Potential of human induced pluripotent stem cells differentiating into corneal epithelial cells in simulated limbal stem cell microenvironment / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the potential of human induced pluripotent stem cells(hiPSCs)differentiating into corneal epithelial cells in the simulated limbal stem cells(LSCs)microenvironment.METHODS: The hiPSC cell lines were established in vitro, and hiPSCs were co-cultured with corneal stromal cells in transwell system, which simulated the LSC microenvironment. Bone morphogenetic protein 4(BMP4)and a specific transforming growth factor β inhibitor(SB431542)were added to improve the differentiation efficacy. The expression of corneal epithelial cell-specific markers CK3 and CK12, corneal epithelial cell precursor CK15, and the limbal stem cell markers ABCG5 were determined by immunofluorescence staining and flow cytometry.RESULTS: The hiPSCs were actively proliferated in vitro, and immunofluorescence staining showed positive stem cell-specific markers OCT4, SOX2, TRA-1-60 and NANOG. Furthermore, hiPSCs co-cultured with corneal stromal cells exhibited LSCs markers ABCG5 and corneal epithelial cell precursor markers CK15 were positive; however, corneal epithelial cell markers CK3 and CK12 were negative. With the addition of BMP4 and SB431542, hiPSCs showed positive expression of CK3, and the CK3 expression increased over the time.CONCLUSION: With the addition of SB431542 and BMP4, hiPSCs cultured in simulated LSCs microenvironment could differentiate into corneal epithelial cells.

Toxicity evaluation of zinc oxide nanoparticles in vitro and in vivo on cornea / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To observe the toxic effects of zinc oxide nanoparticles(ZnO NPs)on cornea by constructing intoxicated model in vivo and in vitro.METHODS:Human corneal epithelial cells(HCEpiC)were cultured in vitro and exposed to different concentrations(0.5, 5, 12.5, 25, 50, 100, 250 μg/mL)of ZnO NPs for 24h. The cell culture medium without nano-solution was used as the blank control group. The viability of the cells was assessed by MTT assay. Three different concentrations(25, 50 and 100 μg/mL)of ZnONPs dispersions were exposed to the conjunctival sac of anesthetized mice three times a day for 7d consecutively. The phosphate buffered saline(PBS)eye group was the PBS control group. Corneal morphology was observed on 1, 3, 5 and 7d, and the eyes were removed on 8d for various laboratory examinations, including corneal pathological changes and expression levels of inflammatory factors(TNF-α, IL-6).RESULTS:After treatment of HCEpiC cells with different concentrations of ZnO NPs for 24h, the MTT results showed that Zno NPs cause damage to cells at 0.5 μg/mL, and the cell survival rate was about 80%(P<0.05). Half of the cells were killed at a dose of 5 μg/mL, the damaging effect on cells in the concentration range of 5～250 μg/mL was concentration-dependent(P<0.0001). After 7d of conjunctival capsule spotting in mice, dot-like staining of fluorescein was seen in the 25 μg/mL ZnO NPs and 50 μg/mL ZnO NPs groups. Localized circular fluorescein stained areas were seen in the corneas of the 100 μg/mL ZnO NPs group. HE staining showed that the corneal epithelial layer, stromal layer thickness and stromal layer immune cell number did not change significantly in the 25 μg/mL and 50 μg/mL ZnO NPs groups(all P>0.05), while the corneal epithelial layer thinned, the corneal stromal layer thickened and the stromal layer immune cells increased significantly in the 100 μg/mL ZnO NPs group(all P<0.05). Immunohistochemical staining showed that the number of corneal stromal immune cells producing TNF-α and IL-6 and the mean integral optical density(IOD)values of TNF-α and IL-6 were significantly higher in the 100 μg/mL ZnO NPs group than in the PBS control group(P<0.05), and the degree of inflammation response was concentration-dependent. Compared with the PBS control group, no significant increase in immune cell count and IOD values in the 25 μg/mL ZnO NPs and 50 μg/mL ZnO NPs groups(P>0.05).CONCLUSION:The toxic damaging effect of ZnO NPs on the cornea was confirmed from both in vitro and in vivo, which provided a theoretical basis for the ocular safety evaluation of ZnO NPs.

