Abstract:New drugs are developed rapidly with novel ideas of action mechanisms for the treatment of glaucoma. The most classic drugs under development are to lower the intraocular pressure (IOP). New agents were invented to lower the IOP through ① induction of metalloproteinases (MMPs), ② contraction of trabecular meshwork cells, ③ inhibition of aqueous humor secretion, and ④ activation of CB-1 receptor, The second class of drugs under development is intended to improve the ocular blood flow (OBF), particularly in retina and optic nerve head (ONH). Drugs that improve the OBF irrespective of the IOP changes could be quite useful for the treatment of normal tension or low-tension glaucoma. Neuroprotection is the latest developed mechanisms of glaucoma treatment. Although the history of neuroprotection research is very short, there are many agents under investigation in this class. They include ① blockade of N-methyl-D-aspartate receptor, ② neurotrophic agents, ③ inhibition of inducible nitric oxide synthases (iNOS), ④ inhibition of apoptosis, ⑤ protective autoimmunity, ⑥ stem cell therapy, and so on. Since all drugs for glaucoma treatment are used to stabilize the disease rather than to cure it, it is critical that an ideal drug with high therapeutic index and low cost price should be invented.

Abstract:To investigate the effect of Z,E-butylidedephthalide (Bdph)on laser-induced experimental choroidal neovasculari- zation (CNV) in rat model and choroid blood flow in rabbits' eyes. · METHODS: Male Brown Norway rats were treated with Nd:YAG laser to break Bruch's membrane. 30mg/kg and 15mg/kg Bdph were given daily through intraperitoneal injection for 4 weeks after laser treatment. Fluorescein angiography (FA) and choroidal flat mount were used to measure the development of CNV. Female New Zealand white rabbits' eyes were instilled with 10g/L Z,E-BdPh solution, and ocular blood flow was measured with colored microsphere technique. · RESULTS: The intensity of fluorescein leakage, indicating the ocular lesion, decreased significantly in group Bdph 30mg/kg and 15mg/kg, as compared to the control at P <0.01. The area of neovascularization checked by FA in both groups of Bdph, at 30mg/kg and 15mg/kg decreased significantly compared to the control group at P <0.05. On the choroid flat mount, the areas of CNV were also smaller in both Bdph groups than that in control group. One percent drug solution instilled into rabbits' eyes could improve the choroid blood flow at 30 and 60 minutes after drug instillation (P <0.05). · CONCLUSION: Z,E-butylidedephthalide can inhibit the development of CNV in the rat eyes and increase the choroid blood flow in the rabbit eyes. These results suggest that Z,E-butylidedephthalide may be a good agent for the treatment of age-related macular degeneration (AMD).

Abstract:To quantitatively detect gene expression level of transforming growth factor-β type 1 receptor (TβR 1) and transforming growth factor-β type 2 receptor (TβR 2) in different stage of diabetic rats’ retina and to observe and analyze the effect of transforming growth factor-β receptors on the retina of rat diabetic animal model. · METHODS: Twenty-eight healthy adult Sprague-Dawley rats were chosen and randomly divided into two groups of normal control (CON) and diabetes mellitus (DM). Diabetes was induced by streptozotocin (STZ) intraperitoneal injection. Gene expression was detected quantitatively with real-time fluorescence quantitative reverse transcription polymerase chain reaction (QRT-PCR). · RESULTS: The mRNA level of TβR 1 and TβR 2 was 0.000493±0.000133 and 0.000166±0.000057 at 4 weeks. The mRNA level of TβR 1 and TβR 2 was 0.000608±0.000232 and 0.000113±0.000049 at 12 weeks. TβR 1 expression was gradually elevated during the progression of diabetic retinopathy. TβR 2 expression was up-regulated at 4 weeks, but down-regulated at 12 weeks. · CONCLUSION: TGF-β and its receptors (TβR 1 and TβR 2) may play important role in the pathogenesis of diabetic retinopathy.

