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Objective:To investigate whether polyethylene glycol hydrogel films (PHFs) can be used as a carrier for the expansion of corneal epithelial cells (CECs) in vitro and whether PHFs can be used in the treatment of limbal stem cell deficiency (LSCD). Methods:Sebacoyl chloride, dihydroxyl PCL and glycerol ethoxylate were used to synthesize PHFs.The thickness, transmittance and mechanical tensile properties of PHFs were measured.Four clean-grade New Zealand white rabbits were selected to culture primary limbal epithelial cells.The expression of keratin marker AE1/AE3 and stem cell marker p63 in the cultured cells were observed under a fluorescence microscope.The cells were divided into negative control group cultured with common cell culture solution, positive control group cultured with cell culture solution containing 100 μmol/L H 2O 2, and PHFs+ CECs group lined with PHFs cultured with common cell culture solution for 24 hours.The proliferation and apoptosis of cells in the three groups were observed by MTT and TUNEL staining, respectively.Fifteen clean-grade New Zealand white rabbits were divided into control group, PHFs group and PHFs+ CECs group by random number table method, with 5 rabbits in each group.LSCD model was constructed in the three groups.The control group was not given any treatment after modeling.In PHFs group, empty PHFs were placed on the corneal surface of rabbits.In PHFs+ CECs group, tissue-engineered grafts constructed with CECs after passage implanted on PHFs were placed on the corneal surface of rabbits.The corneal defect area of rabbits was detected and scored by fluorescein sodium staining.The histological characteristics of rabbits corneal epithelium was observed by hematoxylin-eosin staining.The use and care of animals complied with Guide for the Care and Use of Laboratory Animals by the U. S.National Research Council.The experimental protocol was approved by the Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Harbin Medical University (No.2021006). Results:The synthetic PHFs were with a thickness ≤150 μm, a tensile strength about 6 MPa, and a transmittance over than 99% in the range of 400-700 nm.Most of the cells from primary culture of limbal tissue were positive for AE1/AE3 and p63.MTT test results showed that the A490 value of PHFs+ CECs group, negative control group and positive control group was 0.59±0.01, 0.65±0.07 and 0.06±0.04, respectively, showing a statistically significant overall difference ( F=12.25, P<0.05). The A490 values of PHFs+ CECs group and negative control group were significantly higher than that of positive control group, and the differences were statistically significant (both at P<0.05). TUNEL test results showed that there was a significant difference in the TUNEL-positive cell rate among the three groups ( F=13.45, P<0.05), and the rates of TUNEL-positive cells in PHFs+ CECs group and negative control group were significantly lower than that in positive control group (both at P<0.05). Fluorescein sodium staining results showed that with the extension of postoperative period, the corneal fluorescein sodium staining score of the three groups decreased, which decreased successively in control group, PHFs group and PHFs+ CECs group.Hematoxylin-eosin staining showed fewer irregularly shaped corneal epithelial cells in the control group, and sparse single layer of corneal epithelial cells in some areas of the PHFs group.In PHFs+ CECs group, the corneal epithelium coverage was the largest, and the cell layers increased to 3-5, and the cells were with regular morphology and in close arrangement. Conclusions:PHFs have enough toughness, high transmittance and can expand corneal epithelium in vitro.PHFs are suitable for corneal epithelial transplantation and can promote the repair of corneal epithelium in rabbit model of LSCD.
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Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.
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Humans , Acanthamoeba castellanii , Acanthamoeba , Amoeba , Blindness , Coculture Techniques , Cytokines , Epithelial Cells , In Vitro Techniques , Interleukin-6 , Interleukin-8 , Keratitis , Trophozoites , Vision DisordersABSTRACT
Stem cells are a group of self-renewal cells with the potential to differentiate into a variety of cell lineages. Embryonic stem cells can differentiate into more than 200 types of cell lineages belonging to endodermal, mesodermal and ectodermal tissues. Corneal epithelial cells derive from epidermal ectoderm during embryonic development. When the ocular surface is severely damaged, corneal epithelium with proliferation potential is essential for its reconstruction. Recent studies are focused on differentiation of bioactive corneal epithelial cells. This review summarizes signaling pathways including Notch, Wnt, bone morphogenetc protein or fibroblast growth factor pathways that are involved in regu?lating the development of embryonic ectoderm and corneal epithelial cells revealed in previous studies.