Study on the protective mechanism of Qishen recipe on corneal epithelial cells induced by hypertonic based on JNK1/ AQP5 pathway / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the protective effect of Qishen recipe on corneal epithelial cells induced by hypertonic fluid, and elucidated its mechanism of action in the treatment of dry eye base on JNK1 / AQP5 pathway.<p>METHODS: Human corneal epithelial cells(HCECs)model was created by osmotic pressure at a concentration of 500mOsm/L for 24h. Serum of rats containing drugs in the blank group, model group, Western medicine group, and Qishen recipe low-dose, medium-dose and high-dose groups were treated on the modeled DE HCECs, and the effects of different drug stimulation on the survival rate of HCECs were tested by CCK-8 method. The expressions of inflammatory factors TNF-α, IL-6 in extracellular fluid were explored by ELISA. The expression of apoptosis factors caspase 1 and AQP5 were detected by immunocytochemistry(ICC). The expressions of AQP5, JNK1, p-JNK1 of HCECs after intervention with different drug concentrations were found by Western blotting.<p>RESULTS: Compared with the blank group, the survival rate of HCECs in each group was significantly reduced(<i>P</i><0.01). The extracellular fluid inflammatory factors TNF-α, IL-6 and caspase-1, p-JNK1, AQP5 protein expression levels increased significantly in each group(all <i>P</i><0.01); In comparison to the model group, the survival rate of HCECs in each medication group increased significantly(all <i>P</i><0.01). The expression levels of TNF-α, IL-6 in the extracellular fluid of each drug group, AQP 5 and p-JNK1 protein expression in HCECs, and the expression of caspase-1 and AQP5 protein in the western medicine group and the Qishen recipe high and medium dose group were all reduced(all <i>P</i><0.05). Compared with the western medicine group, the survival rate of HCECs in the Qishen prescription high-dose group was significantly increased(<i>P</i><0.01). The expression levels of TNF-α and IL-6 in each dose group of Qishen recipe were reduced(all <i>P</i><0.05), while the expression levels of caspase-1 in the high-dose Qishen recipe group and the AQP5 protein expression levels of the high and medium-dose Qishen recipe group saw a decrease(all <i>P</i><0.05). However, there was no statistically significant difference in the JNK1 protein expression of HCECs of all the groups detected by Western blotting method(<i>P</i>>0.05). <p>CONCLUSION: Qishen recipe can not only reduce the JNK1 phosphorylation and AQP5 protein expression of HCECs induced by hypertonicity, but also reduce the expression of inflammatory factors TNF-α, IL-6 and the apoptotic factor caspase-1 of HCECs in the extracellular fluid, thus effectively Inhibit inflammation and apoptosis.

Protective effects of Slit2 on corneal epithelium and nerves in diabetic mice and its mechanism / 中华实验眼科杂志

