The amniotic membrane has been shown to have anti-inflammatory,anti-fibrotic,anti-angiogenic properties and ability to provide a substrate for the growth of corneal and conjunctival epithelial cells,and it is an ideal material for ocular surface reconstruction.The treating patten of corneal and ocular surface diseases has changed due to the widespread using of amniotic membrane transplantation,but there are a lot of problems in the application of amniotic memebrane in clinic,such as how to grasp indications and reduce the failure rate,how to apply individual skill for different patients with corneal disease,and how to observe postoperative complicationsm and reasonably use medicine after operation.This paper give some personal experience and opinion in orde to achieve better effects in treatment of corneal and ocular surface diseases using amniotic membrane transplantation.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that zonula occludens-1 (ZO-1) can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds (15 s group) or 40 seconds (40 s group).CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR (RT-PCR) between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2％ hyaluronic acid (HA),antiZO-1 neutralizing antibody (10 mg/L) +0.2％ HA and mouse hypoxia inducible factor-1α (HIF-1α) recombinant protein (5 mg/L)+0.2％ HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group (x2 =6.032,P=0.049),and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group (1.15±0.08 versus 1.53±0.04) (t=4.157,P=0.014).CD31 positive cell number was more and the staining area was larger in the ZO-1 antibody group and HIF-1α positive control group than those in the 0.2％ HA group (cells:t=-129.590,-226.820,both at P=0.000;area:t =-5.310,-8.840,both at P=0.000).The relative expressions level of vascular endothelial growth factor (VEGF) mRNA was 1.33±0.10 and 1.46±0.11 in the ZO-1 antibody group and HIF-1 α positive control group respectively,which were significantly higher than 0.93±0.06 of the 0.2％ HA group (t =-5.820,-7.284,both at P =0.000).The percentages of positive cells in the ZO-1 antibody group and HIF-1α positive control group were significantly increased in comparison with the 0.2％ HA group for F4/80 (t =-16.750,-17.480,both at P =0.000) and for Ly-6G (t =-21.450,-27.680,both at P=0.000).Conclusions Alkali burn induced CNV downregulates the expression of ZO-1 mRNA.Administration of ZO-1 antibody causes the rise of VEGF mRNA in CNV and the infiltration inflammation cells,which suggests that the influence of ZO-1 on CNV is associated with the expression of VEGF.

Background Most anti-inflammation eyedrops are limited in clinical application owing to multiple adverse effects.A novel peptide GC31 derived from human thrombomodulin has a natural anti-inflammatory activity.Compared with conventional anti-inflammatory eyedrops,GC31 possesses more advantages and potential clinical transforming value.However,relevant study is still lack.Objective The purpose of this study was to evaluate the anti-inflammatory effect of GC31 and the possible mechanisms.Methods Sixty SPF male Wistar rats aged 8-10 weeks were randomized into 6 groups using randomized number table.Non-specific keratitis models were established in 40 rats by intrastromal injection of 10 μl of lipopolysaccharide (LPS) dissolved in PBS.Different doses of GC31 (125 μg or 250 μg) or dexamethason soluble in PBS were sunconjunctically injected in the experimental eyes respectively in the low dose GC31 group,high dose of GC31 group and the dexamethason group,and 10 μl of PBS was used in the same way in the PBS control group.No drug was injected in the model group,and the normal rats were employed as the blank control group.The corneas were examined by slit lamp microscope and were scored based on the criteria of Anand 24 hours after injection.Then the corneas were collected for histopathological examination.Expression of nuclear factor-κB (NF-κB) p65 in the corneas was detected using immunochemistry.Expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins were assayed using ELISA.Real-time PCR was used to detect the expressions of IL-6 mRNA and TNF-α mRNA.The use and care of the experimental animals followed Regulation for the Administration of Affair Concerning Experiment animals by State Science and Techonology Commission.Results A significant difference was seen in the ocular inflammatory scores among the six groups (F =301.238,P =0.000).The inflammatory scores were significantly lower in the high dose of GC31 group than those in the model group (1.85 ± 0.36 versus 2.90± 0.43) (t' =-5.144,P =0.000) ; and the scores in the dexamethason group was lower than those in the high dose of GC31 group(t' =-3.931,P=0.000).Infiltration of inflammatory cells in corneal tissue was milder in the high dose of GC31 and the dexamethason group compared with the model group.The positive response for NF-κB p65 was obviously weaker in the rat corneas in the low and high dose of GC31 groups and the dexamethason group in comparison with the model group.The contents of IL-6 and TNF-α proteins in the corneas were significantly reduced in the low and high dose of GC31 group and the dexamethason group compared with the model group (low dose group:t=-2.626,P=0.009;t'=-2.310,P=0.017.high dose group:t =-3.361,P=0.001 ;t'=-3.151,P=0.002),and the contents of IL-6 and TNF-α proteins in the dexamethason group were lower than those in the high dose of GC31 group (t=-3.361,P=0.001;t'=-3.360,P=0.000).In addition,the expression trend and compared results of IL-6 mRNA and TNF-α mRNA among the groups were similar to those of the IL-6 and TNF-α proteins (all at P＜0.01).Conclusions GC31 suppresses LPS-induced corneal inflammation response by downregulating the expression of inflammatory eytokines.The effect is more dominant in the doses of 250 μg than that in the doses of 125 μg.

