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Due to the serious shortage of corneal donor, the development of penetrating keratoplasty and corneal endothelial transplantation is severely restricted in clinical practice.The root cause is the limited proliferation capacity of healthy corneal endothelial cells.With the continuous development of tissue engineering technology and cell engineering technology, the research of tissue-engineered cornea has made some progress. In vitro culture of corneal endothelial cells with high density and healthy endothelial function for transplantation is a hot topic in current tissue engineering research.The keys of tissue-engineered corneal endothelial technology include seed cells, vector materials and the strategy of cell transplantation.At present, many research teams domestic and abroad have reported that the source of seed cells includes human corneal endothelial cells, stem cells, vascular endothelial cells and human amniotic epithelial cells.Vector materials include amniotic membrane, acellular corneal stroma, posterior elastic layer, anterior capsular membrane and various biomaterials.The cultured cells are transplanted by penetrating keratoplasty, corneal endothelial transplantation or anterior chamber injection.This review summarized the latest progress in the research on the source of corneal endothelial seed cells, the selection of vectors and the methods of corneal endothelial transplantation, and summed up the problems faced in the current research and looked forward to its prospects.
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Objective:To investigate the role of nicotinamide (NIC) in the differentiation of neural crest cells from human embryonic stem cells (hESCs), and lay the foundation for the induction of hESC-derived corneal endothelial cells.Methods:hESCs line H1 cultured for 5-7 days was used for induction.According to the different components of the neural crest induction medium, cells were assigned into different groups for 7-days induction, including group treated without NIC cultured in induction medium only, group treated with NIC cultured in induction medium containing 10 mmol/L NIC, NIC+ resveratrol (Res) group cultured in induction medium containing 10 mmol/L NIC and 10 μmol/L Res and Sirtinol group cultured in induction medium containing 10 μmol/L Sirtinol.Res and Sirtinol were used as SIRT1 activity agonist and inhibitor, respectively.The relative mRNA expression levels of hESCs and neural crest cell markers were detected by real-time fluorescence quantitative PCR at 1, 3, 5 and 7 days during the induction.The expression of neural crest cells markers after 7 days of induction was assayed by immunofluorescence staining.The induction efficiency of NIC and the effect of SIRT1 regulation on human natural killer 1 (HNK-1) positive cells expression were evaluated through flow cytometry analysis of percentages of nerve growth factor receptor (P75) and HNK-1 + cells. Results:Compared with the group treated without NIC, the mRNA expressions of totipotent genes octamer transcription factor 4 (OCT4) and homeodomain proteins (NANOG) were significantly decreased, and the mRNA expression levels of neural crest cell markers P75, HNK-1, SRY-related HMG box (SOX) 9 and SOX10 were significantly increased in the group treated with NIC after 5 days of induction (all at P<0.05). In the group treated without NIC, P75 was weakly expressed, and HNK-1 was sporadically expressed, and transcription factor AP-2β (AP-2β) and paired-like homeodomain transcription factor 2 (PITX2) were not detected.In the group treated with NIC, P75, HNK-1, AP-2β and PITX2 were strongly expressed.The proportion of P75 + HNK-1 + cells and P75 + cells were both significantly higher in the group treated with NIC than without NIC ( t=8.481, P=0.001; t=2.987, P=0.041). The percentage of HNK-1 + cells in groups treated without and with NIC, NIC+ Res group and Sirtinol group were (34.267±12.522)%, (89.633±1.358)%, (64.667±6.429)% and (86.300±3.460)%, respectively, with a statistically significant overall difference ( F=36.799, P<0.001). The proportion of HNK-1 + cells in NIC+ Res group was significantly lower than that in the groups treated with NIC and Sirtinol (all at P<0.05). Conclusions:NIC promotes the differentiation of hESCs-derived neural crest cells by inhibiting the activity of SIRT1 to enhance the expression of HNK-1.NIC treatment may provide a new strategy for source of seed cells in the treatment of neural crest cell-related diseases, such as corneal endothelial transplantation.