Abstract:To study the effect of the subretinal fluid (SRF) on proliferation of retinal pigment epithelium (RPE) cells and retinal glial (RG) cells and associated activation and translocation of protein kinase C (PKC) as well as the application of PKC inhibitor. ·METHODS: RPE and RG cells were disintegrated to obtain PKC activity of cytoplasm and cellular membrane after being treated by the subretinal fluid (SRF) from the different stages of PVR patients (grade B and C) or being treated with PKC specific activator [phorbol-12-myris-tate-13-acetate (PMA)] or normal vitreous or DMEM culture medium. PKC activity in cytoplasm and cellular membrane was measured using radioactive isotope 32P labeling in a specific reaction of phosphorylation on PKC substrate. In addition, the PKC inhibitor, dequalinium chloride, was used to pretreat the RPE and RG cells before the cells exposed to SRF or PMA or normal vitreous. 3H-TdR (tritiated thymidine) was used to measure the levels of proliferation of RPE and RG cells with or without the activation and translocation. ·RESULTS: SRF and PMA promoted the proliferation of RPE and RG cells. SRF and PMA activated PKC in the cytoplasm of RPE and RG cells and the activated cytoplasm PKC translocated to the cellular membrane of RPE or RG cells. The cell proliferation or PKC activation or translocation was not equally active in RPE as in RG cells. However, PKC inhibitor which attenuated the cell proliferation did not show significant differenceoninhibitionof RPEandRGcell proliferation( >0.05). ·CONCLUSION: SRF can lead to the activation and translocation of PKC in RPE and RG cells, which promote the proliferation of RPE and RG cells. Dequalinium chloride can inhibit PKC activation and translocation hence slow down the cells proliferation

Abstract:To investigate whether carnosine can inhibit cataract formation and protect Na⁺-K⁺ATPase against inactivation induced by a glucocorticoid. METHODS: Two hundred and twenty clear lenses cultured in vitro were randomly divided into five groups: control group (DMEM), steroid group (DMEM+Dexamethason 10μmol/L), lower concentration carnosine-treated group (DMEM+Dexamethason 10μmol/L+Carnosine 2mmol/L), higher concentration carnosine-treated group (DMEM+Dexamethason 10μmol /L+ Carnosine 5mmol/L) and carnosine group (DMEM + Carnosine 5mmol/L). Progression of cataract formation was evaluated daily using a dissecting microscope. On 1, 3, 5 and 7 days, 10 lenses of every group were homogenized and the activity of Na^{+-K+ATPase was measured by using spectrophotometer. RESULTS: During the incubation, mistlike opacity was observed in the lenses of the control group and carnosine group, but in the steroid group appeared dense nuclear opacity, while both two carnosine-treated groups came out visible demarcation between nuclear and cortical regions on 7 days. A decrease in the activity of Na+-K+ATPase was found in the lens of the steroid group. On 3, 5, 7 days, Na+-K+ATPase activity decreased 22.34% (P =0.002), 47.98% (P <0.001), 75.37%(P <0.001) compared with that at 1 day, respectively. In the carnosine group, the activity of Na+-K+ATPase remained at the level of the control throughout the 7-day incubation, indicating that carnosine itself did not interfere with the original lens enzyme activity. In the lower concentration carnosine-treated group, on 3, 5, 7 days, the activity of Na+-K+ATPase increased 10.8% (P <0.05), 44.6% (P <0.01), 57.4% (P <0.01) of control activity, respectively. In the higher concentration carnosine-treated group, the activity of Na+-K+ ATPase increased 11.3% (P <0.05), 45.7% (P <0.01), 57.6% (P <0.01), respectively on 3, 5, 7 days. The activity of Na+-K+ATPase in both two carnosine-treated groups were only 6.7% and 6.5% lower than that of the control group after 7-day incubation. After the 7-day incubation, the Na+-K+ ATPase activity of the lenses in the steroid group decreased significantly compared with carnosine-treated groups(P ＜0.01). CONCLUSION: Carnosine prevents the cataract formation induced by a glucocorticoid, and significantly inhibits the inactivation of Na+-K+ATPase induced by a glucocorticoid.}

