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Objective To investigate the inhibitory effect of rapamycin,an mammalian target of rapamycin (mTOR) pathway inhibitor,on the proliferation,migration and fibrosis of human pterygium fibroblasts (PFBs).Methods Pterygium tissues were collected from patients with primary pterygium who underwent surgical excision in Shenyang Fourth People's Hospital from May to July 2015.The tissues were cultured in vitro and the PFBs were identified by anti-human vimentin immunofluorescence assay.The 3 to 5 generation cells were used for the experiments.The viability of cells treated with different concentrations of rapamycin was detected by methyl thiazolyl tetrazolium (MTT).The cells were divided into normal control group and rapamycin group,and the scratch wound healing test was used to evaluate migration of the PFBs.The expressions of MKI67,α-smooth muscle actin (α-SMA),fibronectin,caspase3,mammalian target of rapamycin (mTOR) and LC3B mRNA were detected by real-time quantitative PCR.Results The cultured cells showed morphology of long spindle and were vimentin immunopositive.The cell viability in rapamycin treated PFBs demonstrated a dose-dependent decrease.At 24 hours after culture,The cell viability in 30 μmol/L rapamycin group was (76.67±8.84)％ of that in 0 μmol/L rapamycin group (P＜0.001).The relative residual scratch width in 30 μ mol/L rapamycin group was (35.40±11.62) ％ 48 hours after scratch,which was significantly greater than (2.45±0.76) ％ in the normal control group (P＜0.05).Real-time quantitative PCR showed that the mRNA expressions of MKI67,α-SMA,fibronectin and mTOR in rapamycin group were significantly decreased when compared with those in normal control group (all at P＜0.05).The expression of LC3B mRNA in rapamycin group was significantly higher than that in normal control group (P＜0.05).The mRNA expression of caspase3 was not significantly different between the two groups (P=0.861).Conclusions Rapamycin can effectively inhibit the proliferation,migration and fibrosis of PFBs without affecting the cell survival.Detailed mechanism remains to be further studied.Rapamycin may serve as an anti-fibrosis agent to prevent the progression and recurrence of pterygium in the future.
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Objective To investigate the inhibitory effect of rapamycin,an mammalian target of rapamycin (mTOR) pathway inhibitor,on the proliferation,migration and fibrosis of human pterygium fibroblasts (PFBs). Methods Pterygium tissues were collected from patients with primary pterygium who underwent surgical excision in Shenyang Fourth People's Hospital from May to July 2015. The tissues were cultured in vitro and the PFBs were identified by anti.human vimentin immunofluorescence assay. The 3 to 5 generation cells were used for the experiments. The viability of cells treated with different concentrations of rapamycin was detected by methyl thiazolyl tetrazolium ( MTT) . The cells were divided into normal control group and rapamycin group, and the scratch wound healing test was used to evaluate migration of the PFBs. The expressions of MKI67,α.smooth muscle actin (α.SMA), fibronectin,caspase3, mammalian target of rapamycin ( mTOR ) and LC3B mRNA were detected by real.time quantitative PCR. Results The cultured cells showed morphology of long spindle and were vimentin immunopositive. The cell viability in rapamycin treated PFBs demonstrated a dose.dependent decrease. At 24 hours after culture,The cell viability in 30μmol/L rapamycin group was (76. 67±8. 84)％ of that in 0μmol/L rapamycin group ( P<0. 001 ) . The relative residual scratch width in 30μmol/L rapamycin group was ( 35. 40 ± 11. 62 )％ 48 hours after scratch,which was significantly greater than (2. 45±0. 76)％ in the normal control group (P<0. 05). Real.time quantitative PCR showed that the mRNA expressions of MKI67,α.SMA,fibronectin and mTOR in rapamycin group were significantly decreased when compared with those in normal control group (all at P<0. 05). The expression of LC3B mRNA in rapamycin group was significantly higher than that in normal control group (P<0. 05). The mRNA expression of caspase3 was not significantly different between the two groups (P=0. 861). Conclusions Rapamycin can effectively inhibit the proliferation, migration and fibrosis of PFBs without affecting the cell survival. Detailed mechanism remains to be further studied. Rapamycin may serve as an anti.fibrosis agent to prevent the progression and recurrence of pterygium in the future.