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Diabetic keratopathy is one of the common ocular complications of diabetes, and diabetic patients are often accompanied by changes in the morphological structure of the corneal endothelium.Oxidative stress, inflammation, apoptosis, glucose metabolism disorders, mitochondrial injury, and endoplasmic reticulum stress are the main mechanisms of the occurrence and progression of diabetic keratopathy.Studies have shown that advanced glycation end products can activate and induce the formation of a large number of reactive oxygen species (ROS), which in turn causes cell damage and even apoptosis.Mitochondria are the source of ROS, which will be damaged when a large amount of ROS accumulate, and mitochondrial autophagy will be formed when the body removes damaged mitochondria.Mitophagy refers to the process of eliminating aging, dysfunctional, damaged mitochondria through selective autophagy, which is a key mechanism for mitochondria to maintain function.The decrease in the level of mitophagy will lead to the destruction of the hexagonal structure of the diabetic corneal endothelium and its dysfunction, and upregulating the level of mitophagy can play a protective role on corneal endothelium in oxidative stress.The role of mitophagy in diabetic corneal endothelial lesions were reviewed in this article.
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Diabetic keratopathy is a chronic complication of diabetes caused by abnormal metabolites accumulation, oxidative stress, abnormal inflammation and corneal neuropathy.It can result in delayed corneal epithelial healing and decreased corneal sensitivity under the stimulation of ocular trauma or surgery which bring great challenges to clinicians.Activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory is one of the factors that cause chronic complications of diabetes, and is also an important factor for delaying the healing of diabetic wounds.The NLRP3 inflammatory signaling pathway is closely related to corneal oxidative stress, delayed epithelium healing and development of corneal neuropathy.In this paper, the research status and prospects of NLRP3 inflammatory signaling pathway and diabetic keratopathy were reviewed to provide new ideas for studying the mechanism and treatment of diabetic keratopathy.
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Objective:To explore the promoting effects of slit guidance ligand 2 (Slit2) on the repair of corneal epithelium and nerve damage in diabetic mice and possible molecular mechanism.Methods:Sixty SPF C57BL/6 mice aged 5-6 weeks were divided into normal control group, diabetes model group and Slit2 injection group according to the random number table method, 20 for each group.Diabectic model was prepared by intraperitoneal injection of streptozotocin in the diabetes model group and Slit2 injection group.A mouse corneal epithelial injury repair model was established using electric epithelial scraper, and Slit2 recombinant protein was subconjunctivally injected immediately following modeling in the Slit2 injection group.The equal volume of phosphate buffer saline (PBS) was used in a same way in the diabetes model group.No intervention was performed in the normal control group.Corneal epithelial healing were examined at 24, 48 and 72 hours after corneal epithelial defect by corneal fluorescin staining.Real-time fluorescent quantitative PCR was used to detect the expression of Slit2 and its related receptors in the corneal epithelium of normal and diabetic model mice.Fluorescence staining of corneal wholemount with β-tubulin Ⅲ was used to observe the changes in corneal nerve morphology.Immunofluorescence staining was performed to detect the expression and distribution of Slit2 in mouse corneal epithelium in normal control group and diabetes model group, as well as the expression and distribution of Slit2, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), threonine protein kinase (AKT), β-catenin and Ki67 in the healing corneal epithelium of mice after corneal epithelium damage in different groups.The mouse corneal epithelial stem/progenitor cell line (TKE2) was divided into normal control group, high-glucose group and Slit2 treatment group.Western blot was performed to detect the expression of p-EGFR/EGFR and p-AKT/AKT in the TKE2 of the three groups.The expression of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 with 0.01, 0.1 and 0.5 μg/ml Slit2 treatment for 10 minutes, and before and 10, 20, 30, 60, 120 minutes after 0.5 μg/ml Slit2 treatment was detected by Western blot.The effects of Slit2 on the axon regeneration of mouse trigeminal ganglion cells (TGs) were observed by immunofluorescence staining.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Qingdao Eye Hospital of Shandong First Medical University (No.[2020]57).Results:At 48 and 72 hours after corneal epithelial scraping, the speed of corneal epithelial repair was significantly slowed down in diabetes model group in comparison with the normal control group and Slit2 injection group.The relative expression levels of Slit2 and its receptors Robo1, Robo2 and Robo4 mRNA in the normal corneal epithelium in the diabetes model group were significantly higher than those of the normal control group (all at P<0.05). The fluorescence intensity of Slit2 in normal corneal epithelium in diabetes model group was similar to the normal control group, and the fluorescence intensity of Slit2 in damaged corneal epithelium in diabetic mice was significantly weaker than that in normal control group.Corneal nerve plexus was denser at 7 days after corneal epithelial injury and the nerve fibers were increased with more branches in Slit2 injection group compared with diabetic group.The fluorescence intensity of p-EGFR, p-ERK, β-catenin and Ki67 in damaged corneal epithelium in normal control group and Slit2 injection group was stronger than that of the diabetes model group.The relative expression levels of p-EGFR/EGFR, p-AKT/AKT, and β-catenin in TKE2 in high-glucose group were significantly lower than those in normal control group and Slit2 treatment group (all at P<0.05). The relative expression levels of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 after Slit2 treatment were significantly increased in comparison with before Slit2 treatment (both at P<0.05), and the relative expression levels of p-EGFR/EGFR and p-AKT/AKT in TKE2 were elevated as the increase of Slit2 concentration.The activation effect of 0.5 μg/ml Slit2 on EGFR and AKT pathways was most obvious.The synapse length of TGs cultured by high glucose was (40.52±5.44) μm, which was significantly shortened than (72.14±9.48) μm in normal control group and (73.04±4.66) μm in Slit2 injection group (both at P<0.05). Conclusions:Slit2 can protect the corneal epithelium by activating EGFR signaling pathway and play a protective role to neurons by increasing the density of corneal subepithelial plexus and promoting the growth of TGs axons in diabetic mice.
