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Objective:To explore the difference in ocular surface microbiota between patients with and without dry eye.Methods:Forty-two patients (42 eyes) diagnosed with dry eye were enrolled as dry eye group in the First Affiliated Hospital of Xi'an Jiaotong University from June to November 2020, and 37 controls without dry eye (37 eyes) were enrolled as control group in the same period.One eye was selected as the study eye, and the right eye was included when both eyes met the inclusion criteria.Swab samples from the conjunctival sac were obtained and sequenced.Sequencing of the V3-V4 region of the bacterial 16S rRNA gene was performed with Miseq PE301+ 8+ 301 platform.Operational taxonomic species (OTUs) clustering of microflora, comparison of alpha and beta diversity analysis of microflora between the two groups, annotation analysis of species and analysis of microbial markers were performed.The study protocol was approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Jiaotong University (No.XJTU1AFCRC2018SJ-014). Written informed consent was obtained from each subject before any medical examination.Results:A total of 18 586 OTUs were obtained, and 3 674 OTUs were shared between the two groups.Alpha diversity analysis showed that there was no significant difference in observed species index, Chao index, Ace index, Shannon index and Simpson index between the two groups (all at P>0.05), suggesting there was no difference in microbiota richness between them.The PCoA analysis showed that the microbial compositions of the two groups were significantly different ( R2=0.039, F=3.100, P=0.022). The dominant flora of the two groups was similar, with Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Cyanobacteria as the top 5 abundant bacterial phyla, with Pelomonas, Corynebacterium, Propionibacterium, Pseudomonas and Herbaspirillum as the top 5 bacterial genera.LEfSe analysis identified Tissierellaceae, Enhydrobater and Finegoldia as dominant bacterial genera in dry eye group, and Caulobacter and Curvibacter in control group. Conclusions:The composition of ocular surface microbiomes is different between dry eye patients and controls.
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Objective@#To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.@*Methods@#The cultured APRE-19 cells were divided into Avastin group and VAS2870 group, and then the Avastin group was subdivided into the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining, and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.@*Results@#The relative expression of NOX4 was 0.657±0.153, 1.000±0.200, 1.206±0.300, 1.260±0.200 and 1.413±0.273, and the relative expression of VEGF-A was 0.821±0.110, 1.210±0.100, 0.672±0.100, 0.340±0.120 and 0.300±0.130 in the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, respectively, with statistically significant differences among the groups (F=17.631, P<0.001; F=4.777, P<0.05). The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P<0.001). The relative expression of VEGF-A protein in the cells of the 0.25, 0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P<0.05). The expression of NOX4 protein in the cells was 0.970±0.120, 1.060±0.130, 0.880±0.130, 0.567±0.135 and 0.450±0.120, and the relative expression of VEGF-A protein was 0.387±0.135, 0.627±0.125, 0.370±0.140, 0.363±0.140 and 0.160±0.100 in the normoxia control group, hypoxia control group, 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group, respectively, with statistically significant differences among them (F=12.933, P<0.001; F=4.948, P<0.05). The relative expression of VEGF-A protein in the 1, 3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P<0.05).@*Conclusions@#NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.
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Objective To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.Methods The cultured APRE-19 cells were divided into Avastin group and VAS2870 group,and then the Avastin group was subdivided into the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group,3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining,and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.Results The relative expression of NOX4 was 0.657± 0.153,1.000±0.200,1.206 ± 0.300,1.260± 0.200 and 1.413 ± 0.273,and the relative expression of VEGF-A was 0.821±0.110,1.210±0.100,0.672±0.100,0.340±0.120 and 0.300±0.130 in the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,respectively,with statistically significant differences among the groups (F =17.631,P＜ 0.001;F=4.777,P＜0.05).The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P＜0.001).The relative expression of VEGF-A protein in the cells of the 0.25,0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P＜0.05).The expression of NOX4 protein in the cells was 0.970±0.120,1.060±0.130,0.880±0.130,0.567±0.135 and 0.450±0.120,and the relative expression of VEGF-A protein was 0.387±0.135,0.627±0.125,0.370±0.140,0.363±0.140 and 0.160±0.100 in the normoxia control group,hypoxia control group,1 μmol/ml VAS2870 intervention group,3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group,respectively,with statistically significant differences among them (F =12.933,P＜ 0.001;F =4.948,P＜ 0.05).The relative expression of VEGF-A protein in the 1,3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P＜0.05).Conclusions NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.
