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Objective:To prepare specific anti- Fusarium yolk immunoglobulin (IgY) and investigate its tolerance to temperature and pH and verify its antifungal effect against Fusarium. Methods:Eighteen 22-week-old Leghorn laying hens were selected and randomized into negative control group and experimental group according to the random number table method, with 9 hens in each group.The 2×10 7 colony forming units (CFU)/ml suspension of inactivated hyphae of Fusarium and Freund complete adjuvant was mixed in a 1∶1 ratio and emulsified.The hens in the experimental group were injected with 1 ml of the mixture to immunize and received 1 ml of Freund incomplete adjuvant as booster injection at two weeks after the initial injection.The egg yolk was collected from the 5th to 16th week after immunization.Specific anti- Fusarium IgY protein was prepared by salting out method using ammonium sulfate.The obtained protein solution was put into a freeze dryer and made into freeze-dried powder stored at 4 ℃.The hens in the negative control group were injected with 0.9% sodium chloride to prepare the non-specific antibody as the negative control.Bradford method was used to determine the concentration of specific egg IgY protein and the indirect enzyme-linked immunosorbent assay (ELISA) was employed to measure its titer.The 1×10 5 CFU/ml and 1×10 3 CFU/ml Fusarium suspension were cultured with different concentrations of IgY and phosphate buffered saline (PBS) for 4 days, and the absorbance value at 600 nm was measured.The co-incubated PBS/negative IgY with Fusarium solution was set as blank control/negative control accordingly.The concentration-killing curve of anti- Fusarium IgY against Fusarium was obtained.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 7.4, and the diluted specific IgY solution was placed into the water bath for 30 minutes at 30, 40, 50, 60, 70, 80, 90 ℃, respectively, and was cooled down to room temperature.The specific IgY solution was diluted to 0.02 mg/ml with PBS pH 1, pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, pH 12, respectively, and the diluted specific IgY solution was placed at 4 ℃ for one hour.The activity of diluted specific IgY solution by different methods was measured by indirect ELISA, and the tolerance of IgY to various temperatures and pH was evaluated.Twelve 8-week-old SPF female C57BL/6 mice were selected and randomized into the PBS control group and specific IgY treatment group according to the random table method, with 6 mice in each group.The right eyes of the 12 mice were infected with Fusarium to establish mice model of fungal keratitis.One day after modeling, 200 mg/ml of anti- Fusarium IgY was dropped to the right eyes of mice in the specific IgY treatment group, and PBS was dropped to the right eyes of mice in the PBS control group.The corneas of mice in the two groups were observed under the slit lamp microscope at 1, 3 and 5 days following modeling, and the corneal ulcer was scored according to the grading scale for inflammation score.The use and care of experimental animals followed the Association for Research in Vision and Ophthalmology statement.This study protocol was approved by an Ethics Committee of The Affiliated Hospital of Qingdao University (No.QYFYWZLL26168). Results:The IgY protein concentration from the 5th to 16th week after immunization was 1.57, 2.89, 24.98, 25.09, 23.89, 25.78, 21.57, 21.37, 18.98, 15.78, 14.67, 12.67 mg/ml, respectively.The titer of IgY was increased from the 5th week, and it reached the highest titer 1∶10 000 at the 7th week, which could be maintained until the 12th week after immunization before it dropped gradually.The concentration-killing curve showed that compared with the blank control group and negative control group, Fusarium grew slowly in the specific IgY treatment group.The specific IgY with a titer greater than 1∶10 000 had thermal stability below 60 ℃.The activity of specific IgY was highest at pH 4 to 6, which could be maintained above 70% at pH 3 to 9 and was further reduced with the decrease or increase of pH.At 1, 3 and 5 days after Fusarium infection, the inflammation scores were 3.50±0.55, 7.33±0.82, 4.00±0.63 in the PBS control group, and 3.33±0.82, 4.17±0.75, 2.50±0.55 in the specific IgY treatment group.There was a statistically significant overall difference in inflammation scores at various time points between the two groups ( Fgroup=247.35, P<0.05; Ftime=23.19, P<0.05). At 3 and 5 days after Fusarium infection, there was a smaller ulcer area and decreased inflammation scores in the specific IgY treatment group compared with the PBS control group, and the differences were statistically significant (all at P<0.05). Conclusions:The high titer specific IgY can be successfully prepared by salting out method using ammonium sulfate, which is with high stability, tolerance to temperature and pH.Moreover, it can alleviate the severity of corneal ulcers and reduce inflammation scores in the mouse model of fungal keratitis.
