0 05) CONCLUSION:The results of the statistical analysis showed that the two formulations were bioequivalent

Objective To cultivate human retinal capillary endothelial cells (HRECs) and establish two-dimensional model of human retinal vessels in vitro. Methods In a fibronectin-coated raising pound, HRECs were cultured by non-serum human-endothelial-cells substrate and two-dimensional model of human retinal vessels was established. Horseradish peroxidase was used to detect the permeability. Some of the vascular models were cultivated with 5 ng/ml vascular endothelial growth factor (VEGF), whose changes of permeability was compared with which of the models without cultivation with VEGF. The effect of VEGF on vascular permeability was observed. Results Meshy vascular structure came into being due to the confluent HRECs after 2 to 4 days. Comparatively complete two-dimensional vascular model after about 6 days. VEGF increased vascular permeability and promoted the formation of blood vessels. Conclusion HRECs can be cultivated successfully with human-endothelial-cells substrate; standard retinal two-dimensional vascular model in vitro can be established by using cellular raising pound and non-serum human-endothelial-cells substrate.

Background Retinal Müller cells are important gliocytcs and the source of retinal stem cells.Researching the biological behavior of Müller cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases.To establish a stable culture method of Müller cells is a solid basis of relative basic research.Objective This study was to establish a simple and stable method of isolation and culture of human retinal Müller cells and provide sufficient and high-quality Müller cell source.Methods Human retinal Müller cells were isolated from healthy human donor eyes.The mixture solution of hyaluronidase (100 U) and 0.25％ trypsin were used to digest chopped retinal tissue.The DMEM/F12 medium with 20％ fetal bovine serum (FBS) was added to stop the digestion process.RPMI1640 medium with 20％ FBS was used to culture the cell for 72 hours and then replaced the half medium.The cells were passaged by the RPMI1640 medium with 20％ FBS.The morphology of the cells were examied under the optical microscope,and the expressions of glial fibrillary acidic protein (GFAP),a marker of gliocytes,and glutamine synthetase (GS),a special marker of retinal Müller cells,were detected by immunochemistry and immunofluorescence technology.Results Human retinal Müller cells were successfully isolated by enzyme mixture solution of hyaluronidase (100 U) and O.25％ trypsin.The cells were adherent to walls 24 hours after primary culture and completely merged 9-10 days after culture.The cells showed oval in shape with abundant cytoplasm,and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei.After cells passage,the cells were enlarged and grew toward polygonal shape.The positive expression of GFAP was observed in more than 95％ cells and strongly positive expression of GS was observed in more than 90％ cells by immunohistochemstry and immunofluorescent staining.Conclusions Human retinal Müller cells can be successfully isolated by hyaluronidase combined with trypsin digestion.Abundent and pure human retinal Müller cells can be obtained by successively using RPMI1640 medium with 20％ FBS and 10％ FBS.

