ABSTRACT
Background Retinal neovascularization is pathological basis of a variety of fundus diseases,but its pathogenesis is unclear.Studies showed that the expression level of radixin in retina is remarkably increased in retinal neovascularization-related diseases.It is presumed that silencing or down-regulating the abnormal expression of radixin is helpful for curing retinal neovascularization-related diseases.Objective This study was to investigate the inhibitory effect of radixin short hairpin RNA (shRNA) plasmid on expression of radixin gene in retina of oxygen-induced retinopathy (OIR) mice.Methods Sixty-four 7-day-old C57BL/6J mice were randomly divided into normal control group, model control group, radixin shRNA plasmid group and shRNA plasmid group by random number table.There were 16 mice in every group.OIR models were established by exposing the mice in an environment of (75±2) ％ oxygen for 5 days and then returned to the normal air in the model control group,radixin shRNA plasmid group and shRNA plasmid group,while the mice of the normal control group were fed in the normal air environment.Radixin shRNA plasmid or control shRNA plasmid at the dose of 1 μg was intravitreally injected in 12-day-old mice of the radixin shRNA plasmid group or shRNA plasmid group, respectively.Five days later, FD-2000S angiography was performed on the mice of each group and then retinal flatmounts were prepared for the observation of retinal vessels.The mice from various groups were sacrificed and retinal sections were prepared.The vascular endothelial nucleus and new blood vessels extending inner limiting membrane (ILM) were examined by hematoxylin and eosin staining;the expression of radixin in the retinas was detected using immunochemistry;the relative expression levels of radixin mRNA and protein were quantitative assayed by real-time quantitative RCR and Western blot, respectively.The use and care of the animals adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Results The distribution of retinal vessels was normal in the normal control group.Non-perfusion zone at the posterior pole of retina, circuity of blood vessels,leakage of vessel wall and new blood vessels were found in the mice of the model control group.Non-perfusion zone and microaneurysms were also exhibited in the shRNA plasmid group.However,these findings were slight in the radixin shRNA plasmid group.The surface of ILM was in discontinuity in the model mice and shRNA-injected mice with more vascular endothelial cell nucleus and more tubes extending ILM than that in the radixin shRNA plasmid group.The immunochemistry results showed that the expressions of radixin in the normal control group and radixin shRNA plasmid group were weaker than those in the model control group and control shRNA plasmid group.The relative expression levels of radixin mRNA were 1.002±0.043,2.236-±0.093,0.556±0.015 and 2.272±0.096 in the normal control group, model control group,radixin shRNA plasmid group and control shRNA plasmid group,and those in the radixin shRNA plasmid group were significantly reduced in comparison with the normal control group, model control group and the shRNA plasmid group (all at P＜0.01).The relative expression levels were 1.000±0.082,1.193±0.021,0.263± 0.016 and 1.235±0.005 in the normal control group,model control group,radixin shRNA plasmid group and shRNA plasmid,with the lowest expression level in the radixin shRNA plasmid group (all at P＜0.01).Conclusions Radixin shRNA can downregulate the expression of radixin gene in the retinas of OIR mice and further inhibit pathological retinal neovascularization.