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Ophthalmology is interrelated with a variety of disciplines, and its features determine the need for interdisciplinary joint training of postgraduate students majoring in ophthalmology. The joint training can optimize the students' knowledge structures, enhance their innovation abilities and improve their overall qualities. However, there are still some factors restricting the interdisciplinary joint training in the existing education system. We propose to establish a full-time administrative management system, build a multidisciplinary communication platform, attach great importance to tutors' interdisciplinary researches, and establish dual mentor or multi-tutor training system. Moreover, a reasonable interdisciplinary evaluation system and various incentive mechanisms are advocated to improve the quality of postgraduate education in ophthalmology.
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Ophthalmic pathology plays an important role in studing the occurrence,development and pathogenesis of ophthalmic diseases.Ophthalmic pathology can guide the diagnosic and treating process of primary tumor,secondary tumor and inflammatory diseases.Ophthalmic pathology,including pathological morphology,liquid biopsy,and molecular pathology,plays an important role in the connection of basic medicine and clinical medicine and guides the research of pathogenesis and promotes the development of ophthalmology.Combination of ophthalmic pathology and clinical assistant examination can provide an objective basis for precision medicine of eye disease.
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Background Studies have determined that nucleotide binding oligomerization domain 2 (NOD2) plays a key role in innate immune response.However,whether NOD2 participates in the nature defense of fungal keratitis is unclear.Objective This study was to investigate the expression and significance of NOD2 on cornea in the initial of Aspergillus fumigatus keratitis (AFK) in rats.Methods Seventy-two adult clean Wistar rats were randomized into the normal control group,only corneal epithelial scraped group and AFK model group,and the AFK models were established by incubating Aspergillus fumigatus to cornea after corneal epithelium was scraped.All the operations were performed in the right eyes of rats.Reverse transcription PCR (RT-PCR) was carried out to detect the expression of NOD2 mRNA in corneal epithelium 4,8,16,24 hours after operation.Twenty-four hours after operation,the expression of NOD2 protein in rat corneas was examined by immunochemistry and immnunofluorescence technology.Also,the rat corneas were obtained for regular histopathological examination.The use and care of the animals complied with Institutional Animal Care and Use Committee Guidebook by NIH.Results All the models were made successfully.RT-PCR revealed that a fewer NOD2 mRNA were expressed on cornea in the normal control group,but the expressing levels of NOD2 mRNA were increased in the only corneal epithelial scraped group and AFK model group.Compared with only corneal epithelial scraped group,the elevated values of NOD2 mRNA expression in the AFK model group were statistically significant at 4,8,16 and 24 hours after operation (t =-0.409,-0.439,-0.534,-0.618,all at P=0.000).The histopathological examination displayed that the cornel tissue had intact structure in the normal control group,and partly corneal epithelial deficiency,slight corneal swelling and fewer neutrophil granulocytes were seen in the only corneal epithelial scraped group.However,corneal ulcer,severe corneal edema and a lot of neutrophil granulocytes were exhibited in the AFK model group.Immunochemistry and immnunofluorescence staining evidenced that weaker expression of NOD2 was visualized in the corneal epithelial and endothelial layers,and obviously enhanced staining was seen in the AFK model group.The expressing levels (absorbancy) were 0.045 ± 0.005,0.050 ± 0.005 and 0.092 ± 0.006 in the normal control group,only corneal epithelial scraped group and AFK model group,respectively,showing a significant increase in the AFK model group compared with the only corneal epithelial scraped group (t =0.042,P =0.000).Conclusions Expression of NOD2 is upregulated in the corneas with AFK,suggesting that NOD2 participates the natural defense in the initial of fungal keratitis.NOD2 may play an important role in the process of anti-fungal innate immune response in cornea.
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Background Fungal keratitis is a common ocular surface disease and the treatment is dissatisfactory because of short of high efficiency,low toxicity and broad-spectrum medication.Penetrating keratoplasty is an effective therapy,but its clinical applications is limited due to lack of corneal donor.To seek a low-cost and effective management is of an important significance.Objective This clinical trial was to compare the clinical effectiveness between corneal ulcer debridement combined with amniotic membrane cover+drugs therapy and corneal ulcer debridement of combined drugs therapy for fungal keratitis.Methods A prospective cases-controlled study was designed.A total 210 eyes of 210 patients with fungal keratitis were included from January 2001 to January 2013 in Affiliated Hospital of Qingdao University.The patients were randomly divided into observation group (109 eyes) and control group (101 eyes).The combination therapy of corneal ulcer debridement with amniotic membrane cover and drugs was administered in the observational group,and only lesion debridement and drugs therapy was performed on the control group.The curative effect,postoperative visual acuity and therapeutic course were compared between the two groups.Results The effective rates for fungal keratitis were 100.00％,90.41％,77.78％ in the mild,moderate and severe patients,respectively,and those of the control group were 52.38％,80.00％,25.00％ respectively,showing significant differences between them (U =2.211,2.214,2.356,all at P＜0.05).The total effective rate was significantly higher in the observational group than that in the control group (91.09％ versus 75.23％),with a significant difference between them (U=3.040,P=0.002).Postoperative visual acuity was better in the observational group than that in the control group (0.44 ± 0.04 versus 0.22 ± 0.03),with a significant difference between them (t =2.225,P ＜ 0.05).The mean therapeutic course was (22.4 ± 1.8) days in the observational group,which is shorter than (33.2±2.4) days in the control group,with a significant difference between them (t =2.097,P＜0.05).Conclusions Combination therapy of lesion debridement with amniotic membrane coverage and drugs can shorten treating duration and improve vision,and it is superior to only lesion debridement and drugs therapy.
