Group I mGluR-mediated inhibition of Kir channels contributes to retinal Müller cell gliosis in a rat chronic ocular hypertension model.

Müller cell gliosis, which is characterized by upregulated expression of glial fibrillary acidic protein (GFAP), is a universal response in many retinal pathological conditions. Whether down-regulation of inward rectifying K+ (Kir) channels, which commonly accompanies the enhanced GFAP expression, could contribute to Müller cell gliosis is poorly understood. We investigated changes of Kir currents, GFAP and Kir4.1 protein expression in Müller cells in a rat chronic ocular hypertension (COH) model, and explored the mechanisms underlying Müller cell gliosis. We show that Kir currents and Kir4.1 protein expression in Müller cells were reduced significantly, while GFAP expression was increased in COH rats, and these changes were eliminated by MPEP, a group I metabotropic glutamate receptors (mGluR I) subtype mGluR5 antagonist. In normal isolated Müller cells, the mGluR I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) suppressed the Kir currents and the suppression was blocked by MPEP. The DHPG effect was mediated by the intracellular Ca2+ -dependent PLC/IP3-ryanodine/PKC signaling pathway, but the cAMP-PKA pathway was not involved. Moreover, intravitreal injection of DHPG in normal rats induced changes in Müller cells, similar to those observed in COH rats. The DHPG-induced increase of GFAP expression in Müller cells was obstructed by Ba2+, suggesting the involvement of Kir channels. We conclude that overactivation of mGluR5 by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir channels.

[Ginkgolide B promotes axonal growth of retina ganglion cells by anti-apoptosis in vitro].

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.

[Construction of CNTF-GFP plasmid and transfection of Schwann cells by electroporation].

OBJECTIVE: To enhance the neurotrophic effect of Schwann cells, promote optic nerve regeneration, and simplify the means of observation, the CNTF-GFP fusion plasmid was constructed and transferred into Schwann cells by electroporation. METHODS: It was an experimental study. Plasmid pcDNA3. 1/CNTF-GFP was constructed and verified by auto-sequence. Cultured Schwann cells were transfected by electroporation. The transfection efficiency was counted by flow cytometry, the transcription of the gene was evaluated by RT-PCR, and the expression of protein was observed by fluorescence microsphere and cell immunofluorescence. RESULTS: CNTF-GFP plasmid was verified correctly. After electroporation, the green fluorescence of gene-transfected Schwann cells can be seen at 3 hours later, increased at 12 hours later, reached the peak during 24 h to 72 h later, and still could be seen at 7 days later. The transfection efficiency was evaluated at 44.93% +/- 12.92% by flow cytometry. The transcription of CNTF gene and the expression of CNTF protein have been proven by RT-PCR and cell immunofluorescence. CONCLUSIONS: CNTF-GFP plasmid was constructed correctly and Schwann cells were transfected by electroporation successfully and CNTF-GFP fusion protein can be expressed well, which became a good foundation for our future's study on the transplantation of gene-modified Schwann cells to promote optic nerve regeneration.

[Analysis of ophthalmic projects granted by National Natural Science Foundation].

OBJECTIVE: To understand the status of basic research work in the field of ophthalmology by analyzing the projects funded by the National Natural Science Foundation of China (NSFC) from the year of 1986 to 2007, and offer as a reference to the ophthalmologists and researchers. METHODS: NSFC supported ophthalmology projects in the 22 year's period were collected from the database of NSFC. The field of funded projects, the research team and their achievements were analyzed. RESULTS: There were 228 applicants from 47 home institutions were funded in the field of ophthalmology during the past 22 years, 323 projects funded with 66.74 million Yuan in total, in which 165 projects were fulfilled before the end of 2006. The applied and funded projects mainly focus on six different kinds of research area related to retinal diseases, corneal diseases, glaucoma, optic nerve diseases, myopia and cataract, and 70% of them were basic research in nature. As a brief achievement of 165 fulfilled projects, more than 610 papers were published in domestic journals, over 140 papers were published in Science Citation Index journals, more than 600 people were trained, and over 20 scientific awards were obtained. CONCLUSION: The number of funded projects and achievement of fulfilled projects in the discipline of ophthalmology gradually increased over the past two decades, the research fields were concentrated in certain diseases. NSFC has played an important role in promoting the development of ophthalmology research and bringing up specialists in China. However, clinical research, continuously research, transforming from basic research to clinic applications and multidisciplinary cross studies should be strengthened.

[The effect of lentivirus-mediated suicide gene therapy on lens epithelial cells].

OBJECTIVE: To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death. METHODS: a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated. RESULT: the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect). CONCLUSION: GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.

[Construction of bicistronic lentiviral vectors carrying HSV-tk and EGFP gene].

OBJECTIVE: To construct CMV-mediated Lentiviral vectors coexpressing EGFP and HSV-tk gene in order to establish a novel lentiviral vector platform for the suicide gene therapy of eye disease. METHODS: The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. HSV-tk fragments from pcDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site (IRES)-EGFP to construct the bicistronic lenti-HSV-tk-EGFP vector. Human embryonic kidney 293T cells were co-transfected with the lentiviral vector (three plasmids) by calcium phosphate DNA precipitation. HLEC, HXO-Rb(44), SH-SY-5Y and Hela cells were transfected with viral production and the expression of EGFP was examined under fluorescent microscope after transfection. The expression of HSV-tk and EGFP was examined by RT-PCR. RESULTS: Lentivirus mediated stable integration and efficient expression of EGFP and TK genes in the cells tested. Coexpression of HSV-tk and EGFP in HLECs mediated by lentiviral vectors was confirmed by the result of RT-PCR. The transfection efficiency for HLECs was about 100% at MOI = 100, and kept the same level for at least 6 months. CONCLUSION: The bicistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector, it might be used for suicide genes therapy in the treatment some eye disease.