Expression of Cyclin D1 in wound healing of human corneal epithelial cells with high blood glucose level / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To study the effect of high glucose environment on human corneal epithelial cell injury and repair, and to explain the significance of Cyclin D1 protein expression in corneal epithelial cell wound healing in high glucose culture. <p>METHODS: The high-glucose micro-environment of diabetic corneal lesions was simulated. After human corneal epithelial cells were resuscitated, cultured and passaged, a normal control group of DMEM complete medium of equal volume of distilled water and a high-glucose treated group of DMEM complete medium containing 25mmol/L glucose were set respectively. After the cells were overgrown, the cells were stimulated with scratches. The growth conditions and changes of the cells in each group were observed and compared under an inverted phase contrast microscope. Western glucose was used to analyze high glucose at different time points(0, 12, 24, 48, and 72h)Cyclin D1 Protein expression in cultured corneal epithelial cells. The qRT-PCR was used to analyze high glucose at different time points and each group Cyclin D1 mRNA expression.<p>RESULTS: Under the conditions of high glucose treatment <i>in vitro</i>, the repair rate of human corneal epithelial cells was slowed down after injury, floating cells increased, cells reattached less, and cell spacing increased. With the increase of high glucose treatment time, the cell state became worse and the growth rate slow; normal group repaired cell damage faster, increased cell density, regular morphology, and smooth cell membrane. Cyclin D1 expression was up-regulated by Western blot, but the up-regulation effect gradually weakened with time. The highest expression of Cyclin D1 in both groups appeared at 12h. The expression of Cyclin D1 in the high glucose treatment group was lower than that in the normal control group. The qRT-PCR results showed that after high glucose treatment, the expression of Cyclin D1 mRNA was up-regulated, but with the increase of high glucose treatment time, the up-regulation effect weakened, and the mRNA level recovered to the same level as the control group at 48h. <p>CONCLUSION: In the process of corneal epithelial cell wound healing, high glucose negatively regulates and inhibits the expression of Cyclin D1 protein, and is related to the decline of corneal epithelial cell proliferation and apoptosis.

Effect of Diquafosol on Hyperosmotic Stress-induced Tumor Necrosis Factor-α and Interleukin-6 Expression in Human Corneal Epithelial Cells

Study on effect and mechanism of Zhenzhu Mingmu Eye Drops protected HCEC against hypermeability-induced injury / 中草药

Objective: To investigate the effects and mechanism of Zhenzhu Mingmu Eye Drops (ZMED) on human corneal epithelial cells (HCEC) injury model induced by high permeability. Methods: The dye eyes disease model was established using 110 mmol/L NaCl to stimulate HCEC, and different concentration of ZMED was given to the treatment group. MTT assay and lactate dehydrogenase (LDH) leakage were used to evaluate the protection effects of ZMED on HCEC. The effects of ZMED on apoptosis of HCEC were analyzed by flow cytometry. AO/EB double staining and Hoechst 33258 analysis were used as sensitive assays for apoptosis. Moreover, HCEC treated with ZMED were subjected to Western blotting for protein levels of Caspase-9, Caspase-3 and PARP. Results: ZMED significantly increased cell viability of HCEC and decreased the LDH release in a dose-dependent manner. ZMED dramatically decreased the apoptosis rate. Furthermore, ZMED down-regulated the levels of cleaved Caspase-9, cleaved Caspase-3 and cleaved PARP. Conclusion: ZMED could protect HCEC against hypermeability, which may be related to the inhibition of Caspase-9/Casepase-3 signaling pathways.

Research advances in cultured human corneal epithelium cells in vitro / 眼科新进展

The corneal epithelial cells are at the surface of the cornea.Human corneal epithelial cells cultured in vitro contribute to fully understand the biological properties of corneal cells,construct disease models in vitro,and play its clinical significance better.This article reviews the recent advances in human corneal epithelial cells cultured in vitro about extractive fraction,extraction methods,factors of progress,impact identification methods.

Expression of Human beta-defensin 2 mRNA by Lipopolysaccharide in Human Corneal Epithelial Cells

Recently the transcriptional up-regulation of human beta-defensin 2 (HBD-2) by lipopolysaccharide (LPS) was found to be associated with NF-kappaB binding site. Although the general mechanisms of NF-kappaB activation by LPS stimulation are well understood, less is known about the signal transduction pathway leading to LPS-induced NF-kappaB activation in human corneal epithelial (HCE) cells. The aim of this study was to investigate the intracellular signals involved in LPS-induced HBD-2 mRNA expression in HCE cells. Pretreatments of inhibitors for NF-kappaB, protein tyrosine kinase, p38 mitogen activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) attenuated the LPS-induced NF-kappaB DNA binding activity and HBD-2 mRNA expression. Furthermore, pretreatments with inhibitors for protein kinase C (PKC), phosphatidylcholine-phospholipase C, phosphatidylinositol-phospholipase C, or phosphatidate phosphohydrolase prevented LPS-induced HBD-2 mRNA expression and HBD-2 prmoter-driven luciferase activity. However, the increased expression of HBD-2 mRNA and the increased DNA binding activity of NF-kappaB induced by LPS were not changed by the blockage of extracellular signal-regulated kinase (ERK) and of addition of antioxidants. Forskolin, a protein kinase A (PKA) agonist did not induce HBD-2 mRNA expression. These data demonstrate that LPS-induced HBD-2 mRNA expression via NF-kappaB is, at least in part, dependent on PKC, p38 MAPK, JNK, and protein tyrosine kinase status, but appears to be independent on PKA, ERK and ROS in HCE cells. Taken together, there may be more than one signaling pathways that lead to LPS-induced up-regulation of HBD-2 mRNA expression in HCE cells.

The Translocation of Nuclaer Factor Kappa B on Corneal Epithelial Cells after Ultraviolet B Irrradiation

PURPOSE: To seek the role of nuclear factor kappa B (NF-kB) on the corneal epithelial cell death after ultraviolet (UV) irradiation. METHODS: Human corneal epithelial cells transfected by Simian Virus 40 were used in this study. UVB(312 nm) located at 10cm distance from bottom (0.6 mW/cm2 ) was irradiated for 10, 20, 30, and 40 seconds. To measure the cytotoxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was used. Translocation of NF-KB was examined by immunocytochemistry with anti NF-K B p65 antibody and Electrophoretic Mobility Shift Assay (EMSA). To confirm the role of NF-KB , sulfasalazine, a specific inhibitor of NF-KB (0.5 mmole), was pretreated for 30 minutes before irradiatrion, and cytotoxicity and translocation of NF-KB was evaluated. RESULTS: UV irradiation resulted in a significant decrease in viability of cultured human corneal epithelial cells, especially after 20 second duration. When HCECs were irradiated with UVB, the translocation of N F -KB was observed in immunocytochemistry. These translocation was peaked 2 hours after UV irradiation in EMSA. In HCECs pretreated with sulfasalazine, either the cellular death or the translocation of NF-KB was blocked. CONCLUSION: UV irradiation can translocate NF-KB on the cultured human corneal epithelial cells. The cellular death after UV irradiation was blocked by sulfasalazine, a potent inhibitor of translocation of NF-KB. These findings suggest that NF-KB plays an important role in cellular death after UV irradiation.