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Diabetic keratopathy (DK) is a common ocular complication of diabetes.Long-term hyperglycemia impairs many structures of the cornea, leading to corneal opacity and visual dysfunction.A large number of researches focus on the epithelium and nerve abnormities in DK, but the pathogenesis is not completely elucidated.Dendritic cells (DCs) are specialized antigen-presenting cells, linking innate and adaptive immunity, participating in the occurrence and development of diabetes and its complications.To date, there are many myths in relationship between DCs and DK to be solved, and there are a few researches that investigate the relation between DCs and the occurrence and development of diabetes.In this article, the pathogenesis and pathogenic changes of DK, the types and functions of different DCs, the relationship between DCs and chronic inflammation and delayed healing of corneal epithelium in DK, as well as the role of DCs in corneal neuropathy were reviewed in order to provide some references for further investigations about the pathogenesis and treatment of DK.
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Recently, an increasing number of studies on the insulin-like growth factor (IGF) system have been conducted.In addition to its well-known regulation of cell proliferation and differentiation, enhanced metabolic activity, anti-apoptosis, and promotion of cell survival, the role of IGF in tumor growth, autophagy, longevity and aging, and oxidative stress has become a hot spot.Drugs targeting IGF signaling pathways are also progressing into the clinical experimental phase.This article summarized the important role of IGFs and its major signaling pathways in the growth and development, their relationship with the corneal diseases, and discussed the related problems and prospects.
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Objective To explore the occurring and developing characteristics of dry eye syndrome in type 1 diabetic mouse model induced with streptozotocin (STZ)-intraperitoneal injection.Methods Completely randomized design method was performed.Sixty SPF degree male C57BL/6 mice (6-8 weeks old) was randomly divided into diabetic group and control group,which were intraperitoneally injected with citrate buffer and STZ-citrate buffer (50 mg/kg per day),respectively.The average weight,blood glucose level and lacrimal gland weight were examined before injection and 1 month,2 months,4 months after the last injection;meanwhile,phenol cotton thread and rose bengal staining methods were used to check tear formation and ocular surface condition;corneal perception meter was used to test corneal sensitivity;periodic acid-schiff (PAS) staining method was used to test the density of conjunctival goblet cells;histopathological staining and Masson staining methods were used to test the tissue changes of lacrimal gland.Results Compared with before injections,the body weight and lacrimal gland weight in diabetic group were not significantly changed 1 month,2 months and 4 months after injection (all at P> 0.05),but these measurements in diabetic group 1 month,2 months and 4 months after injection were significantly lower than those in control group at corresponding time points (all at P<0.05).Compared with before injections and control group at corresponding time points,the blood glucose level were dramatically higher and the tear formation were significantly decreased in diabetic group at 1 month,2 months,4 months after injection (all at P<0.05).The ocular surface of diabetic model mice showed positive rose bengal staining 2 months after STZ injections.The corneal sensitivities were significantly lower in diabetic model mice 2 months and 4 months after injection than those before injection and in control group at corresponding time points (all at P<0.05).The density of conjunctival goblet cells in diabetic group 4 months after injection was significantly decreased than those before injection in diabetic group and 4 months after injection in control group (all at P<0.05).The apparent collagen fibrosis and inflammatory cell infiltration were observed at lacrimal gland in diabetic model mice 4 months after injection.Conclusions The major early stage manifestations of STZ induced type 1 diabetes mice include retarded growth of lacrimal gland and decreased tear secretion volume,which gradually develop along the course of diabetes;in the later stage,the manifestations include decreased corneal sensitivity,ocular structural damage,structural changes of lacrimal gland and decreased conjunctival goblet cell density.