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Objective To investigate the effect of Wnt1 on the expression of Cyclin D1 in human corneal epithelial cells and its related molecular mechanisms. Methods 12 T25 cell culture flasks were cultured after hu-man corneal epithelial cells anabiosis ,culture and continuous passage for 2 times. Culture flasks were divided into 3 groups with 4 culture flasks in each group. Twenty-five ng/mL and 50 ng/mL recombinant human Wnt1 protein were added in two of the groups,and one group without T-cell culture medium(Wnt1)was used as control. Cells cultured in T25 flask were taken from three groups at different time(6 h,24 h,48 h and 72 h). The total number of corneal epithelial cells in each group was calculated. Expression of Cyclin D1 in corneal epithelial cells was de-tected by Western blot. Results The expression of Cyclin D1 protein in the control group decreased gradually from 0 h to 48 h,and reached the lowest level at 48 h and increased at 72 h. Cyclin D1 protein expression in 25 ng/mL group at 6 h after Wnt1 was added was not detected,and Cyclin D1 protein expression in 50 ng/mL group in-creased. The expression of Cyclin D1 protein in 25 ng/mL group and 50 ng/mL group was significantly higher than that in control group at 24 h,48 h and 72 h,reaching the peak at 48 h and decreased at 72 h. Compared with the control group,the growth rate of corneal epithelial cells in 25ng/ml group and 50ng/ml group increased after Wnt1 was added. There was significant difference in 72 h,but no significant difference in 6h,24h and 48h. Conclu-sions The stimulation of Wnt1 protein can enhance the expression of Cyclin D1 in a certain time range,and has a positive correlation with Wnt1 protein. As one of the target genes of Wnt1 signaling pathway,Cyclin D1 may play an important role in the repair of corneal epithelial injury and its cell proliferation and differentiation.
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Objective To compare and analyze the time of cell eruption formation and the rate of cell eruption of human corneal epithelial cells cultured by different explant culture methods under serum-free conditions.Methods Explant tissues were randomly divided into A,B,C and D groups.In A group,the cornea epithelial cells were put side up after stripping corneal endothelium and scraping the corneal epithelium;in B group,the corneal epithelium was put side down after stripping corneal endothelium and scraping the corneal epithelium;in C group,the corneal epithelium was put side up after only stripping corneal endothelium;and in D group,the corneal epithelium was put side down after only stripping corneal endothelium.The morphological changes of the cells in the 4 groups were observed by phase-contrast microscope,and both aforementioned variable were recorded.Results The average time of cell eruption formation and the rate of cell eruption in A,B,C and D group was (7.00 ± 3.00) d and 47.50％ (19/40),(7.55 ±2.58)d and 45.83％ (11/24),(8.43 ±3.32)d and 63.64％ (14/22),(7.49 ± 2.20) d and 72.22％ (39/54),respectively.The chi-square test showed that there was significant difference in the cell eruption rate between A and D group as well as B and D group (all P ＜ 0.05),but there was no significant difference between the other groups (all P ＞ O.05).Conclusion The time of cell eruption formation in the four different explant culture methods was about one week in free-serum conditions.And D group has higher cell eruption rate,and pure plus stable cultured cells when compared with the other three groups.
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@#AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 <i>in vitro </i>and <i>in vivo</i> and to assess the probability of defensins as a new application for infectious corneal diseases in the future. <p>METHODS: The synthetic rBD-2 DNA fragment was inserted between the <i>Xho</i>I and <i>BamHI</i> restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into <i>E.coli DH5α</i>, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. <p>RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. <p>CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.