BACKGROUND: Embryonic stem cells (ESCs), the seed cells of all mature cells in vivo, are useful tools for nerve transplantation and developmental gene function research. Notch1 signaling pathway is the key pathway to control the ordered neural development and differentiation of many kinds of neural cells, however, there is no report on the dynamic expression of Notch1 signal during the ESC differentiation to date. OBJECTIVE: To investigate the expression of Notch1 protein, transmembrane signal transduction molecule, during directional differentiation of embryonic stem cells into neural cells. DESIGN, TIME AND SETTING: Cell research was carried out between October 2003 and October 2004 at Zhongshan Ophthalmic Center, SUN Yat-sen University, Guangzhou, Guangdong Province, China. MATERIALS: BALB/C mouse embryonic stem cell line Ⅵ (passage 11)was obtained from experimental animal center of SUN Yat-sen University, provided by professor Huang Bing. ESC culture medium was high-glucose DMEM medium with 20% bovine serum and 106 IU/L mouse leukemia inhibitory factor. Induced differentiation medium was high-glucose DMEM medium with 20% fetal bovine.serum and 5×107 mol/L retinoid acid(RA). METHODS: Passage 11 ESCs were resuscitated and incubated by ESC culture medium in incubator at 37℃ with 5% CO2. Passage 11 ESCs were subcultured after 2 or 3 days and RA was added into medium to induce differentiation. Three time points for observation were established: induced for 1, 5 and 9 days. MAIN OUTCOME MEASURES: Morphological changes were observed under inverted phase contrast microscope, MAP-2 antigen expressed in differentiated cells was detected by immunofluorescence method. Immunocytochemistry, Western Blot, flow cytometry assay were used to investigate the Notch1 protein expression. RESULTS: ESCs presented clone-like growth. After induced by RA for 9 days, single neural network was achieved around most of the cell clusters. With the prolongation of induction, MAP-2 positive neural cells increased gradually. Almost all ESC clones expressed Notch1 protein strongly or positively, but Notehl protein expression decreased gradually after induced differentiation (P < 0.01). CONCLUSION: Notch1 signal shuts off progressively during induction of ESCs into neural cells, which suggests Notch1 may play an important role in the differentiation of ESCs into neural cells.

AIM: To establish a simple, rapid and accurate high performance liquid chromatographic method (HPLC) for the determination of cyclosporin A (CsA) in human whole blood, and to study the influence of nicotinylmethylamide (Nic) on the pharmacokinetic parameters of cyclosporin A (CsA). METHODS: Whole blood CsA concentrations were measured by HPLC in 18 healthy volunteers administrated single CsA or co administrated Nic. The data of time blood concentrations of CsA were analyzed by 3p97 Program. The analysis of variance and two one sided t test were used to compare the main pharmacokinetic parameters of CsA in the two administrations. RESULTS: C max and AUC 0～∞ of CsA had statistically significance between the single CsA group and co administration of Nic group (P0.05). CONCLUSION: This HPLC method is simple, sensitive and accurate, and is suitable for routine determination of blood CsA levels in human. Nic can improve the absorption of CsA and increase the C max and AUC of CsA, but has no influence on the metabolism of CsA.

AIM: To study the function of activin receptor-like kinase Ⅰ(ALK1) gene in vascular endothelium.METHODS: The human umbilical vein endothelial cells(HUVECs) were cultured,and the change of expression of ALK1,ALK5 in activation of HUVECs was analyzed.The full-leng coding sequence of ALK1 was cloned into pcDNA3.1+using standard protocols.The constructed pcDNA3.1+ALK1 plasmid were transfected into HUVECs.The proliferation and migration of HUVECs were detected by boyden champer and flow cytometry.RESULTS: The expression of ALK1 was up-regulated in resolution.ALK1 promoted the proliferation and migration of HUVECs.CONCLUSION: ALK1 has an important function in remodeling by promoting the proliferation and migration of endothelial cells.

Background Stem cell transplantation represents a promising treatment option for patients suffering from degenerative disorders.Accumulating evidences indicate that mesenchymal stem cells (MSCs) are able to differentiate into retinal pigment epithelial (RPE)-like cells.However,MSCs are difficult to obtain.Human adipose mesenchymal stem cells (ADSCs) are proved to have similar properties to MSCs,but relevant study is less.Objective This study was to assess the feasibility of human ADSCs differentiating into RPE-like cells and the safety of its application in vivo.Methods The third generation of human ADSCs were incubated into 6-well plate,and 100 ng/ml epithelial growth factor,50 μ mol/L taurine and 5×10-7 mol/L retinoic acid were added into the medium 12 hours after cultured to induce the cells,and conventional cultured cells were used as the control group.Induced cells were traced with PKH26,and Pan-cytoke ratin (Pan-CK) monoclonal antibody was used to identify the cells under the fluorescence microscope.Induced RPE-like cell suspension of 1 μl was intravetreally injected in the right eyes of 6 BALB/c mice,and equal volume of PBS was used in the same way in another 6 mice.The animals were sacrificed 1 month after injection,and the retinal morphology was examined by histopathology under the optical microscope.The ultrastructure of retinal ganglion cells (RGCs) was examined by the transmission electron microscope.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Cultured human ADSCs grew well with the slender polygone shape.Cell membranes showed the red fluorescence for PKH26 after induced.In addition,Pan-CK was expressed in the cell membranes with the red fluorescence in the induced cells,but the response was absent in the control cells.One month after intravitreal injection,induced cells located on the retinal surface,and the retinal morphology was clear under the optical microscope.No abnormality in RGCs was seen under the transmission electron microscope.Conclusions Human ADSCs can differentiate into RPE-like cells after induction.PKH26 can mark induced cells well.There is no adverse effect of induced cells on retina after intravitreal injection in a short-term duration in mice.