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Ophthalmic microsurgery has its own characteristics: high precision , complicated operation , and long learning curve.It is important for young ophthalmologists to master the basic operation skills through standardized and systematic training during graduate studies .The teaching method , integrating theoretical teaching , animal operation and simulative ophthalmic surgery training , have important meanings in promoting the post-graduate students to master the practical and correct ophthalmic microsurgery technique . The aim of this study was to introduce the curriculum design and practice , and to evaluate the model through the examination scores .
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The network teaching platform of ophthalmology of Qingdao University , as the basis construction of the national key discipline , forms a perfect and complete set of teaching system with the aid of part of course information, part of network teaching resources and part of answer and interaction. The network teaching platform focuses on the construction of network teaching resources and answer and interaction. In the college teaching of ophthalmology, through building clinical teaching resource database and discussing on network platform, we carry out interactive and discussion-based teaching, and students can prepare before class and discuss after class. The application of network teaching platform of ophthal-mology in classroom teaching and teaching feedback can improve the teaching methods , deepen the teach-ing content, implement the sharing of teaching resources, and lay a solid foundation for ophthalmology teaching reform.
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Objective To construct subsystem of picture archiving and communicating system (PACS) based on video database of ophthalmology and to evaluate on effectiveness of applying PACS system in postgraduate teaching.Methods PACS subsystem were constructed by collecting audiovisual materials of ophthalmology surgery videos,retinal photography,B-ultrasonography,OCT,UBM of patients.A total of 24 postgraduates were divided into control group (n =12) and experimental group (n=12) by random number table.Students in control group was taught by traditional cases,books and literature while those in experimental by network teaching from PACS subsystem.Two teaching methods were assessed subjectively and objectively.Objective indicators including ophthalmology examination results,operational skill ratings and paper reviewing results as well as subjective indicators including efficiency to accept the knowledge,satisfaction degree of teaching,influencing degree of papers were employed to assess the effectiveness of the two teaching methods.Independent samples t-test and twosample rank sum test were used for statistical analysis,P ＜ 0.05 was considered statistically significant.Results Students in experimental group done better in operation scores and in the assessment of the efficiency of knowledge accepting,satisfaction of teaching and papers effect than control group(P =0.000、0.000、0.000、0.003).The two groups had no significant difference in professional exam achievements and paper review scores (P =0.625、0.354).Conclusions PACS subsystem based on video database of ophthalmology are benefit for postgraduate teaching.
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To investigate the application of seminar teaching on postgraduate education of ophthalmology,postgraduate from Grade 2008 to Grade 2010 were divided into experiment groups and control groups.Seminar teaching and conventional teaching were applied to two groups respectively.The performances of seminar teaching groups at each grade were better than the control groups ( P ＜ 0.05 ).Thus,seminar teaching can enhance theory and operation ability of postgraduate.
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BACKGROUND: Simple drug therapy can not acquire satisfactory effects in treatment of fungal keretitis. At present, penetrating keratoplasy (PKP) has been considered a primary means to cure the fungal keratitis to save eyeball and vision.OBJECTIVE: To evaluate the clinical efficacy of PKP in the treatment of high-risk fungal keratitis.DESIGN, TIME AND SETTING: A retrospective case analysis was performed at the Department of Ophthalmology, Affiliated Hospital of Qingdao University Medical University between January 2000 and January 2007.PARTICIPANTS: A total of 51 patients (51 eyes) with high-risk fungal keratitis who underwent PKP in the Affiliated Hospital of Qingdao University Medical College were recruited into this study. Of these patients, 12 suffered from perfored, 35 from hypopyon, 8 from complicated cataract prior to surgery, and 5 from complicated glaucoma.METHODS: All patients received antifungal and antibacterial treatments prior to surgery and underwent PKP within 4 days following admission. After surgery, antifungal and antibacterial treatments were performed locally and systemically. All patients were followed-up for 6--24 months.MAIN OUTCOME MEASURES: Postoperative visual acuity, recurred fungal infection, rejection of implants, secondary glaucoma, and ulceration of implant.RESULTS: ① Of 51 patients, 18 were followed-up for 6-12 months, 2 for 13-18 months, and 8 for 19-24 months. ② A total of 49 (96.1%) out of 51 patients preserved the eyeballs and the visual acuity improved to different degrees in 48 (94.1%) patients. ③ After surgery, fungal infection recurred in 6 eyes (11.6%), 4 of which were controlled by antifungal medication and 2 was enucleated because of uncontrolled endophthalmitis. Graft rejection was found in 18 (35.3%) eyes, 13 of which recovered transparent by medication and 5 received secondary PKP. Graft ulceration was present in 4 (7.84%) eyes, 3 of which were cured and the remaining one was re-grafted because of severe endothelial cell loss. Secondary glaucoma appeared in 7(13.7%) eyes, and the intraocular pressure was controlled medically and surgically. Complicated cataract occurred in 6 (11.8%) eyes, 3 of which underwent cataract extraction. Most complications were successfully controlled. In the final follow-up period, 45 (88.2%) grafts were transparent.CONCLUSION: PKP is an effective approach to preservation of eyeballs and restoration of visual function in patients with high-risk fungal keratitis, which can not be treated by conservative therapy.