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Objective To evaluate the effect of two polymer membranes, polyhydroxyalkanoates(PHA)and polylactic acid(PLA)during glaucoma filtration surgery(GFS),and to evaluate the morphology of membranous PHA after interlamellar implantation. Methods Twenty-eight New Zealand white rabbits were chosen and twenty-four of them were randomly divided into 6 groups(n=4):the PHA-low group,PHA-high group,PLA-low group,PLA-high group,positive control group(MMC group)and blank control group. The rabbits in each group received GFS. The corresponding polymer membranes were implanted under the scleral flap,while the MMC group was treated with 0.2 mg/mL mitomycin C(MMC) for 3 minutes,and the blank control group was treated without extra drugs. The intraocular pressure(IOP)was examined at 0 d,1 d, 3 d, 7 d, 14 d, 28 d and 84 d after GFS. The corneal layers of four rabbits were implanted with PHA membranes and the corneal morphological changes were observed after 84 d. Results The IOP of the PHA-low and PLA-high groups was lower than that of the blank control group at 84 d after GFS(P < 0.05),and was similar with that of the MMC group(P> 0.05). Morphological studies showed that there were no collagenous fibers filling in the duct, and the collagenous fibers around the membranes were generally arranged in parallel. There were no obvious changes in the peripheral collagen structure after implantation of PHA membranes between the corneal layers. Conclusions Application of PHA and PLA membranes during GFS in rabbits may maintain the level of IOP,and the effect is similar with MMC. The mechanism may be achieved through the mechanical blocking of fibrous tissue.
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Objective To determine the spectrum of conjunctival flora and the antibiotic susceptibility profiles of patients scheduled for penetrating intraocular surgeries.Methods A prospective case control study was performed.A total of 192 patients (192 eyes) scheduled for penetrating intraocular surgeries at the Fourth People's Hospital of Shenyang from February to August 2015 were enrolled.Samples from the conjunctival sac were collected before instillation of any ophthalmic solutions for both aerobic and anaerobic culture.The positive rate and bacterial spectrum were observed.Bacterial isolates were tested for antibiotic susceptibility to 7 commonly used ophthalmic antibiotics using automated drug resistance analyzing system.The research was approved by the Ethics Committee of the Fourth People's Hospital of Shenyang.Results Totally 91 strains were collected from 81 conjunctival samples during aerobic culture,the positive rate was 42.19％.Staphylococcus epidermidis was the most common microorganism (64.84％),followed by Staphylococcus lentus (7.69％) and Staphylococcus aureus (3.30％).Coagulatase negtive Staphylococcus (CNS) accounted for 80.22％ of the positively cultured aerobes.For anaerobic culture,a total of 28 strains were isolated from 28 conjunctival samples,the positive rate was 14.58％ Propionibacterium acnes was the predominant species (71.43％),followed by Finegoldia magna (10.71％).Majority of the CNS were sensitive to gentamycin and vancomycin,with resistance rates lower than 10％,but their resistance rate to erythromycin and ceftazidime was 87.67％ and 63.01％,respectively.Resistance rate of these CNS to levofloxacin,ciprofloxacin,and moxifloxacin was 42.47％,39.73％ and 17.81％,respectively.Multidrug resistance to at least 3 antibiotic classes was present in 38.36％ of the CNS.Conclusions Bacteria in the conjunctiva sac of preoperative patients are resistant to various ophthalmic antibiotics.To follow-up the bacterial distribution and antibiotic resistance is great meaningful in the prophylactic and treatment in ocular surgery-related infections.