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Embryonic stem cells(ESCs)are pluripotential cells,and they can be induced to differentiate into bone cells,cartilage cells,muscle cells,blood vessels and neurogenesis cells.In addition,ESCs are able to participate in the regeneration of ocular surface.At present,many research groups,domestic and abroad,have reported that ESCs can not only differentiate into corneal epithelium-like cells,but also play an important role in the repair of corneal epithelium.However,other unsolved problems in ESCs-related corneal tissue engineering are being concerned,such as the molecular biologic mechanism underlying the directional differentiation from ESCs to corneal cells,and the potential tumorigenicity with grafting of transformed or undifferentiated ESCs.The purpose of this review was to provide a summary of research progress in the differentiation of ESCs into corneal epithelial cells.
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PURPOSE: To investigate the migration and redistribution of rabbit corneal epithelial cells when wearing reverse geometry lens (RGL) or rigid gas permeable lens (RGP). METHODS: In 30 rabbits, the right eyes were fitted with either RGL or RGP and the left eyes were untreated to serve as controls. The rabbits were sacrificed at 1, 3, 7, 10 and 14 days after lens fitting. The central and peripheral corneal thicknesses were measured by microscope and the ratio of right to left corneal thickness was calculated to evaluate the characteristics of change over time. By using the molecular probe 7-nitrobenz-2-ox-1,3-diazolylphallacidin (NBD phallacidin), the samples were examined with light microscope to determine the migration and redistribution of epithelial cells in the rabbit cornea. RESULTS: No consistent changes in the thickness of both central and peripheral corneal epithelium were found. The corneal epithelial cells of both eyes with RGL and RGP reacted positively to NBD phallacidin. The fluorescence was most increased at day 3 of sacrifice in RGL cases and at day 7 in RGP cases, and then decreased in both cases. The corneal epithelium of eyes with RGL exhibited marked increase in the intensity of fluorescence compared to the eyes with RGP. CONCLUSIONS: The corneal epithelium with RGL showed the strongest intensity of NBD phallacidin fluorescence. This result suggests that wearing RGL may induce the migration and redistribution of corneal epithelial cells.
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Rabbits , Cornea , Epithelial Cells , Epithelium, Corneal , Fluorescence , Molecular ProbesABSTRACT
Background Wearing of soft contact lens,as one of the important methods to correct ametropia,has been widely applied at present.The clinically deleterious effects of soft contact lens have been reported,but researches about influence of apoptosis of corneal epithelial cells are scant.Objective This study was to detect and compare the ratio of apoptosis of corneal epithelial cells between the patients with contact lens wearing and without contact lens wearing,and explore the influence of soft contact lens wearing for long term on growth of corneal epithelial cells after injury.Methods A retrospective nest case-controlled study was designed.Forty eyes of 20 myopic patients with wearing of soft contact lens for ≥5 years and 40 eyes of 20 myopic patients without wearing of soft contact lens were included in Affiliated Hospital of Guiyang Medical College.All the patients received the off-flap epipolis laser in situ keratomileusis (Epi-LASIK) from 2010 October to 2011 June.The corneal flaps were collected during the surgery.The apoptosis of corneal epithelial cells was detected by flow cytometry(FCM),and the difference of apoptotic proportion between the two groups was compared using independent sample t test.Results The FCM scatter plot showed that compared to the without contact lens wearing group,the early stage of apoptotic cells with high staining annexin V and low staining proaium iodide (PI) were much more,and survival cells with low staining annexin V and PI were less,and necrotic and late stage of apoptotic cells with high staining of annexin V/PI were more in the with contact lens wearing group.The early stage of apoptosis proportion of corneal epithelial cells was (11.23 ± 5.31)％ in the with contact lens wearing group,and that in the without contact lens wearing group was (7.31± 5.43)％,showing a significant difference between them (t=2.754,P=0.008).Conclusions A long term wearing of contact lens induce more apoptosis of corneal epithelial cells,which can result in a disorder of structure and function of ocular surface during the wound healing stage.
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Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro.MethodsHuman corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells.ResultsRT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells.ConclusionsThe expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.