Objective:To explore the promoting effects of slit guidance ligand 2 (Slit2) on the repair of corneal epithelium and nerve damage in diabetic mice and possible molecular mechanism.Methods:Sixty SPF C57BL/6 mice aged 5-6 weeks were divided into normal control group, diabetes model group and Slit2 injection group according to the random number table method, 20 for each group.Diabectic model was prepared by intraperitoneal injection of streptozotocin in the diabetes model group and Slit2 injection group.A mouse corneal epithelial injury repair model was established using electric epithelial scraper, and Slit2 recombinant protein was subconjunctivally injected immediately following modeling in the Slit2 injection group.The equal volume of phosphate buffer saline (PBS) was used in a same way in the diabetes model group.No intervention was performed in the normal control group.Corneal epithelial healing were examined at 24, 48 and 72 hours after corneal epithelial defect by corneal fluorescin staining.Real-time fluorescent quantitative PCR was used to detect the expression of Slit2 and its related receptors in the corneal epithelium of normal and diabetic model mice.Fluorescence staining of corneal wholemount with β-tubulin Ⅲ was used to observe the changes in corneal nerve morphology.Immunofluorescence staining was performed to detect the expression and distribution of Slit2 in mouse corneal epithelium in normal control group and diabetes model group, as well as the expression and distribution of Slit2, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), threonine protein kinase (AKT), β-catenin and Ki67 in the healing corneal epithelium of mice after corneal epithelium damage in different groups.The mouse corneal epithelial stem/progenitor cell line (TKE2) was divided into normal control group, high-glucose group and Slit2 treatment group.Western blot was performed to detect the expression of p-EGFR/EGFR and p-AKT/AKT in the TKE2 of the three groups.The expression of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 with 0.01, 0.1 and 0.5 μg/ml Slit2 treatment for 10 minutes, and before and 10, 20, 30, 60, 120 minutes after 0.5 μg/ml Slit2 treatment was detected by Western blot.The effects of Slit2 on the axon regeneration of mouse trigeminal ganglion cells (TGs) were observed by immunofluorescence staining.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Qingdao Eye Hospital of Shandong First Medical University (No.[2020]57).Results:At 48 and 72 hours after corneal epithelial scraping, the speed of corneal epithelial repair was significantly slowed down in diabetes model group in comparison with the normal control group and Slit2 injection group.The relative expression levels of Slit2 and its receptors Robo1, Robo2 and Robo4 mRNA in the normal corneal epithelium in the diabetes model group were significantly higher than those of the normal control group (all at P<0.05). The fluorescence intensity of Slit2 in normal corneal epithelium in diabetes model group was similar to the normal control group, and the fluorescence intensity of Slit2 in damaged corneal epithelium in diabetic mice was significantly weaker than that in normal control group.Corneal nerve plexus was denser at 7 days after corneal epithelial injury and the nerve fibers were increased with more branches in Slit2 injection group compared with diabetic group.The fluorescence intensity of p-EGFR, p-ERK, β-catenin and Ki67 in damaged corneal epithelium in normal control group and Slit2 injection group was stronger than that of the diabetes model group.The relative expression levels of p-EGFR/EGFR, p-AKT/AKT, and β-catenin in TKE2 in high-glucose group were significantly lower than those in normal control group and Slit2 treatment group (all at P<0.05). The relative expression levels of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 after Slit2 treatment were significantly increased in comparison with before Slit2 treatment (both at P<0.05), and the relative expression levels of p-EGFR/EGFR and p-AKT/AKT in TKE2 were elevated as the increase of Slit2 concentration.The activation effect of 0.5 μg/ml Slit2 on EGFR and AKT pathways was most obvious.The synapse length of TGs cultured by high glucose was (40.52±5.44) μm, which was significantly shortened than (72.14±9.48) μm in normal control group and (73.04±4.66) μm in Slit2 injection group (both at P<0.05). Conclusions:Slit2 can protect the corneal epithelium by activating EGFR signaling pathway and play a protective role to neurons by increasing the density of corneal subepithelial plexus and promoting the growth of TGs axons in diabetic mice.

