研究目的
To investigate the apoptosis-induced proliferation in UV-irradiated human conjunctival epithelial cells, focusing on changes in apoptosis, ROS generation, JNK phosphorylation, IL-11 levels, AP-1 production, and expression of c-myc, c-fos, and c-jun.
研究成果
UV irradiation induces apoptosis in HCE cells through ROS generation and JNK activation, leading to increased IL-11 production, AP-1 activation, and expression of proliferation markers (c-myc, c-fos, c-jun). This suggests a mechanism of apoptosis-induced compensatory proliferation that helps maintain tissue homeostasis in UV-exposed conjunctival epithelial cells, with implications for understanding pterygium pathogenesis.
研究不足
The study is limited to in vitro cell culture models, which may not fully replicate in vivo conditions. Specific limitations such as sample size or potential biases are not explicitly stated, but the use of a single cell line and specific UV conditions could restrict generalizability.
1：Experimental Design and Method Selection:
The study used UV irradiation to induce apoptosis in HCE cells, with measurements of various biomarkers to assess compensatory proliferation mechanisms. Methods included apoptosis assays, ROS detection, ELISA for protein levels, and immunocytochemical staining.
2：Sample Selection and Data Sources:
HCE cells (Clone-1-5c-4) were purchased and cultured. Cells were irradiated with UV at specific doses (30, 99, or 296 mJ/cm2) and analyzed at various time points.
3：List of Experimental Equipment and Materials:
UV lamp (TFX-20MC, Vilber Lourmat), Muse Annexin V and Dead Cell Assay Kit (EMD Millipore), CM-H2DCFDA (Invitrogen), ELISA kits (e.g., Cell-Based JNK Phosphorylation ELISA kit from Ray Bio, IL-11 Human ELISA Kit from Abcam), immunocytochemical staining reagents (e.g., antibodies from Santa Cruz Biotechnology, Oncogene Research Products, Cell Signaling Technology).
4：Experimental Procedures and Operational Workflow:
Cells were cultured, irradiated with UV, and then processed for apoptosis measurement, ROS generation, JNK phosphorylation, IL-11 levels, AP-1 activity, and expression of c-myc, c-fos, c-jun using specified kits and methods. Statistical analysis was performed using Student's t-test or ANOVA.
5：Data Analysis Methods:
Data were analyzed using statistical tests (Student's t-test for two groups, ANOVA for repeated measurements) with significance at P < 0.05, presented as mean ± standard error.
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UV lamp
TFX-20MC
Vilber Lourmat
Used for UV irradiation of HCE cells to induce apoptosis.
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Muse Annexin V and Dead Cell Assay Kit
EMD Millipore
Used to measure apoptosis in HCE cells by detecting Annexin V binding and dead cells.
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CM-H2DCFDA
5-(and 6-) chloromethyl-2?7?-dichlorodihydrofluorescein diacetate, acetyl ester
Invitrogen
Used to measure ROS generation in HCE cells by fluorescence detection.
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Cell-Based JNK (Thr183/Tyr185) Phosphorylation ELISA kit
Ray Bio (Ray Biotech)
Used to measure JNK phosphorylation in cell lysates using ELISA.
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IL-11 Human ELISA Kit
Abcam
Used to measure IL-11 levels in culture supernatant using sandwich ELISA.
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Transcription Factor ELISA Kit
Panomics (Affymetrix)
Used to measure AP-1 activity in nuclear extracts using ELISA.
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Nuclear/Cytosol Fraction Kit
BioVision
Used to extract cytoplasm and nucleus from cells for fractionation.
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SP600125
Sigma-Aldrich
Used as a JNK inhibitor in pretreatment of cells.
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Recombinant human IL-11
PeproTech
Used to treat cells to study the effect of IL-11 on gene expression.
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c-myc antibody
c-8 mouse monoclonal IgG2a (sc-41)
Santa Cruz Biotechnology
Used as primary antibody in immunocytochemical staining for c-myc expression.
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c-fos antibody
Ab-2 rabbit polyclonal IgG (PC05-100UG)
Oncogene Research Products
Used as primary antibody in immunocytochemical staining for c-fos expression.
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Phospho-c-jun antibody
Ser63 rabbit monoclonal IgG (54B3)
Cell Signaling Technology
Used as primary antibody in immunocytochemical staining for c-jun expression.
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Twinkle LB 970 fluorometer
Berthold Technologies
Used to measure fluorescence intensity for ROS detection.
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Microplate reader
Used to measure absorbance at 450 nm in ELISA assays.
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EnVision staining system
Used for immunocytochemical staining with peroxidase-conjugated dextran polymer reagent.
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Chamber Slides
Chamber SlidesTM
Used for culturing cells for immunocytochemical staining.
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Medium 199
Sigma-Aldrich
Culture medium for HCE cells, containing glutamine, fetal bovine serum, and penicillin-streptomycin.
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HCE cells
Clone-1-5c-4 (Wong-Kilbourne derivative of Chang conjunctiva clone)
DS Pharma Biomedical
Human conjunctival epithelial cell line used as the experimental model.
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