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.

Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair.

Background Dry eye is increasing gradually recently,but its etiology and manifestation are very diverse.Studies showed that menopause of adult females was one of the risk factors of dry eye.In addition,some inflammatory factors also participate in the pathogenesis and development.But the study on the relationship of sex hormone with inflammation and ocular surface damage is still below understanding.Objective This study was to investigate the expressing changes of interleukin (IL) and tumor necrosis factor-α (TNF-α) in conjunctiva and the manifestation of ocular surface in ovariectomized rat model.Methods Twenty clear female SD rats were randomized into the ovariectomized group and the sham operative group according to randomized number table.Ovariectomy was performed in the ovariectomized group,and abdominal myotomy without ovariectomy was performed in the sham operative group.Serum estrogen and androgen levels were detected by radiation immunoassay 3 months after operation.Schirmer Ⅰ test (S Ⅰ t) and corneal fluorescence staining were carried out in the rats before operation and 1 month,2 and 3 months after operation.The morphology of conjunctival epithelial cells was examined by hematoxylin & eosin staining at the 3rd month after operation.The expressions of IL-17A,IL-1 β,IL-6 and TNF-α in conjunctiva were semi-quantitative analyzed by immunohistochemistry and Western blot.The use and care of the animals complied with State Science and Technology Commission Regulations for the Administration of Affair Concerning Experimental Animals.Results Serum estrogen levels were (23.53 ± 1.65) pg/L and (47.89 ± 1.05) pg/L 3 months after surgery in the ovariectomized group and the sham operative group,respectively; the serum androgen levels were (1.84±0.09) ng/L and (2.47±0.12)ng/L in the ovariectomized group and the sham operative group,respectively,showing a significant decline of serum estrogen and androgen levels in the ovariectomized group compared with the sham operative group (t=-35.37,-12.13,both at P＜0.01).No significant differences were seen in S Ⅰ t between the two groups among various time points (Fgroup =0.38,P =0.55 ; Ftime =0.13,P =0.72 ; Finteraction =0.39,P =0.76).No obvious fluorescence staining was seen in the cornea of both the ovariectomized group and the sham operative group.The histopathological examination showed that the layers of rat conjunctival epithelial cells increased with the disordered arrangement in the ovariectomized group.Immunochemistry showed that the expressions of IL-17A,IL-1β,IL-6 and TNF-α (A values) were significantly higher in the ovariectomized group than those in the sham operative group (IL-17A:t=8.22,P＜0.01 ;IL-1β:t=16.43,P＜0.01 ;IL-6:t=13.44,P＜0.01 ;TNF-α:t=16.26,P＜0.01).Western blot assay showed the similar results (IL-17A:t=19.41,P＜0.01 ;IL-1β:t=12.63,P＜0.01 ;IL-6:t=17.17,P＜0.01 ;TNF-α:t=15.19,P＜0.01).Conclusions Serum estrogen and androgen levels drop obviously,and there is an up-regulation of IL and TNF-α expression in conjunctiva tissue in the ovariectromized SD rats.However,no obvious dry eye-related sign occurs.