Protective effects of Resveratrol on inflammation and oxidative stress damage in human corneal epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the protective effects of resveratrol(RSV)on inflammation and oxidative stress damage in human corneal epithelial cells(HCECs).<p>METHODS: The inflammation of HCECs was induced by Tumor necrosis factor-α(TNF-α), and the experiment was divided into: control group, TNF-α group and RSV+TNF-α group. The oxidative stress response of HCECs was induced by H₂O₂, and they were divided into normal group, H₂O₂ group and RSV+ H₂O₂ group. MTT assay was used to detect the viability of HCECs; RT-qPCR and enzyme-linked immunosorbent assay(ELISA)methods were used to detect the expression of IL-1, IL-6 and IL-8; Immunofluorescence staining and Western blot were used to observe the nuclear translocation of NF-κB p65. 2',7'-dichlorofluorescein diacetate(DCFH-DA)fluorescent probe was applied to detect the level of reactive oxygen species(ROS).<p>RESULTS:In the inflammatory response of HCECs, RT-qPCR and ELISA showed that the expression levels of IL-1, IL-6 and IL-8 were increased significantly in the TNF-α group compared with the control group, the above indicators were lower after pretreatment of RSV than those in TNF-α group; Immunofluorescence staining and Western blot showed that the nuclear translocation of NF-κB p65 was increased in TNF-α group, while it was inhibited after pretreatment of RSV. In the oxidative stress response of HCECs, the results of MTT and DCFH-DA fluorescent probe staining showed that H₂O₂significantly decreased the viability of HCECs and increased the production of ROS in HCECs. After pretreatment of RSV, cell viability increased significantly, and RSV inhibited the generation of ROS in HCECs induced by H₂O₂. <p>CONCLUSION: RSV has an inhibitory effect on inflammation and oxidative stress damage in human corneal epithelial cells, and it has been confirmed that RSV inhibits inflammation by inhibiting the activation of the NF-κB pathway.

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

Oxidative damage effect of povidone-iodine on corneal epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the oxidative damage effect of povidone iodine on corneal epithelial cells. <p>METHODS:To study the oxidative damage effect of different concentrations of povidone iodine, the cultured epithelial cells were randomly divided into control group, low concentration group, medium concentration group and high concentration group. To study the oxidative damage effect of disinfection time of povidone iodine, the cultured corneal epithelial cells were randomly divided into control group, short time group, medium time group and long time group. Malondialdehyde(MDA)and superoxide dismutase(SOD)were detected by ELISA, cell viability was detected by CCK-8 method and inverted microscope, and apoptotic rate was detected by flow cytometry. <p>RESULTS: The higher concentration of povidone iodine was associated with the higher MDA content, the lower SOD content, the lower cell activities and the higher apoptotic rate of the corneal epithelial cells, which was in a dose-independent manner. The differences among four groups were statistically significant(all <i>P</i><0.01). The longer disinfection time of povidone iodine was related with the higher MDA content, the lower SOD content, the lower cell activities and the higher apoptotic rate of corneal epithelial cells, which was in a time-independent manner. The differences among four groups were statistically significant(all <i>P</i><0.01). <p>CONCLUSION: The oxidative damage of povidone iodine on corneal epithelial cells were in a dose independent and time dependent manner.

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.

Clinical changes of contralateral unaffected eyes in patients with unilateral infectious keratitis under in vivo confocal microscopy / 中华实验眼科杂志

