AIM:To study azelastine hydrochloride combined with praprofen for the treatment of allergic conjunctivitis in children.METHODS: Totally 80 children with allergic conjunctivitis who were admitted to our hospital from May 2016 to June 2018, they were randomly divided into the control group and the observation group, 40 cases in each group. The control group was given pranoprofen eye drops, on the basis of the control group, the observation group was combined with azelastine hydrochloride eye drops for adjuvant treatment. The two groups children were treated continuously for 1wk. The clinical efficacy, the changes of the clinical symptoms, tear film stability, inflammatory factors and adverse reactions were compared between the two groups.RESULTS: After 1wk of treatment, the clinical efficacy of the observation group was 95.0%, higher than that of the control group, 85.0%(P=0.035); the itching, tearing, foreign body sensation and photophobia scores in the observation group were lower than that of the control group \〖(1.13±0.36)vs (2.28±0.41)scores,(1.05±0.33)vs (2.14±0.49)scores,(1.17±0.46)vs (2.28±0.59)scores(1.29±0.37)vs (1.84±0.55)scores\〗(P<0.05); the tear film rupture time(BUT)in the observation group was significantly higher than that of the control group, and the corneal fluorescein staining test(FL)was significantly lower than that of the control group \〖(11.96±1.14)vs (5.85±0.92)s,(2.85±0.33)vs (6.21±0.68)scores\〗(P<0.05); the tears histamine(HA), eosinophilic cationic protein(ECP)and immunoglobulin E(IgE)in the observation group were lower than that of the control group \〖(31.62±4.51)vs (65.03±6.48)μg/L,(2.20±0.28)vs (7.26±0.72)μg/L,(0.06±0.01)vs (0.18±0.04)mg/L\〗(P<0.05); there was no significant difference in the overall incidence of adverse reactions between the two groups during treatment(χχ2=1.897, P=0.168).CONCLUSION: The clinical efficacy of azelastine hydrochloride combined with pranoprofen in the treatment of allergic conjunctivitis in children is significant, which can effectively improve the level of inflammatory factors, promote the tear film stability recovery, and have good safety, the clinical value is high.

Background Researches showed that transforming growth factor-β2 (TGF-β2) promotes the activity of human Tenon capsular fibroblasts (TFs),which plays a role in the scarring of filtering blebs after antiglaucoma surgery.However,its mechanism is not fully clear.Lysyl oxidases (LOXs) are important extracellular matrix proteases which can catalyze the cross-linking of collagen and elastin.Investigating the impact of TGF-β2 on the expression of LOXs has a great significance for the understanding of the pathogenesis of filtering bleb scarring and its prevention.Objective This study was to investigate the effect of TGF-β2 on the expression of LOXs in cultured human TFs.Methods The TFs at 4-8 generations were divided into normal control group and different concentrations of TGF-β2 treated-groups,and 100,200,400,800 μ1 of TGF-β2 with the final concentration of 2,4,8 and 16 ng/ml was added into the medium to treat human TFs respectively for 24 hours.The LOXs in the cells were detected by Western blot to determine the optimal dose of TGF-β2.The 4 ng/ml TGF-β2(200 μ1) was used to treat human TFs for 6,12,24 and 48 hours respectively,and the change of LOXs expression in the cells over time was assayed by Western blot.The expression and distribution of LOX protein in the normal cells and TGF-β2-treated cells was detected by using immunofluorescence technique.This study was approved by Daping Hospital of Third Military Medical University Ethic Commission.The guardians of the patients who offered the specimen knew the purpose of the study and signed informed consent.Results Western blot assay showed that the expressions of LOX,LOXL1,LOXL2,LOXL3 and LOXL4 in the cells were gradually elevated from the normal control group and 2,4,8,16 ng/ml TGF-β2-treated groups,showing significant differences among the groups (F =37.338,13.438,31.067,11.767,15.167,all at P＜0.01).The expression of LOXL2 protein in the cells was 0.68±0.07,1.09±0.10,1.32±0.07,1.50± 0.06 and 1.89±0.12 in the normal control group and 6-,12-,24-and 48-hour groups respectively after 4 ng/ml TGF-β2 treatment,with a significant increase over time (F =82.832,P=0.000).The expression of LOX was weak in the normal cultured TFs,while the fluorescence intensity of LOX expression was evidently enhanced in the cytoplasm of the cells in the TGF-β2-treated group.Conclusions TGF-β2 upregulates the expressions of LOXs in human TFs in a dose-and time-dependent manner,which probably offers a basis for the further study on the prevention of filtering bleb scarring after glaucoma surgery.

