ABSTRACT
Objective:To explore the difference in the proliferation, migration and ultraviolet radiation effect between double-head and unilateral pterygium fibroblasts (HPFs) cultured in vitro. Methods:Pterygium tissue was obtained from patients who underwent pterygium excision with conjunctival transposition from March 2017 to March 2018 in the Second Affiliated Hospital of Guangzhou Medical University.Nineteen cases with bilateral pterygium and 19 cases with unilateral pterygium were selected for this research.Twelve cases of normal conjunctival tissue were obtained from donor eyes.The fibroblasts were divided into HPFs-nasal (HPFs-N), HPFs-temporal (HPFs-T), unilateral HPFs and human conjunctiva fibroblasts (HCFs), which was taken from the nasal side of the bilateral pterygium, the temporal side of the bilateral pterygium, the unilateral pterygium and the normal conjunctiva, respectively.Human pterygium and normal conjunctival fibroblasts were isolated and cultured by tissue culture method.The fibroblasts were divided into an ultraviolet irradiation group and a normal light illumination group.The cell growth curve was detected by methyl thiazolyl tetrazolium (MTT) assay.The cell scratch healing rate was detected by the cell scratch test after 48 hours.The expression of α-smooth muscle actin αwas detected by immunofluorescence staining, and the number of positive cells in each group was compared.The fibroblasts cultured in vitro were irradiated with ultraviolet irradiation, and the cell scratch healing rate, growth curve and expression of α-SMA were observed.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Guangzhou Medical University.Written informed consent was obtained from each subject. Results:The pterygium and conjunctival fibroblasts were spindle shaped, and the growth rate was gradually increased from day 1 to day 7.The growth rate of HPFs-N was the fastest, and the growth rate of HCFs was the slowest.The growth rate of the four types of fibroblasts was significantly increased after exposure to ultraviolet.There were significant differences in the cell scratch healing rates and α-SMA positive cell expression rates among the four fibroblast types under different lighting conditions ( Fgroup=158.064, P<0.05; Fcell type=326.582, P<0.05. Fgroup=4.731, P<0.05; Fcell type=172.813, P<0.05), of which the scratches healing rate in the HPFs-N cells after 48 hours under the normal light conditions was (79.67±0.86)%, which was significantly higher than HPFs-T ([54.04±0.33]%), unilateral HPFs ([64.12±0.21]%) and HCFs ([58.86±0.41]%), the α-SMA positive cell expression rate in the HPFs-N cells after 48 hours under the normal light conditions was (28.87±1.02)%, which was significantly higher than that in HPFs-T ([13.67±0.23]%), unilateral HPFs ([20.35±1.72]%) and HCFs ([5.12±0.45]%) (all at P<0.05); the cell scratch healing rates and α-SMA positive cell expression rates were significantly increased in the ultraviolet irradiation group than those in the normal light illumination group (all at P<0.05). Conclusions:The nasal side of double-head pterygium fibroblasts is more proliferous and more migratory than that of the unilateral pterygium, the expression of α-SMA is also increased, which can be further enhanced by ultraviolet irradiation.
ABSTRACT
Objective To observe c-Fos expression in visual cortex of infant rhesus monkeys with myopia induced by hyperopic defocus and preliminarily investigate the possibility of visual cortex participating in myopia. Methods Eight SPF grade healthy infant rhesus monkeys aged 20 to 30 days were randomly divided into hyperopic defocused group and control group,4 monkeys for each group. The monkeys in hyperopic defocused group wore -3 D spectacle lenses. The monkeys in control group wore 0 D lenses. The monkeys' refractive error,corneal topography, vitreous chamber depth were measured at the start of lens wear and at 2,4,6,8,12 weeks post-treatment. At 12 weeks post-treatment,the visual cortex tissues were removed for c-Fos protein measurement by immunohistochemistry and Western blot assays. The results were analyzed semiquantitatively to compare the differences of c-Fos expression between hyperopic defocused group and control group. The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. This study protocol was approved by Ethic Committee of Zhongshan Ophthalmic Center ( No. 2013-014). Results After 12 weeks'lens wear,the vitreous chamber elongation amplitude of hyperopic defocused group monkeys was more obvious than that of the control group ([0.93±0.24]mm vs. [0.72±0.09]mm;t=2.292,P=0.047). The decrease of hyperopic degrees of hyperopic defocused group monkeys was more obvious than that of the control group ([-3.23± 1.36]D vs. [-1.55±0.52]D;t=-3.273,P=0.006). The eyes of hyperopic defocused group monkeys appeared a remarkable myopic shift after treatment. The number of c-Fos immunoreactive neurons was less in the hyperopic defocused group than that in the control group,with a statistically significant difference between them ([1 843±191]/mm2vs. [2 296±503]/mm2;t=2.381,P=0.041). Western blot assay showed that the optical density of c-Fos protein in the hyperopic defocused group was significantly less than that in the control group (0.50±0.17 vs. 0.99± 0.22;t=-4.982,P<0.01). Conclusions Hyperopic defocus,as an abnormal visual stimulus,can induce the onset of myopia in infant rhesus monkeys and inhibit c-Fos expression in visual cortex. Visual cortex may participate in myopia induced by hyperopic defocus.