AIM: To observe the effect of ginkgolide B (CB) on the intracellular calcium ion concentration ( [ Ca~(2+) ]_i) and mitochondrial function of cultured rat retinal neurons in vitro. METHODS: in vitro primary culture of rat retinal neurons was used in the experiment. The apoptosis model of glutamate - induced retinal neurons was established and co - cultured with ginkgolide B. The [ Ca~(2+) ]_i and mitochondrial membrane potential of the retinal neurons were detected by laser scanning confocal microscope. RESULTS: Glutamate decreased the survival rate of retinal neurons, increased the apoptosis and the [ Ca~(2+) ]_i, lowered the mitochondrial membrane potential. The [ Ca~(2+) ]_i was clearly diminished and the mitochondrial membrane potential was significantly increased with the GB intervention, and the apoptosis decreased significantly. CONCLUSION: GB protects retinal neurons from glutamate induced neurotoxicity. The effect of GB on retinal neurons might be due to its ability to decrease the [Ca~(2+) ]_i and increase mitochondrial membrane potential.

AIM:To observe the effect of ginkgolide B (GB) on the intracellular calcium ion concentration ([Ca2+]i) and mitochondrial function of cultured rat retinal neurons in vitro.METHODS:in vitro primary culture of rat retinal neurons was used in the experiment. The apoptosis model of glutamate-induced retinal neurons was established and co-cultured with ginkgolide B. The [Ca2+]i and mitochondrial membrane potential of the retinal neurons were detected by laser scanning confocal microscope.RESULTS:Glutamate decreased the survival rate of retinal neurons,increased the apoptosis and the [Ca2+]i,lowered the mitochondrial membrane potential. The [Ca2+]i was clearly diminished and the mitochondrial membrane potential was significantly increased with the GB intervention,and the apoptosis decreased significantly.CONCLUSION:GB protects retinal neurons from glutamate induced neurotoxicity. The effect of GB on retinal neurons might be due to its ability to decrease the [Ca2+]i and increase mitochondrial membrane potential.

Objective To observe the changes of VEGF and Ang-2 expression in streptozotocin(STZ)-induced diabetic rat retina after insulin therapy and explore possible roles of insulin in the development of diabetic retinopathy. Methods Diabetes was induced in 8-week-old male wistar rats by a single intraperitoneal injection of STZ. After three weeks,animals were randomly divided into four groups:(1)diabetic rats received intensive insulin therapy for 20 days;(2)diabetic rats received unregular insulin therapy,which caused the abrupt fluctuation of glycemic level;(3)diabetic control rats; and (4)normal control rats. After treatment,the animals were sacrificed with an overdose of anesthesia,and the eyes were enucleated and fixed in 4% paraformaldehyde immediately. Paraffin sections of retina were prepared.Expression of VEGF and Ang-2 was assessed by immunofluorescence stain and images analysis. Results Quantitative analysis showed VEGF and Ang-2 protein expression was increased by 2.38-fold and 2.41-fold in diabetic rats retinas as compared to non-diabetic rats retinas respectively (P0.05). Conclusion Intensive insulin therapy could decrease VEGF and Ang-2 expression in retina and has protective effect on diabetic retinopathy in STZ-diabetic rats.