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Epidermal growth factor (EGF) is a very important peptide growth factor,which is not only involving in a variety of ocular cell mitosis,proliferation and differentiation,but also participates in mediating the signal transduction between cell and cell,cell and matrix interaction and other biological functions.Corneal injury is a common and frequently occurring disease in ophthalmology,and it would lead to blindness without appropriate treatment.Researches showed that EGF can promote the repair and regeneration of wounded cornea.In recent years,these studies advance from animal experiment stage to clinical stage and also from the morphology evaluation stage to the genetics study stage.This paper provided an overview of EGF concept,its biological effectiveness,its relationship to corneal injury and its clinical application.
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Background The human transforming growth factor beta-induced gene (TGFBI) is the first determined pathogenic gene to corneal dystrophy.But the molecular genetic mechanism is completely unknown.The study of concerning role of TGFBI is very important for us understand the physiological function of cornea,and the pathogenesis of corneal dystrophy.Objective The vector of human transforming growth factor beta-induced gene (TGFBI) in eukaryotic expression was constructed and transfected into the human corneal epithelial cells in order to explore its influence on the growth of human corneal epithelial cells.Methods Total RNA was extracted from normal donor cornea tissue and cDNA was obtained by reverse transcription.TGFBI cDNA was synthesized by reverse transcription-PCR and cloned into pCMV-N-HA vector and identified by sequencing with PCR and EcoRV,XhoI double restriction endonuclease.The cells were grouped into recombinant pCMV-N-HA-TGFBI plasmid group,pCMVN-HA plasmid group,non-transfected group and pGFP-C2 transfected group.The recombinant pCMV-N-HA-TGFBI plasmid was transfected to human corneal epithelial cells and identified by observing the expression of enhanced green fluorescence protein(EGFP) in the cells.The TGFBI mRNA and proteins were harvested from the cells for real-time PCR analysis and Western blot assay respectively in 58 hours after transfection.The growth of the transfected cells was assessed by Cell Counting Kit-8.The expressions of matrix metalloproteinase(MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) proteins and their mRNA in transfected cells were detected using SYBR fluorescence realtime PCR analysis and Western blot assay.Results The sequencing result of pCMV-N-HA-TGFBI positive clone plasmid showed that amplified TGFBI eDNA inserted into the vector at the correct sequence.EGFP was expressed in transfected cells in 48 hours after transfer of pGFP-C2 with the transfer efficacy 70％.The expression intensity of TGFBI mRNA was significantly higher in recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group,and TGFBI protein was expressed in recombinant pCMV-N-HA-TGFBI plasmid group.No significant difference was found in the A450value among recombinant pCMV-N-HA-TGFBI plasmid group,pCMV-N-HA plasmid group and non-transfected group ( F=3.34,P＞0.05 ).The mRNA level of MMP1,MMP3in the transfected cells was significant elevated but that of TIMP1 was declined in the recombinant pCMV-N-HA-TGFBI plasmid group compared with pCMV-N-HA plasmid group and non-transfected group (all P ＜ 0.05 ).Meanwhile,the expressions of MMP1,MMP3 and TIMP1 proteins appeared the same tendency( all P＜0.05).Conclusions Eukaryotic expression vector harboring human TGFBI eDNA can be successfully constructed and efficiently overexpressed in human corneal epithelial cells.TGFBI gene is involved in the physical and pathological conditions of human corneal epithelial cells by regulating the activity of MMP1,MMP3 and TIMP1.The results offer a new approach for the study of the role of TGFBI in pathogenesis of corneal transparency.