Objective To evaluate the clinical changes of corneal epithelial cells,dendritic cells,endothelial cells and corneal nerves in contralateral eyes of patients with unilateral infectious keratitis.Methods A prospective serial case observation study was conducted in patients with unilateral infectious keratitis from January to August 2018 in the First Affiliated Hospital of Harbin Medical University.The corneal epithelial cells density,dendritic cells density,endothelial cells density,total nerve density,total number of nerves and branch nerve density were analyzed with in vivo confocal microscopy (IVCM),slit lamp microscopy was performed on all subjects to observe the conjunctiva,cornea and anterior chamber.Corneal branch nerve density and total nerve density were compared with the control group by homogeneity test of variance.This study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (No.IRB-AF/SC-04/01.0).Results Slit lamp microscopy showed no significant changes in anterior segment of the contralateral uninfected eyes in the 3 groups.The corneal epithelial cells density of uninfected eyes in viral keratitis group,bacterial keratitis group and fungal keratitis group was 1 834 (1 584,2 107),1 905 (1 651,2 332) and 1 859 (1 477,1 995)/mm2,respectively,which were significantly lower than 3 479 (3 080,3 910)/mm2 in the control group,the dendritic cells densities in viral keratitis group and bacterial keratitis group were 175 (139,214)/mm2 and 156 (118,190)/mm2,which were higher than 69(57,76)/mm2 in the control group,the differences were statistically significant (all at P＜0.05).The corneal endothelial cells density of uninfected eyes in viral keratitis group was 1 107(945,1 270)/mm2,which was less than 1 905(1 651,2 332)/mm2 in the bacterial keratitis group and 1 859(1 477,1 995)/mm2 in the fungal keratitis group (both at P＜0.05).The corneal nerve number and total nerve density of uninfected eyes in viral keratitis group were l0(7,11)/mm2 and (1 822.85±622.34) μm/mm2,which were lower than 11 (9,13)/mm2 and (2 340.91±:408.70)μm/mm2 in the bacterial keratitis group,with significant differences between them (P＜ 0.05,P＜ 0.008 3).The morphology of corneal epithelial cells and endothelial cells in each infectious keratitis group was larger than that in the control group,while the morphology and number of dendritic cells in the contralateral eye of patients with viral and bacterial keratitis increased.Conclusions In unilateral uninfected eyes of infectious keratitis,the density of corneal epithelial cells,dendritic cells,endothelial cells and corneal nerves changes correspondingly.There may be a close relationship of corneal immunity and nervous system between the two eyes.

Clinical effect of anterior corneal stroma acupuncture on corneal epithelial cell dysfunction / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the clinical efficacy of anterior stromal puncture(ASP)for the corneal epithelial cells dysfunction(CED).<p>METHODS: Sixteen patients with CED underwent ASP in Wuhan Union Hospital from September 2015 to December 2015 were included. Uncorrected visual acuity, ocular surface disease index(OSDI), corneal fluorescence staining, corneal epithelial thickness, full corneal thickness, corneal subepithelial dendritic cell density, corneal endothelial cell density and corneal epithelial nerve density were observed and recorded at preoperative, 1mo and 3mo postoperatively, respectively.<p>RESULTS: Totally 16 patients compared with preoperatively, there was a significant increase in uncorrected visual acuity and corneal epithelial nerve density(<i>P</i><0.05)or a significant decrease in OSDI, corneal fluorescence staining, corneal epithelial thickness, full corneal thickness and corneal subepithelial dendritic cell density(<i>P</i><0.05)at 1mo postoperatively; while there was no significant difference in corneal endothelial cell density(<i>P</i>>0.05). And compared with 1mo postoperatively, there was a significant decrease in corneal subepithelial dendritic cell density(<i>P</i><0.05)and a significant increase in corneal epithelial nerve density(<i>P</i><0.05)at 3mo postoperatively, while there was no significant difference in uncorrected visual acuity, OSDI, corneal fluorescence staining, corneal epithelial thickness, full corneal thickness and corneal endothelial cell density(<i>P</i>>0.05).<p>CONCLUSION: ASP was effective for CED. Corneal confocal microscopy was essential for the evaluation of ASP efficacy, which can guide the clinical work better and establish the termination of intervention.

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.

Damage Effect of Borneol on Rabbit Corneal Epithelial Cells / 广州中医药大学学报

Objective To observe the damage effect of borneol on rabbit corneal epithelial cells. Methods After the treatment with borneol at 100, 200, 400 μg·mL-1 respectively, the viability of rabbit corneal epithelial cells was determined by methyl thiazolyl tetrazolium ( MTT) assay, cell apoptosis was determined by flow cytometry with Annexin V- fluorescein isothiocyanate/propidium iodide ( FITC/PI) staining, and Caspase-3 mRNA expression was detected with real-time reverse transcription polymerase chain reaction ( RT-PCR) . Results Borneol at the concentrations of 100, 200, 400 μg·mL-1 inhibited the activity of rabbit corneal epithelial cells. Compared with the normal control group, borneol increased the rate of apoptosis, and enhanced the Caspase-3 mRNA expression in rabbit corneal epithelial cells ( P<0.05 or P<0.01) . Conclusion Borneol can inhibit the proliferation of rabbit corneal epithelial cells, induce cell apoptosis through enhancing the expression of apoptosis-related gene Caspase-3 mRNA.