Background Posterior capsular opacification (PCO) following the extracapsular extract of cataract is associated with the proliferation and migration of residual lens epithelial cells (LECs).Study showed that the incidence of PCO is higher in diabetic patients than those of non-diabetes.So if insulin-like growth factor-1 (IGF-1) participates in the pathogenesis of PCO deserve research.Objective This study was to explore the active mechanism of IGF-1/IGF-1 receptor (IGF-1 R) system in the migration of LECs and offer theoretical basis for clinical prevention and treatment of PCO.Methods Human lens epithelial cell lines (HLEC-B3) were cultured and passaged in DMEM.The cells were identified using fluorescence immunocytometry.IGF-1 with the concentrations of 0,30,90 μg/L were added into the medium separately for 48 hours.The numbers of migrated cells were calculated by Transwell test.The cells were cultured in DMEM containing 0,1.5,30,60,90 μg/L IGF-1,and the expressions of IGF-1 Rα and IGF-1Rβ in the cells were assayed and compared by Western bolt.Results The cultured showed the positive response for α-crystallin anibody with red fluorescence in the cellular membrane.Twelve hours after Transwell incubation,the number of migrated cells (Median) was 0(0,1),10(10,11) and 29(27,31) in the 0 μg/L IGF-1 group,30 μg/L IGF-1 group and 90 μg/L IGF-1 group,respectively,showing a significant difference among the 3 groups (Z=12.610,P=0.002).The number of migrated cells in the 30 μg/L IGF-1 group and 90 μg/L IGF-1 group was significantly more than that of the 0 μg/L IGF-1 group (both at P =0.008),and the number of migrated cells in the 90 μg/L IGF-1 group was significantly more than that of the 30 μg/L IGF-1group (P =0.009).Western blot assay showed that the expressions of IGF-1Rα and IGF-1Rβ in the cells were significantly different among the 0,1.5,30,60,90 μg/L IGF-1 groups (F=63.700,130.530,both P =0.000).The expressions of IGF-1 Rα and IGF-1Rβ were gradually elevated as increase of IGF-1 doses when then concentration of IGF-1 was ＞ 30 μg/L,with significant differences among the different concentrations groups (all at P＜0.05).Conclusions IGF-1 can upregulate the expressions of IGF-1R in HLEC-B3 cells in vitro in a dose-dependent manner.Also,IGF-1 enhances the migration ability of HLEC-B3 cells.These results suggest that activation of IGF-1/IGF-1R system may be associated with the pathogenesis of PCO.

Background The primary pathologic mechanism of posterior capsular opacification (PCO) is proliferation,migration and epithelial-mesenchymal transition (EMT) of residuary lens epithelial cells (LECs) after cataract extract surgery.Researches showed that MG132,a proteasome inhibitor,can attenuate the proliferation of bovine LECs,but its effect on human LECs remains unclear.Objective This study was to investigate the inhibitory effect of MG132 on proliferation,migration and differentiation of human LECs in vitro.Methods Human lens capsule were collected during the surgery.Human LECs were primarily cultured by explant method and passaged.The second or third generation of cells were incubated to 96-well plates at the density of 5×105/ml (200 μl/well) for 24 hours.Fibroblast growth factor-2 (FGF-2,10 mg/L),MG132 (10 μmol/L) or MG132+FGF-2 was added into the culture medium for 24 hours separately,and regular cultured cells served as the control group.The proliferation value (absorbance,A490) of the cells was assayed by MTT colorimetric method.A bare area was made by a sterile cotton swab in the cell layer,and migrated cell number in the blank zone was counted to evaluate the migration ability of the cells after 24 hours.Transforming growth factor-32(TGF-β2),MG132 or MG132+TGF-β2 was added into the culture medium for 24 hours separately,and the expression of fibronectin (FN) in the cells was detected using immunochemistry.Results The proliferation values (A490) of the cells were 0.582±0.020,0.723±0.010,0.434± 0.011 and 0.465±0.008 in the control group,FGF-2 group,MG132 group and MG132 + FGF-2 group,respectively,showing a significant difference among the groups (F =110.482,P＜0.01).The A value was significantly higher in the FGF-2 group and lower in the MG132 group and MG132+FGF-2 group than that of the control group (all at P＜ 0.05).The migrated cell number was 8.67 ± 1.08,11.58 ± 1.59,2.67 ± 0.09 and 2.75 ± 0.09 in the control group,FGF-2 group,MG132 group and MG132+FGF-2 group,respectively,with a significant difference among the groups (F=34.301,P＜0.01),and more cells in the blank zone were seen in the FGF-2 group and less cells were in the MG132 group and MG132+FGF-2 group in comparison with the control group (all at P＜0.05).Compared with the control group,the proliferative rate and migrating rate of the cells declined by 25.4％ and 75.0％ in the MG132 group as well as 20.1％ and 68.3％ in the MG132+FGF-2 group,but in the FGF-2 group,they increased by 24.2％ and 33.6％.The expressing levels (A value) of FN in the LECs were 1.242±0.023,2.329±0.113,1.043 ±0.021 and 1.163±0.018 in the control group,TGF-β2 group,MG132 group and MG132 +TGF-β2 group,respectively,with a significant difference among the groups (F =113.752,P＜0.01),a considerably increased expressing value was seen in the TGF-β2 group and decreased value was in the MG132 group and MG132+TGF-β2 group when compared with the control group (all at P＜0.05).Conclusions MG132 can effectively inhibit the proliferation,migration and differentiation of human LECs in vitro.