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Objective To compare the rates and pathological features of diabetic keratopathy in mice induced by single high dose or multiple low dose streptozotocin (STZ) injections.Methods Eighty male C57BL/6 mice (6-8 weeks old) were randomly divided into 4 groups with each group contain 20 mice:normal control group,multiple low dose 1 month group and multiple low dose 3 months group (injected with 60 mg/kg STZ for 5 consecutive times),single high dose 1 month group (injected with 150 mg/kg STZ).The survival rate,model success rate,body weight,glycosylated hemoglobin (HbA1c) content were compared among different modeling group.The percentages of residual epithelial defect area were examined by fluorescein sodium staining after removal of central corneal epithelium.The expression of p-Akt,Sirt1 and Ki67 were evaluated by immunofluorescent staining.The corneal sensitivity were compared among different groups before corneal epithelial curettage,3,7,10 and 14 days after corneal epithelial curettage.The corneal subbasal nerve density at 14 days after corneal epithelial curettage were compared among different groups.This study complied with the declaration of ARVO Results The success rate of diabetic modeling in multiple low dose 1 month group,multiple low dose 3 months group and single high dose 1 month group was 90%,80% and 70%,respectively.The HbA1c levels in the diabetic modeling groups were significantly higher than that in the normal control group (all at P<0.05).The percentage of residual epithelial defect area 24 and 48 hours after corneal epithelial curettage in the multiple low dose 3 months group and single high dosc 1 month group were significantly higher than those in the normal control group (all at P<0.05).The fluorescence intensity of p-Akt,Sirt1 and Ki67 in the multiple low dose 3 months group and single high dose 1 month group were stronger than those in the normal control group.There were no significant differences on corneal sensitivity and corneal nerve density between normal control and multiple low dose 3 months group before and 14 days after the corneal epithelial removal (all at P>0.05).However,the corneal sensitivity and corneal nerve density were dramatically decreased in the multiple low dose 3 months group and single high dose 1 month group before and 14 days after the corneal epithelial removal,and there were significant differences compared with normal control group (all at P<0.05).Conclusions The injection of 60 mg/kg STZ can not induce the features of diabetic keratopathy in mice within 1 month.However,the mice of both 1 month after 150 mg/kg STZ injection and 3 months after 60 mg/kg STZ injection appear the typical epithelial and nerve features of diabetic keratopathy,therefore can be the ideal animal models for research.
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Background Interleukin-6 (IL-6) is a pleiotropic cytokine involving in inflammation and wound healing.Previous report found that IL-6 increases phosphorylated STAT3 (p-STAT3) level and promotes corneal epithelial wound healing by stimulating migration.However,the essential role of IL-6 in corneal epithelial wound healing and the expression changes in diabetic mellitus remains unknown.Objective This study was to explore the roles of IL-6 in corneal epithelial proliferation and wound healing in both normal and diabetic mice.Methods Fifty-two normal C57BL/6 mice were randomized into normal control group (32 mice) and diabetic group (20 mice).Type 1 diabetic mellitus was induced by intraperitoneal injections of 50 mg/kg streptozotocin once per day for consecutive 5 days in the mice of the diabetic group.Whole corneal epithelium was scraped in all mice,and the corneal epithelial defect area was examined by fluorescein staining in 24,48 and 72 hours after corneal epithelium removal.Recombinant mouse IL-6 or anti-IL-6 blocking antibody of 5 μl were subconjunctivally injected according to the grouping and contrasted with PBS injection group or isotype control antibody group,respectively.TKE2 cells,a mouse corneal epithelial stem/progenitor cell line,were trypsinized and incubated in the KSFM with different concentrations of IL-6 or without IL-6,and colony formation efficency (CFE) was examined by crystal violet staining.The expressions of △NP63 and Ki67,specific makers of stem cells,were detected by immunofluorescine technology.The expressions of △NP63,Ki67 and p-STAT3 proteins were assayed in the cells by Western blot,respectively.The expression of IL-6 mRNA and protein in the regenerated corneal epithelium was detected by real time quantitative PCR and ELISA.The use and care of the mice complied with the Statement of Association for Research in Vision and Ophthalmology.Results The percentage of residual corneal epithelium defect area with initial detect area was gradually shrinked over time after PBS and IL-6 injection in both normal control mice and diabetic mice,and the percentage of residual corneal epithelium defect area was significantly reduced in the IL-6 injected group compared with the PBS injected group (normal control group:Fgroup =19.982,P < 0.01;Ftime =589.350,P < 0.01;Diabetic group:Fgroup =25.411,P<0.01;Ftime =334.807,P<0.01).The CFE was (13.23± 1.12)%,(15.87± 1.30)%,(21.69±1.62)%,(25.33±1.28)% and (18.67±1.54)% in the blank control group and 10,20,50,100 ng/ml IL-6-treated groups,respectively,showing a gradual increase of CFE dependent upon IL-6 concetrations (F =35.547,P<0.01).The expressions of △NP63,Ki67,p-STAT3 proteins in the cells were gradually increased over time after 50 ng/ml IL-6 treated for 5,10,15,30 and 60 minutes,and the relative expression level of the cytokines was significnatly higher in the IL-6 cultured groups than that without IL-6 culture group (all at P<0.05).The relative expression of IL-6 mRNA in the regenerated corneal epithelilum was 0.45±0.21 and 1.00±0.16 in the diabetic group and normal control group,respectively,and compared with the normal control group,the expression of IL-6 mRNA reduced by 56% (t=3.42,P=0.03).The content of IL-6 protein in regenerated corneal epithelium of the diabetic group was (257±12) ng/μl,which was significantly lower than (323 ± 17) ng/μl of the normal control group (t =5.60,P<0.01).Conclusions IL-6 promotes the proliferation and regeneration of corneal limbal stem cells to repair defected corneal epithelium by activating STAT3 signaling pathway in both normal and diabetic mice,while the blocking of endogenous IL-6 impairs the corneal epithelial cell activation and wound healing.
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BACKGROUND: Vascular endothelial growth factors are a family of multifunctional cytokines that can enhance vascular permeability, induce angiogenesis, promote endothelial cel growth and migration, and inhibit cel apoptosis. OBJECTIVE:To elaborate the latest progress in the role of vascular endothelial growth factor and its receptors in the corneal tissue. METHODS:A computer-based search of PubMed databases was performed for relevant articles published from 2005 to 2015. The key words were “vascular endothelial growth factor, cornea”. According to the inclusion and exclusion criteria, 43 articles were included in result analysis. RESULTS AND CONCLUSION:Vascular endothelial growth factor and its receptors are involved in the regulation of corneal neovascularization by causing Tip cel activation that affects the Notch signaling pathways. Corneal lymphatic regeneration mainly relies on macrophages to secrete vascular endothelial growth factor-C or vascular endothelial growth factor-D that further activate vascular endothelial growth factor receptor-3 in the lymphatic endothelial cels to cause cel proliferation and migration, and eventualy lead to the formation of new lymphatic vessels. But herpes simplex keratitis HSK induces the corneal lymphatic regeneration by vascular endothelial growth factor-A/vascular endothelial growth factor receptor-2 pathway. Vascular endothelial growth factor family can significantly improve the damaged corneal nerve endings, epithelium and corneal sensitivity, has the function of nerve nutrition and promote restoration of the corneal epithelium.
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Background Recent researches show that oxidative stress is involved in the progress of keratoconus.Nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a critical role in the defense against oxidative stress,but its function in keratoconus is unclear.Objective To investigate the differences of Nrf2-ARE signaling activation and matrix degenerating enzymes between keratoconus and normal corneal stromal cells.Methods Corneal stromal cells were isolated from keratoconus and normal cornea by using dispase and collagenase digestion.The cells were treated with hydrogen peroxide (H2O2) to mimic in vivo oxidative stress condition.Reactive oxygen species (ROS) production was measured by fluorescence substrate DCHF-DA incubation.Nrf2 level and the expression of Nrf2-ARE downstream antioxidant genes were analyzed by Western blot and real-time quantitative-PCR(RT-qPCR).The activity of matrix degenerating enzymes,including urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) system and matrix metalloproteinase-2 (MMP-2) were assessed by Western blot and gelatin zymography respectively.Results In normal culture,keratoconus corneal stromal cells assumed increased basal ROS and Nrf2 level when compared with normal cells(t =18.155,P<0.01).However,after H2O2 treatment,the keratoconus corneal stromal cells showed increased ROS production,while decreased Nrf2 translocation and no significant difference in expression levels of Nrf2-ARE downstream antioxidant genes (Nrf2:t =62.123,P< 0.01 ; (nicotinamide adenine dinucleotide phosphate quinine oxidoreductase-1 [NQO-1]:t =2.209,P =0.092 ; hemo oxygenase-1 [HO-1]:t =0.293,P =0.784 ; superoxide dismutase [SOD2]:t =0.749,P =0.495).The contents of uPA-uPAR and the activity of MMP-2 also showed a higher level in keratoconus corneal stromal cells than normal cells,with significant differences between them (t =19.164,15.458,4.818,all at P<0.01).Conclusions The defect of Nrf2-ARE signaling activation exists in the keratoconus corneal stromal cells,and correlats with the abnormal expression level of stromal degeneration enzymes,which suggests that the defect of Nrf2-ARE signaling activation may be involved in the progression of keratoconus.