The Toxicity of Nonsteroidal Anti-inflammatory Eye Drops against Human Corneal Epithelial Cells in Vitro

This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na+ concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K+ and Cl- concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.

Expression and characterization of recombinant flagellin FlgE of Pseudomonas aeruginosa / 中华微生物学和免疫学杂志

Objective To construct the prokaryotic expression and purification protocols for Pseudomonas aeruginosa type E flagellin (FlgE) and to study its bioactivity.Methods With analysis of Pseudomonas aeruginosa flagellin FlgE sequences,the whole-length FlgE gene was amplified from Pseudomonas aeruginosa genomic DNA by using PCR and primers with proper restriction enzyme sites.The amplified FlgE fragment and prokaryotic expression plasmid pET24a were digested with Nde Ⅰ and Hind ⅢⅣ respectively.The target fragment and vector were recovered and ligated to obtain the recombinant plasmid pET24a-FlgE.DNA sequencing of positive clone confirmed that the target gene and the junctions with vectors were all correct.The plasmid pET24a-FlgE was transformed into BL21 bacteria.The culture conditions like temperature,rotation speed,inducer concentrations,time length were optimized to achieve maximal expression of the target recombinant FlgE with 6×His tag at C terminal.FlgE-His proteins were purified using His-Trap affinity chromatography columns and identified by SDS-PAGE.The purified proteins were further subjected to endotoxin elimination with proper kits.The purified recombinant FlgE was added to cultured corneal epithelial cells for 4 h and the expression of several inflammation-related molecules was examined by using real-time quantitative PCR.Results The recombinant plasmid pET24a-FlgE was successfully constructed and high level FlgE expression was achieved in BL21 with rotation at 16℃ and 1 mmol/L isopropyl-β-D-glucopyranosyl-galactosidase induction for 20 h.Purified recombinant FlgE-His was obtained and used for primary bioactivity assay.After treatment of corneal epithelial cells with 20 μg/ml FlgE for 4 h,the expression of inflammatory cytokines IL-6,IL-8 were significantly increased.Inactivation of the FlgE with ethanol abolished its stimulatory activity.Conclusion The prokaryotic expression and purification system for recombinant Pseudomonas aeruginosa flagellin FlgE was set up,and the recombinant FlgE stimulated the expression of inflammatory factors in corneal epithelial cells.

Inhibition of β1-integrin on apoptosis of rabbit corneal epithelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

· AIM: To investigate the effect of β1-integrin overexpression on the apoptosis of rabbit corneal epithelial cells and the related mechanism. · METHODS: The plasmid expressing β1-integrin-GFP fusion protein was constructed by polymerase chain reaction (PCR), and this plasmid (β1 group) or the empty vector (mock group) was transfected into rabbit corneal epithelial cells, respectively. The expression of β1-integrin-GFP fusion gene was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The adhesion of transfected cells to extracellular matrix (ECM) proteins was determined by adhesion assay. The apoptosis of rabbit corneal epithelial cells was assayed by Hoechst 33342 staining and DNA ladder. The phosphorylation of mitogen-activated protein (MAP) kinase was examined by Western blot. · RESULTS: Rabbit corneal epithelial cells overexpressing β1 -integrin-GFP fusion gene were successfully established. Compared with mock group, β1-integrin transfection significantly promoted the adhesive of rabbit corneal epithelial cells to ECM proteins such as laminin, fibronectin, collagen Ⅰ and collagen Ⅳ. Β1-integrin overexpression inhibited apoptosis and induced MAP kinase phosphorylation in rabbit corneal epithelial cells (P<0.05).· CONCLUSION: These data suggest that overexpression of β1-integrin confers resistance to apoptosis in rabbit corneal epithelial cells, and MAP kinase pathway may play an important role in this process.