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Background Blindness and low vision represent significant public health issues in China.Late diagnosis is the major reason for the irreversible vision impairment.A feasible,cost-effective screening and referral program is very important for the eye health care,prevention and treatment of blindness in China.Objective This study was to evaluate the feasibility and effectiveness of a health examination center-based opportunistic eye disease screening program.Methods This was a cross-sectional study.Subjects undergoing a routine physical examination at the health examination center of the Fourth People's Hospital of Shenyang were invited to attend this program.Presenting visual acuity,intraocular pressure,and nonmydriatic fundus photography were obtained.Optic diso photographs were evaluated independently by two ophthalmologists.Blindness and moderate to severe vision impairment were defined based on the criteria of World Health Organization Visual Impairment Classification in 2009.Glaucoma,diabetic retinopathy (DR) and other suspected eye diseases were diagnosed according to the fundus photography and intraocular pressure.This study was approved by Ethic Committe of the Fourth People's Hospital of Shenyang,the informed consent of each subject was obtained.Results Totally,15 303 subjects were enrolled and 15 197 of them finished the exanimations,giving a response rate of 99.3％.The overall percentage of blindness and moderate to severe visual impairment was 0.08％ (12/15 197) and 2.34％ (355/15 197).Two hundred and twenty-eight (1.50％) subjects were defined as glaucoma suspects and 80 individuals (0.53％) were diagnosed as epimacular membrane.Other suspected eye diseases included DR (0.41％),branchial retinal vessel occlusion (0.24％),macular degeneration (0.09 ％),and macular hole (0.06％).More than 95 ％ of the eye disease suspects have never been previously diagnosed or treated.A total of 358 subjects (2.36％) were defined as ocular hypertension suspects.Conclusions This health examination center-based opportunistic eye disease screening shows a good efficiency and feasibility.It may become an optional program in the national eye health care project,as well as the work of prevention and treatment of blindness.
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Cyclophotocoagulation is one of the efficacious procedures for the treatment of refractory glaucoma.However,its clinical utilization is limited due to severe post-operative complications including phthisis and visual loss.Transscleral cyclophotocoagulation (TCP) is technically simple and less affected by the opening status of the anterior chamber angle.Its effectiveness and complications are mainly related to the energy,spots and scale of the surgery,as well as the ocular pigmentation of the patients.With the advance of technology,both the effectiveness and safety of this procedure gain significant improvement.Meanwhile,it is free from the filtration surgery-related complications due to its noninvasive characteristics.Taken the local economic level,medical resources allocation,as well as the patients' preference and follow-up into account,utilization of TCP on primary open angle glaucoma (POAG) patients with good vision,or as primary surgical treatment for medically uncontrolled chronic angle closure glaucoma (CACG) and neovascular glaucoma (NVG) were recently tried and showed good preliminary results.It provides new insight in glaucoma therapy,although further evidence from prospective,random control trials are still needed.In present review,both the safety and efficiency,as well as related influent factors of TCP were summarized.The possibility and feasibility of this procedure as a primary surgical treatment for glaucoma were discussed.
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Respiratory virus poses a serious threat to human life and health. Airway epithelial cells are the body's first line of defense from a wide variety of foreign pathogens, such as viruses and bacteria. Therefore, successful airway epithelial cell culture can provide a model for investigating the mechanisms underlying respiratory pathogenic diseases following airway virus infection. This respiratory disease model can also be used for the potential development of novel therapeutics. Here we provide a brief review of recent developments on the culture of cells derived from human trachea-bronchial airway epithelium, and the application of this model for studying respiratory virus and disease.
Subject(s)
Animals , Humans , Cell Culture Techniques , Epithelial Cells , Virology , Respiratory Tract Diseases , Virology , Virus Diseases , Virology , Virus Physiological Phenomena , Viruses , GeneticsABSTRACT
Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.
Subject(s)
Animals , Humans , Cell Differentiation , Dependovirus , Genetics , Epithelial Cells , Cell Biology , Metabolism , Green Fluorescent Proteins , Genetics , HEK293 Cells , Lung , Cell Biology , Membrane Potentials , Mucins , Metabolism , Swine , Transduction, Genetic , Tubulin , MetabolismABSTRACT