Abstract:To determine whether hydroxyapatite modified titanium promotes superior adhesion and proliferation of rabbit corneal fibroblast in comparison with pure titanium. · METHODS: We used bioactive hydroxyapatite to modify titanium surfaces. Fourth passage fibroblasts of rabbit cornea were seeded on hydroxyapatite modified titanium surfaces, pure titanium and glass surfaces. Cell adhesion, proliferation and morphology were detected at 24, 48, and 72 hours using a acridine orange stain. Further studies of cell morphology were performed using scanning electron microscopy. · RESULTS: Cell counts were significantly greater on hydroxyapatite modified titanium surfaces at each time point (P＜0.05). At 24 hours, cell spreading was greater on hydroxyapatite-coated titanium and glass than on the pure titanium. At 72 hours, compared with pure titanium and glass surfaces, the cells on hydroxyapatite modified titanium surfaces had greater spreading area and longer stress fibers. · CONCLUSION: Hydroxyapatite modified titanium promotes superior adhesion and proliferation of rabbit corneal fibroblast in comparison with pure titanium.

Abstract:To investigate the effect of TGF-β2 antisense oligode- oxynucleotide on differentiation, proliferation of subconjunctival fibroblast following glaucoma filtration surgery. · METHODS: Glaucoma filtration surgery was performed on both eyes of 28 rabbits. TGF-β2 antisense oligodeoxynucleotide was subconjunctivally injected in the right eyes (group A),and TGF-β2 missense oligodeoxynucleotide (group B) or PBS (group C) was used at the same method in the left eyes as controls. Rabbits were killed at 4, 7, 14 and 28 days after surgery. Intraocular pressure (IOP), bleb characteristics were recorded at different time point. Subconjunctival fibroblasts were examined by immunohistochemistry and electron microscopy. · RESULTS: The IOP of rabbits in group A was significantly lower at 14 days (6.74±1.18mmHg) and 21 days (8.15±1.97mmHg) after operation than the IOP in group B (8.53±1.04, 9.72±1.09mmHg; P <0.01) and group C (8.79±1.21, 9.43±1.27mmHg; P <0.05). The mean bleb survival time was longer (17.2 days) in group A than that of group B (14.5 days) and group C (13.5 days) (P <0.05). The population of the cells expressing α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) was significantly reduced in group A compared with the group B and C. The ultrastructure of fibroblast was not altered by TGF-β2 antisense oligodeoxynucleotide. · CONCLUSION: TGF-β2 antisense oligodeoxynucleotide can prevent the scar formation after glaucoma surgery by inhibiting the differentiation and proliferation of subconjunctival fibroblast. It could be a potentially useful anti-scarring alternative for the prevention of late surgical failure.

Abstract:To study the antiproliferation of vitamin E succinate (VES) on pterygium fibroblasts in vitro and to find a potential agent for prevention and treatment of primary and recurrence pterygium. · METHODS: Primary culture and subculture of pterygium fibroblasts were established in vitro, and different concentrations of VES (0, 10 and 20mg/L) were added to subcultured fibroblasts, respectively. Influence of VES on the growth curve of fibroblast was observed at day 2, 4 and 7 after treatment of VES. 3-[4,5-Dimethylthiazolzyl]-2,5-Diphenyl Tetrazolium Bromide (MTT) assay at 490nm was used to evaluate the effect of the cells proliferation. · RESULTS: The addition of VES to culture caused the marked descent of growth curve in comparison with the control group, and the inhibiting rate of 10 and 20mg/L of VES was 33.2% and 46.7%, 67.9% and 76.8%, 81.7% and 89.3% at day 2, 4 and 7, respectively. VES could obviously inhibit the fibroblast proliferation in dose-dependent manner by MTT assay. · CONCLUSION: VES can significantly inhibit the proliferation of pterygium fibroblast in vitro.