ABSTRACT
Background Hypoxia is the main factor of retinal neovascularization and is closely associated with retinal ganglial cells (RGCs) degeneration.However, the study of retinal neural tissue lesions is rare.Objective This study was to investigate the influence of hypoxia environment on the expression of annexin A2 (ANXA2) in mouse RGC-5 cells and explore the mechanism of RGCs damage induced by hypoxia.Methods Immortalized mouse RGC-5 cells were cultured in high glucose DMEM with 10％ fetal bovine serum.The cells were identified by detecting the expression of Thy-1 ,a specific biomarker of RGCs.CoCl2 was added into the medium at the final concentrations of 50,100,200 and 300 μmol/L, and the cells without CoCl2 served as the control group.Cell viability (absorbance) was assayed by cell counting kit-8 (CCK-8) method in 12,24 and 48 hours after addition of CoCl2.The hypoxic cell models were established in DMEM with 100 μmol/L CoCl2 and divided into the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group,with the normal cultured cells as the normal control group.Apoptotic cells were determined by using hoechst 33342 stain.The expression levels of ANXA2 mRNA and protein in the cells were detected by real-time quantification PCR and Western blot,respectively.The expression and location of ANXA2 in the cells were examined by using immunofluorescence technique.Results The cultured cells grew well and showed the fusiform and polygonal shape,with positive expression of Thy-1 protein.Compared with the normal control group, the viabilities of the cells were insignificantly changed in the 50 μ mol/L CoCl2 group and 100 μmol/L CoCl2 group (all at P＞0.05) ,but the cell viabilities were significantly reduced in the 200 μμmol/L CoCl2 group and 300 μmol/L CoCl2 group in various time points (all at P＜0.05).Hoechst 33342 staining showed that the apoptotic cells with nuclear condensation and high green fluorescence intensity were obtained in the hypoxia groups.The relative expression levels of ANXA2 mRNA were significantly lower in the hypoxic groups than those in the normal control group (all at P ＜ 0.05).The relative expression levels of ANXA2 protein were significantly lower in the hypoxia 3-,6-, 12-and 24-hour group than those in the normal control group (all at P＜ 0.05).Apoptotic cells were seen in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group compared with the normal control group, showing the bright blue fluorescence in cellular nucleus for hoechst 33342.The relative expressing levels of ANXA2 mRNA in the cells were 0.80±0.14,0.67±0.33, 0.49±0.17 and 0.39±0.02 in the hypoxic 3-hour group,hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were significantly declined in comparison with the normal control group, with a statistically difference among the groups (F=434.354, P =0.000).The relative expression values of ANXA2 protein were 0.552 6±0.012 3,0.425 9± 0.033 4,0.344 9 ± 0.017 8 and 0.382 7 ± 0.022 1 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group,which were remarkably lower than 0.602 1 ±0.001 4 in the normal control group, showing considerably difference among the groups (F =3.057, P =0.000).ANXA2 proteins were highly expressed in the cellular nucleus and less expressed in the cell membrane and cytoplasm in the normal cells.Compared with the normal control group, the ANXA2 protein showed weak expression in the hypoxia group and primarily in the cytoplasm.Conclusions The expression of ANXA2 down-regulates in hypoxic mouse RGC-5 cells,which may participate in the apoptosis process of RGCs in high glucose environment.
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Objective: To observe the clinical therapeutic effects of supplementing Qi and activating blood circulation method(益气活血法) for critically ill patients.Methods: Ninety critically ill patients with(Qi-deficiency)(气虚) and blood stasis(血瘀) syndromes who diagnosed according to standard in a book named clinical diagnosis and treatment nomenclature of traditional Chinese medicine were randomly divided into the the treatment group(n=60) and the control group(n=30).The general therapy of the two groups was the same.Additionlly,the treatment group was administered Shenmai injection(丹参注射液) and Danshen power(丹参粉针剂),15 days were as one therapeutic course.Results: In the treatment group,the total effective rate of clinical therapeutic effects was 85.00 %;before and after treatment,traditional Chinese medical scores was 38.63?9.08 vs.24.27?7.43,acute physiology and chronic health evaluationⅡ(APACHEⅡ) 18.11?4.54 vs.12.47?1.64,platelet(PLT) count(198.00?54.16)?10~9/L vs.(174.00?40.82)?10~9/L,(haematocrit)(HCT) 0.340?0.049 vs.0.440?0.057,mean cell hemoglobin(MCH)(34.00?3.10)pg(vs.(31.00?1.83) pg).The differences of above parameters were significant between the two groups,and they were superior in the treatment group to those in the control group(all P
ABSTRACT
Specific surface polysaccharide antigen mannan was extracted from a strain ofCandida albicans,isolated from blood of a case of disseminated candidiasis,employingmodified method of Sakaguchi.The final product contained 99% sugar and 0.06% protein.Acid hydrolysis of the sugar revealed it consists of mannose only.In bot himmunodiffusion and immunoelectrophoresis,with anti-Candida albicans serum,it formeda single precipitin band.The above data indicates that surface mannan extracted fromCandida albicans is of high purity.