BACKGROUND: Cholinergic system projected in brain and hippocampal structure is relevant with learning and memory. Piracetam acts on protecting and repairing cerebral neural cell, resisting cerebral functional injury due to physical and chemical factors and improving learning-memory capacity.OBJECTIVE: Chronic epilepsy in childhood animal and learning-memory complex animal model were self-prepared to observe the changes in content of acetylcholine and activity of cholinacetyltranslase in cerebral hippocampus and the intervention of piracetam.DESIGN: Randomized control experiment and non-blind evaluation were designed.SETTING: Department of Pediatrics of Second Hospital of Hebei Medical University and Institute of Traditional Chinese Medicine of Hebei Medical UniversityMATERIALS: The experiment was performed in Hebei Medical University and College of Life Sciences of Hebei Normal University from July to December 2004, in which, 50 Wistar childhood rats of clean grade and either sex were employed.METHODS: Coriamyrthin injection was administrated muscularly to duplicated chronic epileptic grand mal model in rats. Muscular injection was repeated once every three days. During modeling, those with general paroxysmal convulsion with posterior extremities standing or falling with standing or general stiffness-paroxysmal attack continuously for 3 times, the injection was changed to be once every 14 days. Ten rats were selected to be in normal control without modeling. The rest 40 rats after 3 months of modeling were randomized into 4 groups, named piracetam of 2.4 g/'L group (Group A), piracetam of 4.8 g/L group (Group B), dilantin 6 g/L +piracetam 4.8 g/L group (Group C) and model group (Group D), 10 rats in each. In each group, gastric infusion was performed continuously in 3 months after modeling, once per day, 10 mL/kg. In Group A and Group B,piracetam mixed solution of 2.4 g/L and 4.8 g/L was administrated for infusion respectively. In Group C, dilantin 6 g/L and piracetam 4.8 g/L were infused. In group D and the control group, normal saline 10 mL/kg was administrated. Relevant index determination was done 1 month after medication. Morris water maze test was performed to discover platform time and searching distance of epileptic rats, continuously for 3 days, twice per day. After test, the rats were sacrificed to collect brains to determine the content of acetylcholine in bilateral hippocampus. The activities of cholinacetyltranslase and acetylcholinesterase were determined with radioimmunity method.of acetylcholine in bilateral hippocampus and the activities of cholinacetyltranslase and acetylcholinesterase of rats in each group.ing platform time of rats in every group: the corresponding average searching time in Group D was increased compared with the control group [(63±11) s, (40±8) s; (61±9) s, (38±7) s; (57±8) s, (36±9) s; (55±11) s,(33±10) s; (52±7) s, (30±9) s; (49±9) s, (27±6) s, P ＜ 0.01]. In Group C and Group B, the searching time of 6 tests was decreased of various degrees compared with Group D [(44±9) s, (45±9) s;(43±9) s, (42±8) s; (42±7) s,(42±7) s; (40±9) s, (39±9) s; (38±7) s, (35±9) s; (35±6) s, (34±8) s,t=2.352-4.029, P ＜ 0.05-0.01]. In every medication group, the average searching time was decreased gradually by the increased frequency of erage searching distance in Group D was remarkably increased compared with the control [(793±74) cm, (420±81) cm;(763±89) cm, (418±57) cm;(690±67) cm, (382±69) cm; (623±81) cm, (356±71) cm;(592±98) cm,(330±69) cm;(550±54) cm,(301±97) cm,P＜ 0.01]. In Group C and Group B, the average searching distance of 6 tests was decreased of various degrees compared with Group D [(586±91) cm, (510±89) cm;(566±70) cm,(497 ±76) cm; (521 ±84) cm, (455 ±56) cm; (480 ±74) cm, (421 ±63) cm;(437±51) cm, (396±79) cm;(392±79) cm, (385±48) cm, t=2.364-4.230, P ＜ 0.05-0.01]. In every medication group, the average searching distance tent of acetylcholine in brain hippocampus and the activities of cholinacetyltranslase and acetylcholinesterase of rats in each group: those in Group D were all remarkably reduced compared with the control [(2.2±0.7) nmol/g,(3.8±0.9) nmol/g;(503.3±103.3) pkat/g, (778.3±125.0) pkat/g;(190.0±51.7) μkat/g, (368.3±86.7) μkat/g, P ＜ 0.01]. In mixed group and Group B, the content of acetylcholine and activity of acetylcholinesterase were remarkably higher than the Group D [(2.7±0.6) nmol/g, (2.9±0.6) nmol/g;(256.7±58.3) μ kat/g, (306.7±88.3) μkat/g, t=3.445-4.148, P ＜ 0.01]. In Group B, the activity of cholinacetyltranslase [(668.3±118.3) kat/g] was remarkably higher than those in the Group D(P ＜ 0.01). Every index in group A was basically same as model group.CONCLUSION: Grand mal of chronic epileptic rat model is characterized as declined capacity of spatial learning and memory and associated with decreased content of acetylcholine and the activities of cholinacetyltranslase and acetylcholinesterase in brain hippocampus, explaining the successful complex model of learning and memory disturbance. Piracetam 4.8 g/L may increase content of acetylcholine and the activities of cholinacetyltranslase and acetylcholinesterase in brain hippocampus and improve learning-memory capacity, but its effect at 2.4 g/L was not remarkable.