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@#Objective To observe the application of amniotic membrane in conjunctival sac plasty.Methods 24 cases with different eye diseases were performed with conjunctival sac plasty,in which 11 eyes were performed with amniotic membrane transplantation,5 eyes with symblepharon which adhered with cornea or emerged neovascularization were performed with amniotic membrane transplantation and lamellar keratoplasty,other 8 eyes were performed with amniotic membrane transplantation and conjunctival fornixe.They were followed up for 6~12 months.Results There was no inflammation observed after operation.The transplanted amniotic membrane turned transparent and the conjunctiva at sewing location crawled to amnion 10 d after operation.One month later,the neonatal conjunctiva covered transplanting region completely.Among the 24 eyes,9 eyes were healed,13 eyes were improved,and 2 were ineffective.Conclusion Amniotic membrane is a kind of effective material during conjunctival sac plasty for narrow conjunctival sac.
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@#Objective To observe the effect of small-interfering RNA(siRNA) targeting c-Met,the receptor of hepatocyte growth factor(HGF),on the proliferation of human lens epithelial cells(LECs).Methods siRNA was transferred into LECs cultured in vitro by HiperFect Transfection Reagent.Real-Time PCR was applied to observe the expression of c-Met mRNA in LECs after gene transfer,and MTT assay was used to detect the proliferation of LECs induced by HGF.Results The expression of c-Met mRNA in LECs was significantly decreased in the experimental group,compared to that in the controls(P<0.01).Proliferation of LECs induced by HGF was inhibited,compared with the single HGF stimulated group(P<0.01).Conclusion The RNA interference targeting c-Met can effectively inhibit the expression of c-Met mRNA,and the proliferation of LECs induced by HGF.
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At present, mesenchymal stem cell treatment mainly applies in local and systematic transplantation, gene therapy combining stem cells, as well as tissue engineering. Based on mesenchymal stem cells are easily separated, cultured, have potentials of fast proliferation, easy exogenous gene's induction and expression, and can maintain multi-differentiation potential throughout long-term culture in vitro, and the quite stable genetic background. Mesenchymal stem cells have widely application prospect in tissue engineering of ophthalmology. At present, domestic and foreign researches have had massive evidences that mesenchymal stem cells can differentiate into cornea and retina cells. The application of mesenchymal stem cells in the anterior eye is usually by dint of a kind of carrier, such as amniotic membrane, fibrin gel membrane and so on. Moreover, the temperature sensitive culture dish has made the non-carrier transplantation of ocular surface stem cells. The application of mesenchymal stem cells in the posterior eye mostly concentrates in seeking for the feasible treatment of retinitis pigmentosa. Researches are mature on mesenchymal stem cells as seed cells acting on damaged tissue in ocular repairs and regeneration. But its oncogenicity and the security are waiting for further solving.
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<p><b>OBJECTIVES</b>To observe the expression of betaig-h3 in normal cornea and keratoconus and to elucidate the role of extracellular matrix in keratoconus.</p><p><b>METHODS</b>In situ hybridization was used to detect the expression of betaig-h3 in the cornea. The cDNA library was screened with human betaig-h3 cDNA probe to locate betaig-h3 mRNA in cells.</p><p><b>RESULTS</b>Expression of betaig-h3 was found mainly in the stroma of the normal cornea and keratoconus, but decrease depending on the degree of keratopathy. In some serious cases, no expression signal was detected. The strongest expression was seen at the border of the normal region and keratoconus.</p><p><b>CONCLUSIONS</b>betaig-h3, the structural component of the extracellular matrix, can affect cell adhensiveness in the development of corneal fibrous interstitial organization. During the development of keratoconus, decreasing levels of betaig-h3 cause the diminution of corneal steadiness, which is related to formation of keratoconus.</p>