Background Cataract was directly associated with the damage to the structure and function of lens epithelial cells (LECs).In those patients who suffer from cataracts,morphologic changes of LECs is the most compelling evidence confirming loss of cellular structure and function of LECs.So,learning about the morphological changes of LECs of the different types of cataracts is very important for study on biological behaviors of LECs in different environments or diseases.Objective This study was to evaluate the morphological and ultrastructural changes of the LECs in different types of cataracts.Methods Anterior capsular member from age-related cataracts,diabetic cataracts and high myopia complicated cataract were obtained during the cataract surgery and 15 pieces for each.Trypan blue and alizarin red (TB-AR) stain,haematoxylin and eosin stain were performed in the samples to assess the viability and morphology of LECs.The ultrastructural change of LECs was observed under the transmission electron microscope.The features of the LECs were compared among the different types of cataract.Results TB-AR stain showed that LECs were polygon in shape with the mosaic arrangement and round cell nucleus,and a few dead cells were seen in the samples age-related cataract.In the diabetic samples,LECs largened from swelling with different sizes.More dead cells were found in the high myopia complicated cataract.Haematoxylin and eosin stain exhibited that the anterior capsular membrane presented a homogeneuous membrane,and monolayer LECs attached firmly anterior capsular membrane in the samples of related cataract.Majority of the cells had the intact structure.However,the interspaces between cells and capsular membrane were found in diabetic cataract.Also,smaller LECs were seen in high myopia complicated cataract with the irregular cell morphology.Under the transmission electron microscope,LECs presented the normal shape,intact intercellular tight junction and well attachment between cells and capsular membrane in the samples of the age-related cataract.In the samples of the diabetic cataract,edema of LECs and large intercellular spaces were seen.In addition,the jogged pump and vacuolar degeneration of cytoplasm were revealed in the high myopia complicated cataract.Conclusions The degeneration,necrosis and apoptosis was a common pathological basis of age-related cataract,diabetic cataract and high myopia complicated cataract.However,the damage of LECs was more serious in diabetic cataract and high myopia complicated cataract than that of agerelated cataract.

Background Mitomycin C (MMC) is still irreplaceable until now.While it's current administration method has proved less than effective in the treatment of refractory glaucoma.To construct a MMC sustained release system which can maintain effective concentration and reduce toxicity is important for postoperative scarring regulation after glaucoma filtration surgery.Objective To evaluate the postoperative effect of use PTMC-F127-PTMC thermosensitive hydrogel as a new drug delivery carrier to sustained release MMC in rabbit trabeculactory.Methods Sixty rabbits,aged 10 to 14 weeks,were divided into 5 trabeculectomy groups in accordance with the random number table,including surgery only group,blank PTMC-F127-PTMC group and three sustained groups with 0.1 ml PTMC-F127-PTMC loaded with 0.05,0.10,0.20 g/L MMC injected after surgery.The MMC concentration of anterior chamber aqueous in three sustained release group with 0.1 ml PTMC-F127-PTMC loaded with 0.05,0.1,0.2 g/L MMC injected after surgery were tested by Guangzhou Analysis and Testing Center using high performance liquid chromatograph-mass spectrometer (HPLC-MS).At 3,7 days postoperatively,0.1 ml aqueous humor from 2 random selected rabbits in each group was extracted using 1 ml syringe with 30G needles from corneal limbus.At postoperative 1,3,5,7,10,14,28 days,bleb width and depth were calculated with caliper measurements and height was graded semiquantitatively by slit-lamp examinations,intraocular pressure (IOP) was measured with Tonopen.And corneal endothelial cell densities were examined by corneal endothelial counting before and 28 days after surgery.Sequential sections of the operative region were prepared and stained with hematoxylin and eosin and proliferating cell nuclear antigen (PCNA) after taking off the eyeballs from dead rabbits at 28 days later.Results MMC could be sustained released from PTMC-F127-PTMC hydrogel for more than 20 days.The mean postoperative bleb survival time in trabeculectomy surgery only group,blank hydrogel group and three sustained release groups were (5.3 ± 0.4),(5.5 ± 0.4),(12.2 ± 1.0),(25.1 ± 0.9),(26.7 ± 0.7) days respectively,the difference between each group was significantly (F =123.200,P =0.000).0.05 g/L MMC sustained release group has a better bleb survival time than that of surgery only group and blank hydrogel group (P =0.000).Compared with other groups,0.10 g/L and 0.20 g/L MMC sustained group has the longest bleb survival time (P =0.000),and more obvious IOP downtrending (F=53 010.000,P＜0.01).But the difference between the two groups was not significant.There was no difference in cornea endothelia cells counts between each group and no MMC was detected in aqueous humors.Histopathology test shows that the inflammatory response and fibrosis were lighter in MMC sustained release group,with stronger proliferation inhibition.Conclusions PTMC-F127-PTMC thermosensitive hydrogel can be a new drug delivery carrier to sustained release MMC.Sustain release MMC can extent bleb survival time with low toxicity.