ABSTRACT
Objective To explore the clinical effect of lacrimal endoscope assisted minimally invasive lacrimal duct recanalization combined with silicone tube intubation in the treatment of lacrimal duct obstruction. Methods 3 cases(4 eyes)of the superior canaliculus obstruction,5 cases(6 eyes)of the inferior canaliculus,20 cases (29 eyes) of common canaliculus and 25 cases (36 eyes) of nasolacrimal duct were enrolled. They were randomly divided into two groups:Group A(40 eyes)treated with lacrimal endoscope assisted laser lacrimal forming combined with silicone tube implantation and Group B(36 eyes)with lacrimal endoscope assisted laser lacrimal forming combined with silicone tube implantation. The cases were all followed up for 3-6 months after treatment. Results Except the intraoperative bleeding,there were no significant differences in the incidence of various complications. All the eyes could be recanalized under the aid of lacrimal endoscope. The total effective rates were 87.50% and 91.43% in Group A and B,respectively. The difference was not statistically significant. Conclusion Lacrimal endoscope assisted minimally invasive lacrimal duct recanalization combined with silicone tube intubation is a safe,effective,and minimally invasive method to recanalize the lacrimal obstruction.
ABSTRACT
Objective To explore the clinical effect of lacrimal endoscope assisted minimally invasive lacrimal duct recanalization combined with silicone tube intubation in the treatment of lacrimal duct obstruction. Methods 3 cases(4 eyes)of the superior canaliculus obstruction,5 cases(6 eyes)of the inferior canaliculus,20 cases (29 eyes) of common canaliculus and 25 cases (36 eyes) of nasolacrimal duct were enrolled. They were randomly divided into two groups:Group A(40 eyes)treated with lacrimal endoscope assisted laser lacrimal forming combined with silicone tube implantation and Group B(36 eyes)with lacrimal endoscope assisted laser lacrimal forming combined with silicone tube implantation. The cases were all followed up for 3-6 months after treatment. Results Except the intraoperative bleeding,there were no significant differences in the incidence of various complications. All the eyes could be recanalized under the aid of lacrimal endoscope. The total effective rates were 87.50% and 91.43% in Group A and B,respectively. The difference was not statistically significant. Conclusion Lacrimal endoscope assisted minimally invasive lacrimal duct recanalization combined with silicone tube intubation is a safe,effective,and minimally invasive method to recanalize the lacrimal obstruction.
ABSTRACT
Background Researching the pathological characteristics and components of cells in internal limiting membrane of idiopathic macular hole (IMH) has an important clinical significance for the prevention of IMH.However,the study results are still disputable.Objective This study was to investigate the histopathological features of internal limiting membrane of IMH and the types of cells inside it, and explore the pathomechanism of IMH.Methods Seven specimens of internal limiting membrane were obtained during the vitrectomy with IMH patients in the Second Affiliated Hospital of Guangzhou Medical University from February 2012 to August 2013 under the informed consent of patients.The histopathological examination was performed for the structural observation and cellular identification of internal limiting membrane.The expression and location of glial fibrillary acidic protein (GFAP), CD45 and CD44 in internal limiting membrane were examined by using immunochemistry and immunofluorescence technology.Results All the seven specimens showed continuous undulating membrane with red staining.Two specimens appeared to be uniform in thickness and few cells were distributed in the specimens.The internal limiting membranes were uneven in thickness in the other specimens with retinal pigment epithelial cells,neuroglia cells,fibrocytes,macrophages and lymphocytes in them.Immunochemistry showed the positive expression of GFAP in the outer layer of the specimens.CD45 positive cells were detected in the internal limiting membranes, and CD44 was detected in the inner layer of the specimens.Conclusions Few cells exist in the internal limiting memranes of IMH.However,neuroglia cells and CD45 positive cells emerge in the internal limiting memranes of stage 3 or above IMH eyes, indicating the proliferation of cells and immuno-inflammation response exist during the IMH development.The up-regulation of CD44 expression promotes inflammatory response of internal limiting memranes.