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BACKGROUND:After cerebral tissue ischemia and anoxia in young rats,the cerebral edema gets serious, and the levels of nitric oxide (NO) and malondialdehyde (MDA) decrease. Radix Astagali seu Hedysari has the pharmacological effects of enhancing immunity, anti-anoxia and improving myocardial ischemic reinfusion injury.OBJECTIVE: To investigate the influences of Radix Astagali seu Hedysari (huangqi) on contents of NO and MDA in brain tissue of young rats with cerebral injury after cerebral ischemia and anoxia.DESIGN:A randomized and controlled trial.SETTING: Department of Pediatrics, Second Hospital, Hebei Medical University; Department of Internal Medicine, Institute of Traditional Chinese Medicine, Hebei Medical University; Department of Pathophysiology, Hebei Medical UniversityMATERIALS:The experiment was conducted from January to April 2004at Department of Pathophysiology, Hebei Medical University. Total 40 SD rats, 7-day old, were at random divided as normal control group, model group, humgqi low-dose group and huangqi high-dose group, with 10 rats in each group. Huangqi injection (The content in 10 mL injection is consistent with 20 g raw drug) was provided by Institute of Traditional Chinese Medicine of Hebei Medical University (produced in Chengdu Di'ou Jiuhong Pharmaceutical Factory, Batch No. 0005028).METHODS:Except rats in normal group, those in the rest groups, under conscious and local anesthesia, were all given common carotid artery ligation, establishing cerebral injury model due to ischemia and anoxia. Rats in normal group were intraperitoneally injected 0.1 mL normal saline; rats in model group were intraperitoneally injected 9 g/L normal saline, 0.1 mL each day; rats in huangqi low-dose group and huangqi high-dose group were respectively given 0.1mL, 0.5 mL huangqi injection, once a day, intraperitoneally. Cerebral blood flow was detected immediately, 2 and 4days after injection. Then the rats were decapitated for collecting the brains to measure the water content in brain, the contents of NO and MDA.MAIN OUTCOME MEASURES: [1] Water contents in brains of rats in every group. [2] Cerebral blood flow, and the contents of NO and MDA.RESULTS:Totally 40 rats were involved in the trial and all entered in the final result analysis. [1] The water content in brain of each group: Compared with normal group, the content in model group was increased immediately after model establishment [(87.316±0.275)%, (88.259±0.297)% ,P ＜ 0.05 ],and did not return to the normal level at the second day [(86.973±0.265)%,(88.173±0.445)%,P ＜ 0.05]; compared with model group, the content in huangqi high-dose group was obviously decreased at second day[(88.173±0.445)%, (86.542±0.141)% ,P ＜ 0.05]. [2] Measurement of cerebral blood flow: compared with control group, the blood flow in model group was obviously decreased immediately after model establishment[(231.88±13.33), (139.54±10.58)mV,P＜ 0.05], and did not return to normal level till the 4th day [(234.57±14.38), (145.38±13.33)mV,P ＜ 0.05];compared with model group, the blood flow in huangqi low-dose group and huangqi high-dose group, at day 4, was obviously increased [(145.38±13.33),(288.45±12.89), (313.82±21.74)mV,P ＜ 0.01]. [3] The contents of NO and MDA: The contents in model group, immediately after model establishment, were obviously higher than those in normal control group [(26.55±5.23 ), ( 19.67±7.17 )μmol/L,P ＜ 0.05; (7.88±2.55), (4.22±0.12) μmol/L, P＜ 0.01], and at day 4, were significantly higher than those in normal control group [(48.65±17.06), (18.65±2.12)μmol/L,P ＜ 0.01; (5.29±0.68),(4.06±0.39)μmol/L,P ＜ 0.05]; compared with model group, the contents in huangqi low-dose group and huangqi high-dose group were obviously decreased at day 4 [(48.65±17.06), (23.77±12.79), (24.67±11.54)μ mol/L,P＜ 0.01; (5.29±0.68), (4.51±2.30), (3.68±0.39)μmol/L,P ＜ 0.01].CONCLUSION:Huangqi could obviously reduce cerebral edema from ischemia and anoxia, increase cerebral blood flow. It could decrease the contents of NO and MDA that is metabolite of free radical injury, thus playing its role to inhibit lipid peroxidation injury.