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Objective To measure the tear film lipid layer thickness (LLT) in dry eye patients and investigate the correlations of LLT with ocular surface signs.Methods One hundred and thirty dry eye patients (130 eyes),including 64 meibomian gland dysfunction (MGD) patients and 66 non-MGD patients,were included in this study.LLT,break-up time (BUT),fluorescein staining (FL),Marx's line (ML) score and Schirmer I test were performed and examined.The distribution of LLT in different age groups and the correlations between LLT and other examinations were analyzed.Results There was significant difference in LLT among different age groups (P =0.007),while LLT was not significantly different between male and female in each age group (P ＞ 0.05).LLT was positively correlated with age (r =0.334,P ＜ 0.001) and was not correlated with sex (r =0.107,P =0.226).LLT was positively correlated with upper eyelid ML score (r =0.295,P =0.001) and lower eyelid ML score (r =0.233,P =0.008).There was no significant correlation of LLT with BUT,FL or Schirmer Ⅰ test (all P ＞0.05).In the MGD group,there were positive correlations of LLT with upper eyelid ML score and lower eyelid ML score (all r =0.306,P =0.014),and no correlation of LLT with other examinations (all P ＞ 0.05).In the non-MGD group,there was no correlation of LLT with other examinations (all P ＞ 0.05).In a multivariate linear regression analysis,age and upper eyelid ML score were significantly related to LLT (β =0.254,P =0.005 for age and β =0.207,P =0.022 for upper eyelid ML score) in all dry eye patients.Age was the only factor related to LLT (β =0.382,P =0.002) in the MGD group.Upper eyelid ML score and lower eyelid ML score were higher in the MGD group than the non-MGD subgroup (all P ＜ 0.001).Conclusion LLT is positively correlated with age and ML score in dry eye patients.The measurement of tear film LLT,as an auxiliary examination in the diagnosis of dry eye disease,should be analyzed with the influential factors including age.
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Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro,and acellular porcine corneal stroma (APCS) was a good saffold material.However,whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied.Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet.Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured,and the cultured medium was centrifuged and mixed with 75％ human corneal endothelium medium (CEM)at a proportion of 1 ∶ 3 to prepare the 25％ ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane,and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution,and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1)and Na+-K+-ATPase in the cell sheet were detected by immunofluorescence staining.Results The second generation of HCECs cultured with 25％ ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent,and no corneal cells were seen,the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the cell density of (2 694±143)/mm2.ZO-1 and Na+-K+-ATPase were positively expressed on the HCECs sheet.Conclusions Twenty-five percent ESC-CM promotes the proliferation and maintains the normal morphology of HCECs.APCS provide a good scoffold and microenvironment for the formation of HCEC sheet.The HCEC sheet Possesses the pump function of HCECs and is a good corneal donor for transplantation.
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Background The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells (ESCs) micro-environment enhance the stemness of human limbal stem cells (LSCs),but its mechanism has not been elucidated.Objective This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs.Methods Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20+20％ ES culture supernatant (ESC-CM),respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase (TERT) siRNA (19-25nt siRNA) or siRNA (sc-37007) was transfected into the cells of ESCCM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry,and the expressions of telomerase and reactive oxygen species (ROS) in TERT siRNA-or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR,immunofluorescence staining and Western blot were employed to determine the expressions of p63,ATP-binding cassette transporer G2 (ABCG2),integrin β1 mRNA and proteins and cytokeratin 3 (C K3) in the cells.The levels of focal adhesion kinase (FAK),Akt,glycogen synthase kinase 3β (GSK3β) and p21 protein and phosphorylation proteins in the cells were detected by Western blot.Results The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency (CFE) of (7.6±0.6) ％ in ESC-CM group,but the cells to the sixth generation with the CFE of (5.6±0.6)％,showing a significant difference between them (t =4.454,P =0.011).The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group (all at P＜0.05).The apoptotic rate of the cells was (7.67± 1.31)％ in the siRNA-F transfected group,which was significantly lower than (32.33 ±3.13)％in the siRNA-TERT transfected group (t =-12.588,P =0.000).No significant differences were seen in the expression levels of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the primary cells between the ESC-CM group and the CnT-20 group (all at P＞0.05),but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).However,the expressions of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).The telomerase activity was (4.83±0.67) ％ in the siRNA-TERT transfected group,which was significantly lower than (46.71±1.22) ％ of the siRNA-F transfected group (t =52.116,P =0.000).The expression of pFAK,pAkt,pGSK3β proteins were weakened,but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor,GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P＜0.01),and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P＜0.01).Conclusions ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways.
