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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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Limbal stem cells (LSCs),the source of corneal epithelial cells,play an important role in the ocular surface. In recent years, with the development of somatic stem cell application and tissue engineering, biomaterials and cell culture technology,progress has been made on the basic researches and clinical applications of ocular surface reconstruction with ex vivo cultured limbal epithelial and oral mucosal epithelial cell sheet transplantation. However, there are several issues, including the successful clinical outcomes for ocular surface reconstruction,and the in vivo tracking of donor stem cells,remained indefinitive. This article introduced and compared recent advancements of tissue engineering techniques ex vivo cultured autologous or allogeneic limbal,oral mucosal epithelial cells in ocular surface reconstruction,so as to provide a useful direction for the future research of ocular surface reconstruction.
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Background Researches showed that cystic fibrosis transmembrane conductance regulator protein(CFTR) is a channel secreting anion and water,and it plays an important role in tear secreting.Traditional conception thought that CFTR pathway is cAMP-PKA-dependent without the participation of intracellular calcium.However,studies disclosed that elevating intracellular cAMP could not open the CFTR channel.So whether calcium signal is associated with CFTR-related corneal epithelial secretion is controversial.Objective This study was to investigate the association between CFTR secretion and intracellular calcium signaling in rabbit corneal epithelium.Methods Sixteen New Zealand white rabbits were randomized into odd number group and even number group by computer randomized number method.The corneas were obtained under the general anesthesia and placed in Ussing Chamber for the record of short cricuit current (Isc).The right eyes of rabbits in the odd number group were stimulated with ATP and served as ATP stimulating group.The left eyes were pretreated with CFTRinh-172 prior to ATP stimulation and served as CFTRinh-172 pretreated group.In the even number group,the left eyes of rabbits were pretreated with BMPTA/AM before ATP stimulation and served as BMPTA/AM pretreated group,and the right eyes of the rabbits were used to isolate and culture corneal epithelial cells by explant adherent method,the level of intracellular calcium were evaluated using Leica SP5 laser scan confocal microscope.Results The ATP-induced ΔIsc of corneal epithelium was (5.73 ± 1.36),(1.30 ± 0.95) and (2.47 ± 0.55) μA/cm2 in the ATP stimulating group,CFTRinh.172 pretreated group and BMPTA/AM pretreated group,respectively,and the AIsc was significantly reduced in the CFTRinh.172 pretreated group and BMPTA/AM pretreated group compared with ATP stimulating group (t=11.201,5.508,both at P ＜ 0.001).The fluorescence intensity of intracellular calcium release after ATP stimulation was 3.25 folds more than that before ATP stimulation.Conclusions ATP promotes rapid short circuit current of corneal epithelium.CFTRinh-172 depresses the ATP-induced corneal epithelium AIsc,and BMPTA/AM suppresses intracellular calcium release.It is suggested that intracellular calcium signaling secretion probably participates in the functional CFTR activity in rabbit corneal epithelium.