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Background Diclofenac sodium eye drops,pranoprofen eye drops and bromfenac sodium hydrate eye drops are three clinical commonly used nonsteroidal anti-inflammatory drugs(NSAIDs).The variation of cytoxicity among these drugs and whether the cytoxicity is related to the supplements are also unknown.Objective This study was to compare the cytotoxicity of three non-steroidal anti-inflammatory eye drops and their active components with cultured human corneal epithelial cells (HCECs) in vitro,and to discuss toxic origins of these drugs.Methods HCECs were cultured in different drugs with the final concentration of 0.10%,0.05%,0.02% and 0.01%.Cell proliferation was evaluated by MTT assay.Then,0.002% eye drops (1∶50) was added,and the migration and damage of the cells were deceted by transwell migration assay and lactate dehydrogenase (LDH) assay.Results The cytotoxicity of three nonsteroidal anti-inflammatory eye drops on HCECs was concentration-dependent (all at P=0.00).Diclofenac sodium eye drops showed the most dominant effects on the proliferation,migration and damage of HCECs among the three eye drops,while bromfenac sodium eye drops showed the least effect on the cell damage (proliferation:Fdrug =20.25,P=0.00;migration:F =103.43,P =0.00;damage:Fdrug =164.16,P =0.00).Compared with the eye drops,their active components showed less cytoxicity.Pranoprofen appeared the least effects on the proliferation,migration and damage of HCECs (proliferation:Fdrug =332.27,P =0.00;migration:F =332.27,P =0.00;damage:Fdrug=154.83,P=0.00).Conclusions The cytotoxicity ofdiclofenac sodium eye drops is more obvious than that of pranoprofen eye drops or bromfenac sodium hydrate eye drops.The cytotoxicity of the three eye drops originates from their supplements or the interaction between the supplements and active components.
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Background Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency.Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis,such as human embryonic stem cells and karatin epithelial cells.Objective This study was to investigate the improving effect of Y-27632,a ROCK inhibitor,on the activity of rabbit LSCs in corneal preservation medium.Methods Corneal preservation solution was prepared by adding 12.5% chondroitin sulfate,10.0% low molecular dextran,20.0 mg/L dexamethasone,100 mg/L tobramycin sulfate,9.5 g/L Hepes and 0.375 mg/L L-glutamine in MEM.The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4,7,14 days,and the density and morphology of corneal endothelial cells were examined by using 0.25% trypan blue staining and 0.2% alizarin red staining.Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation.Colony shape of LSCs was observed under the light microscope,and colony-formation efficiency was analyzed after Giemsa staining by Image J software.Results The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days.In the seventh day after preservation,the cells remained the regular hexagon in shape in the preservation solution with Y-27632,however,the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632.The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262-± 75) cells /mm2,while in the preservation solution with Y-27632 was (2 425 ±95) cells/mm2(P<0.001).The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside.But in 7 days and 14 days after preservation,the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group.No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P>0.05).In 7 days and 14 days after preservation,the active rates of corneal epitheli.al cells were (73.00±2.12)% and (56.00±0.71)% in the preservation solution with Y27632,which were significantly higher than (66.00 ± 4.00) % and (49.00 ± 0.71) % in the preservation solution without Y-27632,showing statistically significant differences between them (t =3.098,P =0.018;t =9.798,P =0.000).In addition,the cloning-formation rates of LSCs were (11.05±0.21)% and (3.10±1.97)% in the preservation solution with Y-27632 in 7 days and 14 days after preservation,revealing significantly elevation in comparison with (2.05 ± 1.20) % and (0.40 ±0.14) % in the preservation solution without Y-27632 (t =18.107,P =0.000;t=3.184,P=0.017).Conclusions Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium,suggesting its potential use during storage of cornea.