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Objective To observe the long-term clinical efficacy of intravitreal ranibizumab (IVR) in patients with idiopathic choroidal neovascularization (ICNV),and to explore the indicators that affect curative effect.Methods A retrospective self-controlled study was performed.The clinical data of 61 ICNV patients (61 eyes) from January 2013 to May 2014 in the First Affiliated Hospital of Xi'an Jiaotong University were Collected.The best corrected visual acuity (BCVA),the central retinal thickness (CRT),the height of pigment epithelium detachment (PED) and the defect length of ellipsoidal zone (EZ) before and after treatment were analyzed,the baseline clinical indicators were compared among different IVR treatment times and ICNV types.Results The mean follow-up time was (41.5±4.6) months.The mean BCVA (LogMAR) were 0.59±0.32 and 0.17 ± 0.12,the mean CRT were (331.18±80.42) μm and (245.07±44.67) μm,the mean height of PED were (246.73±104.75) μm and (205.78±117.01) μm and the mean defect length of EZ were (2 315.10± 1 233.77) μm and (1 325.98± 1 157.30) μm before and after treatment,respectively,with significant differences between before and after treatment (all at P＜0.05).Fifty-three patients (86.89％) completed the treatment in the first year or within three times.The mean CRT and the height of PED in the IVR ≥ 3 times treatment group were significantly higher than those in the IVR 1 times treatment group (all at P＜0.05).There was no significant difference in the average treatment times among inferior lateral of macular fovea,side of macular fovea and outside of macular fovea (F =1.982,P ＞ 0.05).Conclusions IVR therapy for ICNV is well tolerated with an improvement in BCVA,CRT,height of PED and defect length of EZ.The majority of patients can complete the treatment within 1 year,and most patients can be cured within 3 times treatments.The number of treatments may be associated with the CRT and the height of PED at baseline.
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OBJECTIVE:To screen the formulation of 5-fluorouracil (5-FU) polylactic acid (PLA) sustained-release discs (5-FU-PLA-DS),and study its in vitro drug-release mechanism. METHODS:UV spectrophotometry was used to determine the 5-FU content in the release medium. Using simulate body fluid as release medium,in vitro drug-release test was conducted under 37℃water bath. Using PLA with molecular weight of 3000,6000,10000,15000,20000,15 species of round 5-FU-PLA-DS with drug containing of 1.5,2.5,3.0 mg/piece and 3.0 mm in diameter and 1.0 mm in thickness were prepared. Using effective con-centration sustained release time and cumulative release rate as indexes, the optimal formulation was screened. The form of 5-FU-PLA-DS was observed by scanning electron microscopy after release,and its release mechanism was evaluated. RESULTS:In the optimal formulation, the PLA molecular weight was 20000 and drug containing was 3.0 mg/piece. The prepared 5-FU-PLA-DS can release for 119 d,with cumulative release degree of 100.63% and effective concentration sustained for 91 d. Scanning electron microscopy showed that the microspheres at the surface were degraded to the release medium first,and then the microspheres of inner layer exposed and release drug gradually after PLA degraded. The main mechanism of drug-release was melt-ing and diffusion. CONCLUSIONS:5-FU-PLA-DS is successfully prepared,with long release time in effective concentration,can be degraded step by step from outside to inside and achieve non-synchronous drug-release of microspheres at different layers.