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Background Biomaterials for corneal tissue engineering must demonstrate several critical features for potentialutility invivo, includingtransparency, mechanicalintegrity, biocompatibilityand slow biodegradation. Silk film biomaterial had been characterized to meet these functional requirements. ObjectiveThis study was to investigate the feasibility of physico-crosslink regenerated silk fibroin film as tissue engineered corneal scaffold. MethodsHuman corneal epithelial cells(CECs) links were cultured by regular method and CECs in logarithmic phase were than incubated on physico-crosslink regenerated silk fibroin film membrane. The shape of cultured human CECs was observed after 24,48 and 72 hours under the inverted microscope and scanning electron microscope( SEM ) ,and the CECs were cultured on culture plates as controls. The growth state of CECs on regenerated silk fibroin film was observed daily for 7 days by MTT, and cell cycle analysis and the presence of apoptosis of human CECs were examined by flow cytometry after incubation on regenerated silk fibroin film. Regenerated silk fibroin filmCECs (4 mm×3 mm) were implanted into the corneal stroma of the right eyes of New Zealand white rabbits. At the end of 4 and 8 weeks after implantation, the appearance of the ocular surface was examined using slit lamp and corneal neovascular area was measured. Corneal histopathological examination was carried out to assess the degradation of graft materials and immunohistochemistry was performed to detect the expression of CD34 in the corneal tissue after operation. ResultsThe morphology and structure of CECs were identical using the two cultured Methods when observed under the inverted microscope and SEM after 24,48 and 72 hours. No significant difference was found in the A490 value 1,2,3,4,5,6 or 7 days after incubation on regenerated silk membrane and in culture plates ( Fmethod =0. 641 ,P＞0.05 ). The apoptosis rates of CECs on regenerated silk membrane or culture plates were 1.8％ and 2.0％ and the amount of cells in G2/G1 phase was 1. 956 and 1. 945, respectively. Histopathological examination showed that the regenerated silk membrane material degraded and was replaced by regular collagen tissue 2 months after implantation,and the presence of neovascular area and inflammatory cells were less prominent in 2 months than 1 month post-implantation. The expression level of CD34 in corneal tissue was evidently lower 1 and 2 months after operation than the Ad-VEGF165-induced positive control group (P＜0. 05), and no significant differences were seen when compared with normal CECs(P＞0.05). ConclusionsPhysico- crosslink regenerated silk fibroin film is an excellent biomaterial for tissue engineered corneal scaffold with good biocompatibility.
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PURPOSE: To investigate the biologic effects of topical calf serum on corneal epithelial cells in vitro. METHODS: The effects of calf serum on the corneal epithelial cells were evaluated using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, and the concentration of IL-1alpha, TGF-beta1 and MMP-9 in the cells was measured. Cell damage was determined using lactate dehydrogenase (LDH), and cellular morphologies were examined by transmission electromicroscopy. RESULTS: Metabolic activity of the corneal epithelial cells decreased at higher concentrations and longer exposure durations. IL-1alpha, TGF-beta1 and MMP-9 titers were lower in calf serum-treated cells than in the control. LDH and cellular damage to the corneal epithelial cells, such as chromatin margination and cytoplasmic organelle swelling, were prominent in cells treated with 30% calf serum. CONCLUSIONS: Cellular metabolic activity was higher and cellular toxicity was lower in cells treated with 10% calf serum compared to those treated with the 20% and 30% concentrations. Furthermore, inflammatory cytokines were sufficiently inhibited in cells treated with the 10% solution. These results indicate that 10% calf serum could be used clinically.
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Humans , Chromatin , Cytokines , Cytoplasm , Epithelial Cells , L-Lactate Dehydrogenase , Organelles , Transforming Growth Factor beta1ABSTRACT
PURPOSE: To investigate the biologic effects of preservative-free artificial tear drops on cultured human corneal epithelial cells in vitro. METHODS: Efficacies of the preservative-free artificial tear drops-Kynex(R), Hyalein Mini 0.3%(R), and Refresh Plus(R)-were evaluated using the MTT assay. Cell damage was determined using the lactate dehydrogenase (LDH) assay. Cellular proliferation was determined using a migration and wound-healing assay. The ingredients of the drugs were analyzed. Apoptotic response was evaluated with flow cytometric analysis. RESULTS: Metabolic activity of the corneal epithelial cells showed similar activity to that of the control. Cellular migration and proliferation also were not significantly different between the preservative-free artificial tear drop groups and the control. The LDH titers tended to increase for up to 24 hours after exposure to the preservative-free artificial tear drops, but there was no significant difference in LDH titers between the control groups and the artificial tear drop-treated groups. Apoptosis and necrosis were observed using flow cytometry at 24 hours in all groups. The electrolyte levels, pHs and osmolarities of the three drugs were not significantly different. CONCLUSIONS: The clinically available preservative-free artificial tear drops Kynex(R), Hyalein Mini 0.3%(R), and Refresh Plus(R) had no significant toxic effects on corneal epithelial cells and thus can be used safely.
