OBJECTIVETesting the protein-removing effect of the protein-removing care solution on the protein precipitation of the soft contact lenses.

METHODSoak the lenses into the artificial-tears to simulate the protein absorption, test the absorbency of cleaned protein at the wavelength of 280 nm by UV spectrophotometer, and compute the percentage of protein.

RESULTTesting results of the percentage of the cleaned protein are repeatable.

CONCLUSIONThis experimental method can be used to evaluate the cleaning effect of the protein- removing care solution, but still needs much improvements.

Contact Lens Solutions ; Contact Lenses, Hydrophilic ; Eye Proteins ; analysis ; Humans ; Spectrophotometry, Ultraviolet ; Surface-Active Agents ; pharmacology

BACKGROUNDPigment epithelium-derived factor (PEDF) is expressed in several normal organs and identified as an inhibitor of neovascularization. In the present study, we investigated the effect of PEDF in an in vitro model of ocular choroidal neovascularization.

METHODSMicrodissection was used to isolate the human choroidal endothelial cells (CECs), followed by the use of superparamagnetic beads (Dynabeads) coated with the CD31 antibody, which selectively binds to the endothelial cell surface. The mitogenic and motogenic effects of vascular endothelial growth factor (VEGF) on cultured choroidal capillary endothelial cells were examined in the presence or absence of PEDF (1, 10, 100, and 1000 ng/ml) using cell counts and migration assays.

RESULTSCells bound to the beads were isolated using a magnetic particle concentrator and they were successfully cultured and characterized to be endothelial cells that possessed greater than 95% immunoreactivity to von Willebrand factor. PEDF suppressed the proliferation and migration of VEGF-induced choroidal capillary endothelial cells. However, the concentration of PEDF which we used has little effect on normal CECs.

CONCLUSIONSPEDF played an important role on the growth and migration of VEGF-stimulated choroidal endothelial cell. These findings suggest that PEDF may be an effective approach to the treatment of choroidal neovascular disorders.

Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Choroid ; blood supply ; drug effects ; Choroidal Neovascularization ; drug therapy ; Endothelial Cells ; cytology ; drug effects ; Eye Proteins ; pharmacology ; Humans ; Nerve Growth Factors ; pharmacology ; Serpins ; pharmacology ; Vascular Endothelial Growth Factor A ; pharmacology

BACKGROUNDThe common pathological characteristics of corneal injury include inflammatory factors activation, vascular endothelial cells or inflammatory cells infiltration into lesions, corneal edema, corneal neovascularization (CNV), and scar formation. PEDF-34 is the functional fragment of pigment epithelium-derived factor (PEDF) that has anti-angiogenic and anti-inflammatory properties and contains an N-terminal 34-amino acid peptide. This study was to investigate the anti-inflammatory effects of PEDF-34 on H2O2-induced corneal injury in vitro.

METHODSAfter cultured in H2O2 (0.1 mmol/L) for 2 hours, human corneal fibroblasts (HCFs) and human umbilical vein endothelial cells (HUVECs) were treated with PEDF-34-nanoparticles (NPs) at different concentrations (0.1, 0.5, 1.0, 2.0 µg/ml) or 2.0 µg/ml control-NPs for 24 hours. The viable cells were quantified using the MTT assay. Western blotting or ELISA analysis was performed for measuring the human vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) expression of both HCFs and HUVECs. VEGF and nuclear factor κB (NF-κB) mRNA levels of HCFs were semi-quantified by RT-PCR.

RESULTSThe survival rates of HCFs or HUVECs stimulated by H2O2 did not decrease significantly (P > 0.05) compared to those in the normal conditions. As compared to control-NP group, PEDF-34-NPs had dose-dependent inhibitive effect on HUVECs with the MTT assay, but not HCFs. Western blotting analysis showed that the VEGF and ICAM-1 levels in the HCFs and HUVECs stimulated by H2O2 were significantly higher than those in the normal conditions, which were decreased dramatically in those treated with PEDF-34-NPs. RT-PCR analysis revealed that the VEGF mRNA and NF-κB mRNA levels increased in H2O2-stimulated HCFs, while both of them decreased in PEDF-34-NP groups dose dependently.

CONCLUSIONSPEDF-34-NPs may play an important role in regulating the NF-κB pathway, inhibiting inflammatory activity. PEDF-34-NPs may be a potential new drug for treating corneal injury in the future.

Anti-Inflammatory Agents ; chemistry ; pharmacology ; Cells, Cultured ; Corneal Injuries ; chemically induced ; metabolism ; Eye Proteins ; chemistry ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Hydrogen Peroxide ; pharmacology ; Nerve Growth Factors ; chemistry ; Peptides ; chemistry ; pharmacology ; Serpins ; chemistry

OBJECTIVETo express and purify the recombinant human pigment epithelium-derived factor (PEDF) which inhibits the proliferation of the endothelium cells from blood vessel in E.coli.