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OBJECTIVE:To screen the formulation of 5-fluorouracil (5-FU) polylactic acid (PLA) sustained-release discs (5-FU-PLA-DS),and study its in vitro drug-release mechanism. METHODS:UV spectrophotometry was used to determine the 5-FU content in the release medium. Using simulate body fluid as release medium,in vitro drug-release test was conducted under 37℃water bath. Using PLA with molecular weight of 3000,6000,10000,15000,20000,15 species of round 5-FU-PLA-DS with drug containing of 1.5,2.5,3.0 mg/piece and 3.0 mm in diameter and 1.0 mm in thickness were prepared. Using effective con-centration sustained release time and cumulative release rate as indexes, the optimal formulation was screened. The form of 5-FU-PLA-DS was observed by scanning electron microscopy after release,and its release mechanism was evaluated. RESULTS:In the optimal formulation, the PLA molecular weight was 20000 and drug containing was 3.0 mg/piece. The prepared 5-FU-PLA-DS can release for 119 d,with cumulative release degree of 100.63% and effective concentration sustained for 91 d. Scanning electron microscopy showed that the microspheres at the surface were degraded to the release medium first,and then the microspheres of inner layer exposed and release drug gradually after PLA degraded. The main mechanism of drug-release was melt-ing and diffusion. CONCLUSIONS:5-FU-PLA-DS is successfully prepared,with long release time in effective concentration,can be degraded step by step from outside to inside and achieve non-synchronous drug-release of microspheres at different layers.
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Background Studies indicate that the decline of androgen level affects the secretion function of epithelial cells of meibomian glands and lacrimal glands ,which results in the abnormality of tear quality and quantity.However,whether androgen level regulates the mucin adhesion to ocular surface is still not elucidated.Objective This study was to investigate the tear functional changes and corneal ultrastructural changes in castrated male mice.Methods Fifty-six SPF male BALB/c mice were devided into normal control group, sham group, and orchectomy 1-week group, orchectomy 2-week group, orchectomy 4-week group, orchectomy 6-week group, orchectomy 8-week group according to random number table.Orchectomy was performed in the mice of the orchectomy 1-,2-,4-,6-and 8-week group,and only incision rather than enucleation of testis was carried out in the mice of the sham group.Peripheral venous blood samples were collected during eyeball enucleation in all the mice and serum androgen concentration was detected by radioimmunoassay.Tear secretion level was measured with phenol red thread.Break-up time of tear film (BUT) was tested,and corneal inflorescence staining was scored based on the criterion of Toshida.The corneas of all the mice were collected for ultrastructural examination under the transmission electron microscope.The study complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results Mouse serum androgen concentration was (573.87±6.74) ng/μl in the normal control group and (579.74-±8.24)ng/μl in the sham group, and there was no significant difference between these two groups (t =1.432, P =0.251) , however, the serum androgen concentration was 0 ng/μl in all castrated mice after orchectomy.The tear secretion levels were (5.26 ± 0.99), (4.89 ± 0.56), (4.62 ± 0.83), (4.28 ± 0.63), (3.36 ± 0.56),(2.60±0.27) and (2.08 ±0.35) mrn/5 minutes in the normal control group, sham group and orchectomy 1-, 2-, 4-,6-and 8-week groups, respectively, and the tear secretion levels were significantly lower in the orchectomy 2-, 4-,6-and 8-week groups than those in the normal control group (all at P＜0.05).The BUT were (68.33±12.86), (62.47± 3.75),(58.67±10.03), (47.17±7.64) ,(39.51±3.39),(32.67±3.88) and (31.00±2.36) seconds in the normal control group, sham group and orchectomy 1-, 2-, 4-, 6-and 8-week groups, respectively, and the BUTs were significantly shorter in the orchectomy 2-,4-, 6-and 8-week groups than those in the normal control group (all at P＜0.05).The fluorescein stain score of corneal epithelium was gradually increased with the lapse of castrated time.The decrease of number,shortening and edema of microvilli as well as separation of desmosomes of corneal epithelial cells were found under the transmission electron microscope in castrated mice.Conclusions Decreasing of serum androgen level, declining of aqueous tears,instability of tear film and damage of corneal epithelial cell ultrastructures occur in castrated male mice, and these manifestations are similar to dry eyes.