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Humans , Apoptosis , Cell Proliferation , Epithelial Cells , Eye, Artificial , Flow Cytometry , Hydrogen-Ion Concentration , L-Lactate Dehydrogenase , Necrosis , Osmolar Concentration , TearsABSTRACT
PURPOSE: To investigate the biologic effects of topical ocular artificial tears used in patients wearing contact lens on in vitro corneal epithelial cells. METHODS: The efficacies of the topical artificial tears Iris(R), Irisplus(R), Eyemiru contact pure(R), and Eye2O(R) were evaluated using the MTT and wound healing assays. Cell damage was determined using lactate dehydrogenase (LDH), and the solution ingredients were analyzed. Cellular morphologies were examined by inverted light microscopy and transmission electromicroscopy. RESULTS: Metabolic activity of corneal epithelial cells, as determined by the MTT assay, decreased in the Iris(R) eye drop group, but those of the other groups were similar to that of the control. The LDH titers increased up to one hour after Iris(R) eye drop use, and the increased level was maintained for 24 hours. The other three artificial tears showed similar low LDH titers to that of the control. Cellular migration was not observed, although cellular damage to the corneal epithelial cells, such as chromatin margination and cytoplasmic organelle swelling, was prominent with Iris(R) use. CONCLUSIONS: Among four brands of topical artificial tear drops used among patients wearing contact lens, Iris(R) caused markedly more severe damage to cultured human corneal epithelial cells than did Irisplus(R), Eyemiru contact pure(R), or Eye2O(R).
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Humans , Chromatin , Cytoplasm , Epithelial Cells , Eye , L-Lactate Dehydrogenase , Light , Microscopy , Ophthalmic Solutions , Organelles , Tears , Wound HealingABSTRACT
AIM: The study was undertaken to investigate the effort of Dexamethasone (DEX) on cultured rabbit corneal epithelial (RCE) cells and rabbit corneal epithelial wound healing.METHODS: For the in vitro experiments, primary cultures of RCE cells were used. DEX in different concentrations was added to cultured RCE cells. The effects were measured with tetrazolium salt (MTT)method and flow cytometry. For the in vivo wound-healing experiments, a central corneal deepithelialization was created and were treated with 0.1g/L DEX eyedrop randomly explain how randomly. Epithelial wound healing was evaluated clinically and analyzed histopathologically using light microscopy along with immunohistochemical staning and electronic microscopy.RESULTS: Less than 0.1g/L DEX didn't influence survival rate in cell culture conditions by MTT assay. Flow cytometric studies revealed that 0.1g/L DFX had no effect on cellular growth phase in cultured rabbit corneal epithelial cells. The mean time of the epithelial healing was significantly shorter in the DEX-treated group than in the control group at 24h. There were strong proliferative-cell-nuclear-antigen(PCNA) expressions in newly generated epithelial cells of both groups. The Dex-treated group had a more regular architecture of stromal lamella and significantly less inflammatory response than the control group under electronic microscopy.CONCLUSION: Less than 0.1g/L DEX had no inhibiting effect on cultured rabbit corneal epithelial cell growth.0.1g/L DEX eye drops can effectively promote epithelial growth and reduce inflammatory response, which may have useful clinical application at the early stage of corneal wound healing process.