β1-integrin overexpression induces adhesion and migration of rabbit corneal epithelial cells in vitro / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM:To investigate the effect of β1-integrin overexpression on the adhesion and migration of rabbit corneal epithelial (RCE) cells.·METHODS:Eukaryotic expression vector encoding β1-integrin-GFP fusion DNA was transfected into RCE cells,and the β1-integrin-GFP fusion gene was examined by RT-PCR and Western blot.The adhesion to Matrigel and the migration of the transfected cells were determined by adhesion and mobility assays.The phosphorylation of focal adhesion kinase (FAK) was examined by Western blot.·RESULTS:The overexpression of β1-integrin-GFP fusion gene by RCE cells was successfully established.β1-integrin transfection significantly promoted the adhesion of RCE cells to Matrigel ( P < 0.05 ).β1-integrin overexpression also promoted the migration ability of RCE cells and induced FAK phosphorylation in them (P < 0.05).·CONCLUSION:These data suggest that overexpression of β1-integrin promotes the adhesion and migration of RCE cells and that the FAK pathway may play an important role in this process.

Transplantation of in vivo Cultivated Limbal Corneal Epithelial Cells with Total Limbal Stem Cell Deficiency

PURPOSE: To treat limbal stem cell deficiency, we investigated the epithelial characteristics in rabbits with total limbal stem cell deficiency (LSCD) after reconstruction with autologous limbal corneal epithelial cells in vivo expanded on amniotic membrane. METHODS: The right eyes of 10 rabbits were rendered total LSCD, as verified by impression cytology. In the left eyes of 10 rabbits, in vivo limbal corneal epithelial cells culture was achieved. In group A (n=4), we evaluated the characteristics of the cultured limbal corneal epithelial cells and in group B (n=6) we evaluated the characteristics of the transplanted limbal corneal epithelial cells at postoperative 1, 2, and 4 weeks. RESULTS: Successful epithelial growth was observed on amniotic membrane in all eyes of group A. Transplanted epithelial cells were well remained in five eyes of group B. The epithelial cells of group A and B were confirmed with corneal specificity by immunohistochemical staining (AK-2, AE-5). CONCLUSIONS: The corneal surface with unilateral total LSCD can be successfully reconstructed by transplantation with autologous limbal corneal epithelial cells in vivo expanded on amniotic membrane.

The Study of the Effects of Glucose on Cultured Human Corneal Epithelial Cells

PURPOSE: In this study, we evaluated the effects of glucose on the proliferation, migration, morphological characteristics, and proteinase expressions in human corneal epithelial cells to discover the cause of diabetic corneal epithelial complications. METHODS: Human corneal epithelial cells transfected by SV40 were cultured in media containing 17.5 mM (control), 25 mM, and 100 mM D-glucose to stimulate a diabetic condition. In some experiments, 25 mM or 100mM D-mannitol was added to the media to control for the osmotic effect of glucose. We performed MTT analysis of the proliferation assay, cell count of the migration assay, scanning electron microscopic (SEM) examination for morphologic change, and RT-PCR for the expression of mRNA of metalloproteinase (MMP). RESULTS: Raising the concentration of glucose from 25 mM to 100 mM significantly decreased in the cellular proliferation and migration. These findings were not observed in the presence of 25 mM or 100 mM mannitol. In SEM, the pseudopods of the cells were markedly decreased in the 100mM glucose media. Although the expression of MMP-2 was not significantly different, that of MMP-9 was significantly increased with increasing glucose concentration. CONCLUSIONS: Higher levels of glucose show a significant effect on cellular proliferation, migration, morphology, and the expression of MMP-9 in corneal epithelial cells. This study may be an initial step in understanding the mechanism of corneal epithelial wound healing in diabetic patients.

Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells / 华中科技大学学报（医学）（英德文版）

In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner.50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did.Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.