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Background Statins has prominent roles in regulating lipids,anti-inflammation,autoxidation and protecting vascular endothelial cells.Sartans can promote cell growth and the expression of cytokines.Since the pleiotropic effects of statins and sartans on a variety of cell types,it is inferred that the two medicines can delay retinal aging.Objective This study was to explore the anti-aging effect of simvastatin and telmisartan on the physiological aging of retina.Methods Sixty-six three-month-old healthy SD rats were selected in this study,and 6 of them served as the youth group and the right eyeballs were immediately enucleated.The other rats were raised until 9-month-old in the same conditions and then randomly divided into the simvastatin group,telmisartan group and the control group with 20 rats for each group.The simvastatin of 5 mg/kg and telmisartan of 8 mg/kg were given by intragastric administration once a day in the simvastatin group and the telmisartan group until 17-month-old,and the equal amount of normal saline was used in the control group in the same way.The number of survival rats was 12 in the simvastatin group,10 in the telmisartan group and 8 in the control group.The right eyes were enucleated after heart perfusion of 4％ paraformaldehyde solution for the preparation of retinal paraffin sections.Retinal thickness was measured by pathological examination,and the expressions of the retinal neuron markers,including Thy-1,protein kinase C-α (PKC-ot),opsin and rhodopsin,were detected by immunofluorescence technique to evaluate the morphology of retinal ganglion cells (RGCs),bipolar cells as well as the thickness of the outer segment of photoreceptors.Results The retinal structure was clear in the rats of the youth group.However,the RGCs arrangement and inner segment (IS) and outer segment (OS) structure were abnormal in the simvastatin group,the telmisartan group and the control group.Compared with the rats of the youth group,the thickness of outer nuclear layer (ONL),outer plexiform layer (OPL),inner nuclear layer (INL),inner plexiform layer (IPL) and the total thickness of the aging rats were decreased,and the IS/OS thickness was increased in the simvastatin group and the telmisartan group (all at P＜ 0.01).Thy-1 stain showed that the number of RGCs was reduced in the simvastatin group,telmisartan group and the control group compared with the youth group,and that in the simvastatin group was increased in comparison with the control group (all at P＜0.01).PKC-αt stain exhibited that the density of bipolar cells was increased but the axon terminal bouton was declined in the simvastatin group,telmisartan group and the control group compared with the youth group,and the axon terminal bouton was declined in the simvastatin group compared with the youth group and the control group (all at P=0.000).Opsin and rhodopsin stains displayed that the OS thickness was increased in the simvastatin group,telmisartan group and the control group compared with the youth group,and that in the telmisartan group was reduced in comparison with the control group (all at P＜0.01).Conclusions As SD rat aging,retinal thickness is gradually attenuated and the number of RGCs is gradually declined.Although the density of bipolar cells seem to be unchanged,their synaptic connections are decreased and the OS is thicken.Simvastatin and telmisartan can delay retinal senescence by protecting retinal neurons against aging and thinning thickened OS.
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Objective To explore the effect of Ligustrazine on neurogenesis in cortex after focal cerebral ischemia in rats. Methods Focal cerebral ischemia was induced by left middle cerebral arteryocclusion with asuture. Two hours later, injection of Ligustrazine (80 mg/kg, 1 time/d) was performed peritoneally. Four hours after the ischemia,5-bromodeoxyuridine (BrdU) (50 mg/kg, 1 time/d) was injected peritoneally. At 7 d, 14 d and 21 d after ischemia,BrdU positive cells in the cortex were observed by immunohistochemical staining. Results In ischemic model group, at 7 day, sparsely-distributed BrdU positive cells were observed in the Ⅱ - Ⅵ layers of the ipsilateral cortex, with a band-like distribution in ischemic penumbra. With the prolongation of ischemia, the number of BrdU positive cells increased.In Ligustrazine group, BrdU positive cells were also observed in the Ⅱ - Ⅵ layers of the cortex, with an intense distribution in ischemic penumbra. The numbers of BrdU positive cells at 7 d, 14 d and 21 d were more than those in ischemic model group respectively. Conclusion Ligustrazine increases the proliferated cells in cortex after focal cerebral ischemia in rats. The results suggest that it may be useful for promoting self-repair after ischemia.
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Objective To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium )or 10%FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin(13.33%)in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1(30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.
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Objective: To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods: RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium) or 10% FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results: RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin (13.33%) in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1 (30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion: Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.