Background Our previous study demonstrated that the aptamer S58 specifically targeted transforming growth factor-β receptor Ⅱ (TβRⅡ) and inhibited the transdifferentiation of human Tenon capsule fibroblasts (HTFs) mediated by transforming growth factor-β (TGF-β).Chitosan-nanoparticles (CS-NP) are good drug carriers,but the efficacy and safety of CS-NP/aptamer complexes deserve attention.Objective The aim of this study was to synthesize a novel CS-NP/aptamer complex called CS (S58)-NP and investigate its properties and applicability.Methods Human Tenon capsule tissue was obtained from patients during strabismus surgery,and HTFs were cultured and passaged using the explant culture method.The fourth to tenth generations of cells were used in the experiment.Different concentrations of CS-NP were used to prepare the CS(S58)-NP by the ionic cross-linking method with a surface charge rate (N/P) for S58 of 10,20,30 or 40.The particle size and Zeta potential were measured by the Zeta analyzer.The shape and distribution of CS (S58)-NP particles were examined under the scanning electron microscope.The binding of CS-NP with S58 and resistance of CS (S58)-NP to DNase Ⅰ were examined by agarose gel eletrophoresis.The release rate of S58 from CS (S58)-NP in PBS was quantitatively analyzed by a ultraviolet spectrophotometer.The cytotoxicity of CS(S58)-NP to HTFs was evaluated by detecting the production of lactate dehydrogenase (LDH).Results The Zeta analyzer showed that the particle size of CS (S58)-NP was 130-270 nm and its electric potential ranged from + 16 to +28 mV.The CS (S58)-NP particles appeared spherical with an even distribution under the scanning electron microscope.The mean encapsulation efficiency of CS(S58)-NP was 88.9％,89.3％,91.7％ or 90.5％,respectively,when the N/P was 10,20,30 or 40.After being encapsuled by CS-NP,S58 could resist the degradation from DNase I.Its total releasing level in PBS increased with the lapse of time,with a maximum releasing speed at 24 to 36 hours.The total releasing level reached 100％ at 96 hours.With increaseing concentrations of CS(S58)-NP,the relative releasing level of LDH in HTFs suspension gradually elevated with a significant difference among the groups (F =588.018,P =0.000),with the highest released LDH level at 50 nmol/L of CS(S58)-NP (12.853％ ±0.375％).Conclusions CS-NP provides a protective and slow-releasing effect on the S58 aptamer.CS (S58)-NP shows a good biocompatibility with HTFs with a low cytotoxicity at a concentration of ＜50 nmol/L.CS(S58)-NP could be used to inhibit TGF-β induced transdifferentiation of HTFs in the future.

Background Research showed that transforming growth factor-β2 (TGF-β2) promotes scar formation.But its mechanism in scarring after glaucoma filtration surgery is worthy of studying.Objective This study was to investigate the effect of TGF-β2 on myofibroblast transition of human Tenon fibroblasts (HTFs) and scarring after glaucoma filtration surgery.Methods Tenon capsular tissue was obtained from 3 patients with strabismus during the surgery and was incubated in DMEM with 10％ fetal bovine serum (FBS).The cells were collected and passaged in the free-serum medium for 24 hours,and then 1,2,5,10,20 μg/L TGF-β2 was added into the medium respectively,to induce the transformation of HTFs,and 2 μg/L or 5 μg/L TGF-β2 was used to treat the HTFs for 6,24,48 and 72 hours.The control group was not treated with TGF-β2.The expressions of α-smooth muscle actin (α-SMA) and phosphorylation of the signaling proteins (pSmad2) in HTFs were detected by Western blot assay.The expressions of α-SMA and F-actin were located by cell immunofluorescine technique under the confocal immunofluorescence microscopy.Cell contractility was determined by collagen gel contraction assays.This study was approved by Ethic Committee of Institute of Surgery Research of Daping Hospital,and informed consent was obtained from each patient or custodian initial of the study.Results The expression of α-SMA protein in the HTFs was increased significantly after the treatment of TGF-β2 in comparison with the control group and reached a peak at 24-48 hours.The α-SMA expression was gradually weakened in the 10 μg/L TGF-β2 groups.Little of α-SMA and F-actin were expressed in the control group.However,strong staining for α-SMA and F-actin were observed in the 1,2 and 5 μg/L TGF-β2 groups and then the staining weakened at the concentration of 10 μg/L.In addition,pSmad2 showed a stronger expression in the 2 μg/L TGF-β2 group than that in the PBS group and FBS group,with the strongest expression in 30 minutes through 2 hours.The untreated gel contracted (78.00±3.13)％ from its initial size,and contraction in the 1,2,5,10 μg/L TGF-β2 group were (63.88±1.78)％,(20.69±0.65)％,(19.49-±0.54)％,(16.24±0.84) ％,respectively,TGF-β2 increased HTFs contraction significantly (Fgroup =859.400,P =0.000).Conclusions TGF-β2 can induce transdifferentiation of Tenon fibroblast into myofibroblast and increase cell contractility,with a concentration-dependent and time-dependent pattern to an extent.It may be the mechanism of scar formation after glaucoma filter surgery.