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Background Studies indicate that the decline of androgen level affects the secretion function of epithelial cells of meibomian glands and lacrimal glands ,which results in the abnormality of tear quality and quantity.However,whether androgen level regulates the mucin adhesion to ocular surface is still not elucidated.Objective This study was to investigate the tear functional changes and corneal ultrastructural changes in castrated male mice.Methods Fifty-six SPF male BALB/c mice were devided into normal control group, sham group, and orchectomy 1-week group, orchectomy 2-week group, orchectomy 4-week group, orchectomy 6-week group, orchectomy 8-week group according to random number table.Orchectomy was performed in the mice of the orchectomy 1-,2-,4-,6-and 8-week group,and only incision rather than enucleation of testis was carried out in the mice of the sham group.Peripheral venous blood samples were collected during eyeball enucleation in all the mice and serum androgen concentration was detected by radioimmunoassay.Tear secretion level was measured with phenol red thread.Break-up time of tear film (BUT) was tested,and corneal inflorescence staining was scored based on the criterion of Toshida.The corneas of all the mice were collected for ultrastructural examination under the transmission electron microscope.The study complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results Mouse serum androgen concentration was (573.87±6.74) ng/μl in the normal control group and (579.74-±8.24)ng/μl in the sham group, and there was no significant difference between these two groups (t =1.432, P =0.251) , however, the serum androgen concentration was 0 ng/μl in all castrated mice after orchectomy.The tear secretion levels were (5.26 ± 0.99), (4.89 ± 0.56), (4.62 ± 0.83), (4.28 ± 0.63), (3.36 ± 0.56),(2.60±0.27) and (2.08 ±0.35) mrn/5 minutes in the normal control group, sham group and orchectomy 1-, 2-, 4-,6-and 8-week groups, respectively, and the tear secretion levels were significantly lower in the orchectomy 2-, 4-,6-and 8-week groups than those in the normal control group (all at P＜0.05).The BUT were (68.33±12.86), (62.47± 3.75),(58.67±10.03), (47.17±7.64) ,(39.51±3.39),(32.67±3.88) and (31.00±2.36) seconds in the normal control group, sham group and orchectomy 1-, 2-, 4-, 6-and 8-week groups, respectively, and the BUTs were significantly shorter in the orchectomy 2-,4-, 6-and 8-week groups than those in the normal control group (all at P＜0.05).The fluorescein stain score of corneal epithelium was gradually increased with the lapse of castrated time.The decrease of number,shortening and edema of microvilli as well as separation of desmosomes of corneal epithelial cells were found under the transmission electron microscope in castrated mice.Conclusions Decreasing of serum androgen level, declining of aqueous tears,instability of tear film and damage of corneal epithelial cell ultrastructures occur in castrated male mice, and these manifestations are similar to dry eyes.
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Background As the main cellular constituent of corneal epithelium,corneal epithelial cells play critical roles in regulating and controlling the migration, proliferation and differentiation of cells during the repair of damage.MicroRNA (miRNA) is endogenously expressed small non-coding RNAs, which participates in a variety of biological processes.Previous studies demonstrated that miR-204 is highly expressed in normal corneal epithelium,but its function is still unclear.Objective This study was to investigate the function and mechanism of miR-204 in corneal epithelial cell proliferation.Methods Corneal epithelial tissue was collected during the corneal refractive surgery from the patients with refractive error under the informed consent, and human corneal epithelial cells(HCECs) were cultured and passaged.The relative expressing levels of miR-204 mRNA in the normal corneal epithelium and HCECs were detected by real time quantitative PCR.Cultured HCECs were evenly divided into three groups.The liposome with miR-204 mimic was transfected into the cells of the miR-204 mimic group, and blank liposome was transfected in the cells of the positive control group,and regularly cultured cells served as the normal control group.Cell proliferation capability was evaluated by colony-forming assay, and the percentage of the cells in different cell cycles was analyzed by flow cytometry.Western blot assay was employed to detect the expression levels of p-RB, E2F1 ,p27,CyclinA, CDC2, p-CDC2 and CDK2 proteins in the cells.Results The relative expression levels of miR-204 mRNA were 1.077 ±0.268 in the normal corneal epithelium and 0.041 ±0.018 in the HCECs, showing a significant difference between them (t =7.700,P＜0.001).The cloning cell number was evidently decreased in the miR-204 mimic group in comparison with the positive control group and normal control group.The percentage of cells in the G1 phase was 47.75％ in the miR-204 mimic group, which was significantly higher than 37.23％ in the positive control group and 40.72％ in the normal control group.The expression levels of E2F1 and p27 proteins in the cells were elevated (t=14.87,25.11;both at P＜0.01) and those of CDK2 and p-CDC2 proteins were decreased (t=5.39,10.65;both at P＜0.01) in the miR-204 mimic group in comparison with the positive control group.Conclusions The overexpression of miR-204 in the normal corneas probably is associated with non-proliferation status of corneal epithelial cells.Transfection of miR-204 into corneal epithelial cells can inhibit the proliferation of corneal epithelial cells probably by up-regulating the expression of E2F1 and p27 and suppressing the expression of CDK2/CyclinA and p-CDC2/CyclinA,which lead to cell arrest in G1 phase.