-PM classification and ATN classification, the consistency of physician A, B, C, D, E and physician F were 73.01%, 77.19%, 81.28%, 81.28%, 88.89%; κ value were 0.472, 0.538, 0.608, 0.610, 0.753, respectively. In the ATN classification, the T0, T1, T2, T3, T4, and T5 were in 109, 18, 11, 12, 9, and 8 eyes, respectively; TX was in 4 eyes. ConclusionsThere are differences in the consistency of classification of severe PM macular lesions among physicians with different clinical experience, and the consistency will gradually improve with the accumulation of clinical experience.