METHODSPEDF gene was inserted into the prokaryotic expression vector pGEX-4T-2. The recombinant protein PEDF was expressed in E.coli BL-21, and purified by the GST Sepharose 4B affinity column. The recombinant human PEDF protein was identified by Western blot and mass spectrum. The biological activity of the recombinant human PEDF protein was measured by using MTT.

RESULTSThe 46 kDa recombinant human PEDF protein was obtained. It significantly inhibited the proliferation of the human umbilical vein cell line HUVEC.

CONCLUSIONThe recombinant human PEDF with anti-angiogenesis activity protein may be successfully purify through prokaryotic expression.

Angiogenesis Inhibitors ; pharmacology ; Apoptosis ; drug effects ; Base Sequence ; Cell Division ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Vascular ; cytology ; Eye Proteins ; pharmacology ; Humans ; Molecular Sequence Data ; Nerve Growth Factors ; pharmacology ; Prokaryotic Cells ; metabolism ; Recombinant Proteins ; pharmacology ; Serpins ; pharmacology ; Umbilical Veins ; cytology

Animals ; Apoptosis ; drug effects ; genetics ; COS Cells ; Cercopithecus aethiops ; Cytoskeletal Proteins ; genetics ; metabolism ; Eye Proteins ; genetics ; metabolism ; Glaucoma, Open-Angle ; genetics ; metabolism ; Glycoproteins ; genetics ; metabolism ; Humans ; Mutation ; Ubiquinone ; analogs & derivatives ; pharmacology

In this study, we investigated whether retinal soluble proteins, such as interphotoreceptor retinoid-binding protein(IRBP), play a role in the induction of nitric oxide by macrophages in vitro. Cells from the murine macrophage cell line RAW 264.7 and rat and rabbit peritoneal macrophages were incubated in the presence of retinal soluble protein. The nitrite level in the cultured supernatant was evaluated for nitric oxide production using the Griess reaction. IRBP induced significant, dose-dependent nitrite production in both RAW 264.7 and rat peritoneal macrophages. Induction of inducible nitric oxide synthase (iNOS) by retinal proteins was inhibited by the iNOS-specific inhibitor, aminoguanidine, and the tyrosine inhibitor, genistein. These results show that soluble retinal proteins significantly induce nitric acid production by macrophages. Increased production of reactive oxygen species by macrophages in the presence of this soluble retinal protein in vivo may accelerate photoreceptor degeneration in uveitis.
Animal ; Cell Line ; Comparative Study ; Enzyme Inhibitors/pharmacology ; Eye Proteins/pharmacology* ; Guanidines/pharmacology ; Macrophages, Peritoneal/metabolism ; Macrophages, Peritoneal/drug effects* ; Macrophages, Peritoneal/cytology ; Nitric Oxide/biosynthesis* ; Nitric-Oxide Synthase/metabolism ; Nitric-Oxide Synthase/antagonists & inhibitors ; Rabbits ; Rats ; Rats, Inbred Lew ; Retinol-Binding Proteins/pharmacology*

BACKGROUNDRetinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.

METHODSHuman primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence.

RESULTSThree pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P < 0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).

CONCLUSIONT7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

Base Sequence ; Cells, Cultured ; Choroidal Neovascularization ; therapy ; DNA-Directed RNA Polymerases ; metabolism ; Humans ; Molecular Sequence Data ; Pigment Epithelium of Eye ; cytology ; metabolism ; RNA Interference ; RNA, Small Interfering ; biosynthesis ; pharmacology ; Transcription, Genetic ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics ; Viral Proteins ; metabolism

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-inflammatory, antioxidant and antithrombotic properties and plays a protective role against atherosclerosis (AS). The purpose of the present study is to explore the effects of oxidized low density lipoprotein (ox-LDL) on the expression of PEDF in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and incubated with ox-LDL at different concentrations (6.25, 12.5, 25, 50, 100 and 150 mg/L) for 24 h. Apoptosis of endothelial cells were assayed by morphological staining and flow cytometry. The intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Cell viability was assayed by MTT assay. PEDF protein and mRNA expressions in HUVECs were analyzed by Western blot and quantitative real-time PCR, respectively. The results showed that ox-LDL significantly induced apoptosis, reduced cell viability, increased intracellular ROS levels and decreased the PEDF expression in HUVECs in a concentration-dependent manner. Ox-LDL at 50 mg/L obviously decreased the PEDF protein expression compared with control group (P < 0.05), whereas 25 mg/L ox-LDL already markedly reduced the PEDF mRNA expression (P < 0.05). In conclusion, the results suggest that ox-LDL down-regulates the PEDF expression through an increased ox-LDL-induced intracellular production of ROS.
Apoptosis ; Cells, Cultured ; Down-Regulation ; Eye Proteins ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Lipoproteins, LDL ; pharmacology ; Nerve Growth Factors ; metabolism ; Reactive Oxygen Species ; metabolism ; Serpins ; metabolism