In our previous study, a dominant sequence called aptamer S58 antagonized TGF-β-induced myofibroblast transdifferentiation in human Tenon's capsule fibroblasts (HTFs) through sealing the targeting site of TGF-β receptor II (TβR II). However, rapid degradation by ubiquitous nucleases limited the aptamer's efficacy. Chitosan-nanoparticles (CS-NP) are good drug carriers. Herein we synthesises novel chitosan nanoparticle-aptamer S58 complexes called CS(S58)-NP in order to preserve and prolong S58's efficacy. We synthesised CS(S58)-NP at various molar ratios of CS-NP to S58 using an ionic gelation method. Then, the properties of the CS(S58)-NP including particle size, zeta potential, protection capacity, slow-release effect and cytotoxicity were studied. The targeting effect of the CS(S58)-NP was also studied. CS(S58)-NP at molar ratios of 20 and 30 showed high aptamer encapsulation efficiency, powerful aptamer protection, stable sustained-release ability and low cytotoxicity. FITC-labelled CS(S58)-NP could successfully bind to TβR II. As a result, TGF-β-induced cell proliferation and α-SMA expression were both inhibited. Furthermore, the CS(S58)-NP could inhibit TGF-β-induced α-SMA expression for a longer time than naked S58, even in serum. This research applied CS-NP as the aptamer carrier. The research results demonstrate that CS-NP are potentially able to preserve and prolong aptamer S58's efficacy. This study reveals that the use of CS-NP is promising for aptamer delivery and CS(S58)-NP can be a potential anti-scarring therapeutic approach after glaucoma filtration surgery.

PURPOSE:To isolate aptamers that were bound to the extracellular segment of TGF-β receptor II (TβRII) and evaluate their effect on the TGF-β-induced transdifferentiation of fibroblasts.METHODS:TβRII-binding aptamers were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) from a single stranded DNA (ssDNA) library. Human Tenon's fibroblasts (HTFs) were cultured and treated with TGF-β2, TGF-β2 and aptamer S58/68, or aptamer S58/68 alone. Western blot analysis was performed to determine levels of α-smooth muscle actin (α-SMA) and the signaling protein phosphorylated Smad2 (p-Smad2). α-SMA and p-Smad2 subcellular distribution and fibrous actin (F-actin) with rhodamine-phalloidin staining were evaluated by confocal immunofluorescence microscopy. Cell contractility was assessed in collagen gel contraction assays.RESULTS:Twenty-one sequences were obtained after eight rounds of selection. Two preferential sequences, aptamer S58 and S68, were isolated and used in the following experiments. Aptamer S58 significantly inhibited α-SMA expression and incorporation into actin stress fibers, as induced by TGF-β2. Aptamer S58 also suppressed TGF-β2-induced cell contraction. Furthermore, aptamer S58 inhibited the TGF-β2-induced phosphorylation and nuclear translocation of Smad2. However, we did not find any effect of aptamer S68 on TGF-β2 activity in vitro.CONCLUSIONS:Our study revealed that a novel aptamer binding TβRII inhibited TGF-β2-induced myofibroblast transdifferentiation in HTFs.

Objective To culture fibroblasts from different human Tenon capsule in vitro and to compare their growth property.Methods Get human tenon capsule tissue from normal people,glaucoma patients and pterygium patients respectively,fibroblast cells were grown in DMEM medium containing 10% fetal bovine serum at 37 ℃,5 %CO2,and the medium was changed every two day.Cell proliferation was assayed by counting the cell number and MTT method,then the cell growth curves were drawn.Results The cells were in the logarithm growth period from 48 hours to 6 days after seeding the plate.The cells cultured from normal people,glaucoma patients and pterygium patients tissues showed no significant difference.Conclusion The proliferating characteristic of fibroblasts of glaucoma and pterygium showed no obvious difference in that of normal Tenon capsule tissue in vitro.

本文观察一例蛇毒溅入患者双眼的症状、体征、治疗及痊愈的过程,急救冲洗双眼是救治的关键.