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BackgroundAn ideal animal model is very important for the investigation of the immune mechanism of high-risk rejection corneal transplantation.ObjectiveThis study was to compare three methods of creating a high-risk corneal transplantation model in rabbits to study high-risk rejection corneal transplantation.MethodsForty-five New Zealand white rabbits were utilized and assigned randomly to three groups of different modeling methods,with 15 rabbits for each group.The high-risk corneal transplantation models were created by suturing with 5-0 silk thread in 4 quadrants,inducing alkali burn with 1 mol/L NaOH or corneal xenotransplantation.In the suturing group and alkali burning group,the rabbits received a unilateral 7.25 mm diameter corneal allograft after corneal neovascularization was induced,and in the xenotransplantation group,corneas from cats were used as donors.Rabbits were followed-up for 4 weeks in all groups.Corneal neovascular area was calculated and compared among the three groups.The amount of rejection,inflammatory index ( IF),neovascularization and histology of grafts were clinically scored to calculate the reject index (RI).ResultsThere were 14,15 and 15 rabbits that survived the high-risk penetrating corneal transplantation,respectively,in the suturing group,alkali burning group and xenotransplantation group.Two weeks after operation,the IF scores were 0.543 ± 0.103,0.811 ± 0.054 and 0.191 ±0.087,and the RI were 2.111±0.928,7.0±0.816 and 3.182±0.751 in the suturing group,alkali burning group and xenotransplantationgroup,respectively,showingstatisticallysignificantdifferencesamongthethreegroups (x2 =25.736,22.432,P =0.000).The IF value was lower in the xenotransplantation group compared with the suturing group and alkali burning group (Z =3.841,3.993,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.568,P =0.000).The RI value of the xenotransplantation group was significantly raised in comparison with the suturing group and declined in comparison with the alkali burning group (Z =2.373,P =0.018;Z =3.936,P =0.000),and that of the suturing group was lower than the alkali burning group (Z =3.729,P =0.000 ).The survival times of the grafts were ( 17.9±2.0 ) days,( 13.4 ±2.4) days and ( 15.5 ±2.0 ) days in these three groups with a significant difference among them ( F =9.474,P =0.001 ).The neovascularization area in the xenotransplantation group was smaller than the suturing group and alkali burning group (P＜ 0.05 ).Histological examination revealed a large number of inflammatory cells infiltration in the grafts 2 and 4 weeks after transplantation in the suturing group and alkali burning group,but less inflammatory cells were seen in the xenotransplantaion group.Immunofluorescence staining showed abundant CD4+ T positive cells in the grafts in the three groups.Conclusions The cat-rabbit corneal xenotransplantation can induce stable and moderate immune rejection.This animal model has milder inflammatory response and less corneal neovascularization than the suture and alkali burn models.This method therefore is an ideal model for high-risk corneal transplantation.
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AIM:To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS: Embryonic body induced by retinoic acid and retinal m?ller cells were selected in neural stem cell-defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocytochemically with anti-nestin, anti-BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESULTS:Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn-3 genes. CONCLUSION:Neural stem cells could be derived from embryonic body induced by RA and m?ller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degeneration in the future.
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AIM: To investigate the inducing action of retinal cells of rat neonates on the adult rat bone marrow stromal cells (BMSCs).METHODS: The full marrow from adult Wistar rat were cultured and passaged repeatedly to harvest the pure BMSCs. In vitro, the BMSCs were induced by retinal cells of rat neonates in transwell.The morphological changes of the BMSCs were observed under phase contrast microscope, the specific markers of the induced cells were ~identified immunocytochemically with nestin, Map-2, neuron-specific enolase(NSE),neurofilament(NF), Thy 1.1 and glial fibrillary acidic protein(GFAP) antibodies. The mRNA of nestin, NF, NSE, Thy 1.1 and Ran were detected in the induced cells by RT-PCR.RESULTS: The BMSCs were induced by reinal cells of rat neonates into the retina-like neurons, which showed the typical neural morphology and expression of the specific retinal neural antigens.CONCLUSION: The BMSCs from the adult rat can be induced into retina-like cells by retinal cells of rat neonates. [