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PURPOSE: Recently, 20% ethanol has been used to make a corneal epithelial flap effectively. We investigated the toxicity of ethanol using the corneal epithelial cell line and rat cornea. METHODS: Cultured corneal epithelial cells were exposed to different concentrations (5~60%) of ethanol for different exposure time (20~120 seconds). And then cell viability was measured with MTT assay. In vivo, rat central corneas (3mm in diameter) were exposed to 20% ethanol for 30 seconds. The eyes were enucleated, fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM) at indicated interval (0, 4, 8 and 12hours, 1, 3 and 7 days). TUNEL assay was used to detect apoptotic cells. RESULTS: Cultured epithelial cells showed survival rate of over 50% in ethanol concentration of below 20% (76.2 +/- 7.4%) and exposure time of below 1 minute (54.8 +/- 6.5%). In vivo experiment, after 20% ethanol exposure for 30 seconds, vacuoles were detected in the nuclei of epithelial cells and gradually disappeared. Hemidesmosomes were detected in the basement membrane at all times. TUNEL positive cells were detected in the epithelial layer and anterior stroma at the edge until 8hrs. CONCLUSIONS: Our results showed 20% ethanol exposure for 30 seconds did not seems to induce significant damage to the cornea. Corneal epithelial damage could occur by physical force during epithelial flap making.
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Animals , Rats , Basement Membrane , Cell Survival , Cornea , Epithelial Cells , Ethanol , Hemidesmosomes , In Situ Nick-End Labeling , Keratectomy, Subepithelial, Laser-Assisted , Microscopy , Microscopy, Electron, Transmission , Survival Rate , VacuolesABSTRACT
PURPOSE: To investigate the difference in healing of the corneal epithelial cells between in LASEK and in PRK and the factor to induce the difference. METHODS: The rate of corneal epithelial recovery was measured after mechanical removal of corneal epithelium (PRK) or after removal with alcohol (LASEK) in rabbits. PCNA (proliferating cell nuclear antigen) staining was performed to compare the degree of cellular proliferation. Immunocytochemical staining was also carried out to examine the expressions of fibronectin and EGFR (epidermal growth factor receptor), which were known as the factors participating in corneal epithelial regeneration. In cultured porcine corneal epithelial cells, the degree of epithelial proliferation was compared between the control group (group I without alcohol treatment) and alcohol treated groups with various alcohol concentrations (groups II, III, IV and V) after cell culture for 2 weeks. RESULTS: The rate of epithelial healing was faster in LASEK group than in PRK group. These epithelial cells recovered to normal cells faster in LASEK group than in PRK group according to light and electron microscopic findings. PCNA expression and fibronectin expression were higher in LASEK group than in PRK group, whereas no difference was seen in the expression of EGFR. Although the proliferation of corneal epithelial cells increased, it was not proportional to the amount of added alcohol-treated epithelial cells. CONCLUSIONS: We showed that the corneal epithelia recovered to normal shape faster in LASEK than in PRK group when alcohol treated corneal epithelial cells were treated to the site of the wound. Higher expression of fibronectin in LASEK group suggest that fibronectin could be a factor promoting corneal epithelial cell regeneration.
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Rabbits , Cell Culture Techniques , Cell Proliferation , Epithelial Cells , Epithelium, Corneal , Fibronectins , Keratectomy, Subepithelial, Laser-Assisted , Proliferating Cell Nuclear Antigen , Regeneration , Wounds and InjuriesABSTRACT
Objective To observe the effect of lentivirus vector-medicated enhanced green fluorescence protein (EGFP) transfection on in vitro corneal epithelial cells.Methods Primary corneal epithelial cells of rabbits were cultured and transfected with lentivirus vector into different multiplicity of infection (MOI) in order to find the best transfection dose.After 24,48,72,and 96 h of transfection,expression of EGFP was observed under microscope and detected by RT-PCR.The transfection rate of corneal epithelial cells was calculated in different MOI.Results EGFP was expressed in corneal epithelial cells 48 h after transfection,which increased with the increasing transfection time.The transfection rate of corneal epithelial cells was 4.5%?0.6%,30.4%?0.9%,51.9%?1.4%,and 75.4%?0.7%,respectively,when MOI was 1,10,50,and 100(P