ABSTRACT
Background The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells (ESCs) micro-environment enhance the stemness of human limbal stem cells (LSCs),but its mechanism has not been elucidated.Objective This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs.Methods Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20+20％ ES culture supernatant (ESC-CM),respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase (TERT) siRNA (19-25nt siRNA) or siRNA (sc-37007) was transfected into the cells of ESCCM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry,and the expressions of telomerase and reactive oxygen species (ROS) in TERT siRNA-or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR,immunofluorescence staining and Western blot were employed to determine the expressions of p63,ATP-binding cassette transporer G2 (ABCG2),integrin β1 mRNA and proteins and cytokeratin 3 (C K3) in the cells.The levels of focal adhesion kinase (FAK),Akt,glycogen synthase kinase 3β (GSK3β) and p21 protein and phosphorylation proteins in the cells were detected by Western blot.Results The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency (CFE) of (7.6±0.6) ％ in ESC-CM group,but the cells to the sixth generation with the CFE of (5.6±0.6)％,showing a significant difference between them (t =4.454,P =0.011).The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group (all at P＜0.05).The apoptotic rate of the cells was (7.67± 1.31)％ in the siRNA-F transfected group,which was significantly lower than (32.33 ±3.13)％in the siRNA-TERT transfected group (t =-12.588,P =0.000).No significant differences were seen in the expression levels of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the primary cells between the ESC-CM group and the CnT-20 group (all at P＞0.05),but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).However,the expressions of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).The telomerase activity was (4.83±0.67) ％ in the siRNA-TERT transfected group,which was significantly lower than (46.71±1.22) ％ of the siRNA-F transfected group (t =52.116,P =0.000).The expression of pFAK,pAkt,pGSK3β proteins were weakened,but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor,GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P＜0.01),and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P＜0.01).Conclusions ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways.
ABSTRACT
Background Recent studies indicated that human amnion mesenchymal stem cells (hAMSCs) can be induced to differentiate into multiple types of cells in vitro,but whether the hAMSCs can differentiate into ocular surface cells has not been reported yet.Objective This study was to investigate the feasibility of inducing differentiation of hAMSCs into ocular surface cells by co-culturing with human bulbar conjunctiva fibroblasts (hBCFs).Methods This study was approved by Ethic Committee of Affiliated Second Hospital of Guangzhou Medical University.HAMSCs were isolated from placenta under the informed consent of healthy delivery women.hAMSCs were cultured,passaged and identified by detecting the expressions of CD44,CD45,CD73,CD90 in the cells with flow cytometer,osteogenesis and adipogenic differentiation experiments.Human conjunctival tissue was obtained during the eye operation under the informed consent of patients and hBCFs were isolated and cultured with explant culture.The cells were divided into the hAMSCs culture group and the hAMSCs and hBCFs co-culture group and cultivated in Transwell chambers for 7 days.The expressions of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in the cells were assayed by immnofluorescence technique.Results Cultured hAMSCs showed the slender shape and cell body enlarged with passage.CD44,CD73 and CD90 were expressed in the cells,and the expression of CD45 was absent.After 3-4 weeks of osteogenesis and adipogenic induce,the cells showed red staining for alizarin and oil red O.In the co-culture group of hAMSCs and hBCFs,hAMSCs presented the epithelioid cell-like in shape and showed the positive response for CK19 and weaker response for α-SMA.However,in the hAMSCs culture group,the cells showed the positive response for α-SMA and absent response for CK19.Conclusions The hAMSCs can differentiate into ocular surface cells after being induced by hBCFs.And the differentiation mechanism is possibly relevant to mesenchymal cells epithelium.
ABSTRACT
Internet based problem-based learning (PBL) mode was used in teaching of ophthalmology in order to increase the quality of ophthalmology teaching and to raise the student's ability of problem solving.The method and requirements of teaching were discussed based on the teaching schema.Examples of cataract teaching using internet based PBL mode was performed.The characteristics and merit of the mode were summarized in the end.