BACKGROUND: The anti-tumor effect of pigment epithelium-derived factor (PEDF) has been widely confirmed. However, the anti-tumor effect of its peptides is rarely reported. This study aims to investigate the effects of PEDF and its peptides on the apoptosis and migration of non-small cell lung cancer (NSCLC). METHODS: In this study, A549 cells and H1299 cells were selected as the research object, and the cells were divided into normal group, PEDF treatment group, 34 peptide treatment group, 44 peptide treatment group and 34+44 peptide treatment group by administering different drugs at the same concentration to the cells. The proliferation activity of cells in each group was detected by CCK-8 method; the migration ability of cells was detected by scratch test; the expression levels of apoptosis related proteins such as protein kinase 3 (RIP3) and cleaved-caspase-3 were detected by Western blot; the expression levels of epithelial mesenchymal transition (EMT) markers in each group, such as cadherin (E-cadherin) and α-smooth muscle actin (α-SMA) were detected by Western blot; the apoptosis rate of each group was detected by flow cytometry. RESULTS: The results of CCK-8 showed that PEDF and its peptides could inhibit cell proliferation, and the inhibitory effect of 34+44 peptide was the strongest (P<0.05); Observation under the microscope found that PEDF and its peptides can inhibit the proliferation and mesenchymal transformation of A549 cells and H1299 cells, and the inhibitory effect of the 34+44 peptide group is the most obvious; Western blot indicated that compared with other groups, the expressions of cleaved-caspase-3 and RIP3 in 34+44 peptide group were significantly higher (P<0.05), and the expressions of EMT protein E-cadherin were higher, the expression of α-SMA decreased (P<0.05); The results of flow cytometry showed that the apoptosis rate of 34+44 peptide group was significantly higher than those of other groups (P<0.05); The scratch test showed that compared with all the other groups, the healing rate of 34+44 peptide group was the lowest (P<0.05). CONCLUSIONS 34+44 combination peptide can better promote the apoptosis of NSCLC, inhibit the migration of NSCLC, and thereby inhibit the growth of NSCLC.
Apoptosis ; Cadherins/genetics* ; Carcinoma, Non-Small-Cell Lung/genetics* ; Caspase 3 ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Epithelial-Mesenchymal Transition ; Eye Proteins ; Humans ; Lung Neoplasms/genetics* ; Nerve Growth Factors ; Peptides/pharmacology* ; Serpins ; Sincalide

OBJECTIVETo investigate the functions of promoter hypermethylation of secreted frizzled-related protein (sFRP) genes in colorectal tumorigenesis and progression.

METHODSThree colorectal cancer cell lines, RKO, HCTll6 and SW480, were treated hy 5-aza-2'-deoxycytidine and trichostatin A for demethylation. The promoter hypermethylation and expression of sFRP genes in colorectal tumor tissue and colorectal cancer cell lines were detected hy methylation-specific PCR and reverse transcription PCR, respectively.

RESULTSNone of the normal colorectal mucosa tissues showed methylation of sFRP genes. sFRP1, 2, 4 and 5 were frequently methylated in colorectal adenocarcinoma, adenoma and aberrant crypt foci (ACF) (sFRP1 > 85%, sFRP2 > 75%, sFRP5 > 50%), the differences between any two of them were not significant (P >0.05). Methylation was more frequent in colorectal tumors than in normal mucosa and adjacent normal mucosa from patients with tumor. Hypermethylation of sFRP genes was present in three colorectal cancer cell lines. When sFRP genes were methylated, their corresponding mRNA expression was absent. After cells were treated by DAC/TSA combination, the silenced sFRP expression could be effectively re-expressed.

CONCLUSIONHypermethylation of sFRP genes is a common early event in the evolution of colorectal tumors that occurs frequently in ACF. Methylation of sFRP1, 2 and 5 genes might serve as biomarkers for the early detection of colorectal tumors. Demethylation can effectively reverse gene expression that appears possibly to be an effective way for tumor therapy.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Adenoma ; genetics ; metabolism ; pathology ; Adult ; Aged ; Azacitidine ; analogs & derivatives ; pharmacology ; Biomarkers, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Eye Proteins ; genetics ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; HCT116 Cells ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Hydroxamic Acids ; pharmacology ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; RNA, Messenger ; metabolism