Abstract:To construct and identify LEDGFp52 eukaryotic expression vector for RNA interference. · METHODS: Recombinants were designed and established by targeting gene LEDGFp52 and plasmid pGensil-1 based on LEDGFp52 cDNA sequences of Genomes. Two pairs of oligonucleotides were synthesized according to the Tuschl principle and inserted into plasmid pGenSil-l to generate siRNA eukaryotic expression vector. DH5α strains were transformed, plasmids were extracted, and recombinant vectors were identified by the restriction map and the sequence analysis. The cultured cells were transfected by the recombinant plasmid (pGensil-1-RNA.LEDGFp52-1). At 48 hours after transfection, the whole cell protein was extracted, and the protein level was detected using Western blotting with mouse anti-human LEDGFp52 monoclonal antibody.　 · RESULTS: Recombinant plasmids completely concord with the designs by the restriction map and the sequence analysis, the protein level of LEDGFp52 was down-regulated at 48 hours after transfecting pGensil-1-LEDGFp52-1 expression vector into HeLa cells, the recombinant eukaryotic expression vectors were successfully constructed. · CONCLUSION: siRNA recombinant can be successfully constructed by RNAi technique to inhibit the expression of LEDGFp52.

Abstract:To express the DT₃₈₉-hbFGF (389 amino acid residues of the N-terminus of diphtheria toxin (human basic fibroblast growth factor) fusion protein for potential targeting therapy towards posterior capsule opacification (PCO) after cataract surgery. METHODS: The DNA of inactivated diphtheria bacillus and RNA of 12-week fetal brain cortex were extracted, respectively. The fragments of truncated diphtheria toxin (containing 389 amino acids of N-terminus, DT₃₈₉) and full-length human basic fibroblast growth factor (hbFGF) sequence (encoding 18kDa protein) were amplified by PCR. The two fragments were inserted into pGEX-4T-1 prokaryotic expression vector to obtain pGEX-DT₃₈₉-hbFGF prokaryotic expression plasmid. After sequence analysis, the expressing plasmid was transformed into Escherichia Coli BL21 strain and expression was induced under IPTG. The expressed fusion protein was purified and identified. RESULTS: The gene fragments encoding DT₃₈₉ and hbFGF were amplified and their gene sequences were confirmed. Hybrid gene expression plasmid pGEX-DT₃₈₉ (hbFGF) was constructed. The fusion protein DT389-hbFGF was expressed and purified. CONCLUSION: The successful cloning and expression of DT₃₈₉-hbFGF immunotoxin provides a foundation for targeting therapy towards posterior capsule opacification.

Abstract:AIM:b To determine the aqueous, vitreous, serum levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in patients with proliferative diabetic retinopathy (PDR), and to speculate on the source of the change in concentration and to discuss its clinical significance. METHODS:b Forty-one eyes with proliferative diabetic retinopathy were included in the study, 16 of which were complicated by neovascularization of iris (NVI). Twenty-one eyes with idiopathic macular hole (MH) were as controls. The aqueous,vitreous, serum levels of PEDF and VEGF of all the groups were determined with ELISA. PEDF, VEGF and the levels in the three groups were compared with analysis of variance(ANOVA). The PEDF, VEGF concentrations in aqueous,vitreous and serum were analyzed with Pearson correlation test, and the correlation of PEDF and VEGF levels was also analyzed with Pearson correlation test. RESULTS:b The aqueous levels of PEDF decreased significantly in sequence in groups of control, PDR without NVI,PDR with NVI. VEGF levels increased coordinately. The similar findings existed in vitreous samples. The PEDF, VEGF levels in aqueous were not correlated significantly with those in serum, but correlated positively with those in vitreous. The intraocular levels of PEDF had a negative correlation to those of VEGF. CONCLUSION:b The reduction of intraocular PEDF level and elevation of intraocular VEGF level may play an important role in the occurrence and progression of PDR. In the development of PDR, the PEDF,VEGF levels in aqueous may be mainly effected by local pathological changes, as anti-angiogenic and pro-angiogenic factors, their unbalanced intraocular distribution may promote the angiogenesis of the iris and retina.