ABSTRACT
Objective:To study the biocompatibility of fibrin sealant (FS) and human corneal fibroblasts (HCFs) obtained by small incision lenticule extraction (SMILE).Methods:The human corneal stromal tissues were selected from corneal stromal lens in 24 eyes of 12 patients underwent SMILE in the First Affiliated Hospital of Guangxi Medical University from March to April 2018.HCFs were isolated and cultured in vitro within 1 hour after the corneal stromal lens were extracted and the growth status of HCFs on FS surface was observed.HCFs were divided into 2-fold leaching solution group and normal control group, and the cells in the two groups were treated with 2-fold leaching solution or complete medium according to grouping, respectively.The apoptosis of HCFs in the two groups was observed by acridine orange (AO)/ethidium bromide (EB) double staining.The proliferation of HCFs in the two groups was assayed by methyl thiazolyl tetrazolium (MTT) method.HCFs in logarithmic phase were divided into 2-fold leaching solution group, normal control group, and the cells were treated with 2-fold leaching solution or complete medium according to grouping, respectively.In addition, a blank control group without HCFs was also set and treated with complete medium.The absorbance value and relative growth rate of HCFs in the three groups were compared.HCFs in logarithmic phase were divided into 1-fold leaching solution group, 2-fold leaching solution group and normal control group, and the cells were treated with 1-fold leaching solution, 2-fold leaching solution or complete medium culture according to grouping, respectively.The apoptosis of HCFs in the three groups was compared by Annexin V-FITC/PI flow cytometry, and the cytotoxicity of the three groups was graded.Written informed consent was obtained from each patient before the operation.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (No.2018[022]). Results:HCFs grew well on FS surface and the morphology was normal.MTT assay showed that HCFs in the 2-fold leaching solution group and the normal control group had a similar proliferation tendency, and the toxicity index of HCFs in the 2-fold leaching solution group was graded 0-1 at 0-72 hours after changing solution.After AO/EB staining, the HCFs in the 2-fold leaching solution group and the normal control group were normal, and only a small amount of early apoptotic cells were observed.Flow cytometry showed that the apoptosis rates of the normal control group, once leaching solution group and the double leaching solution group were (4.96±1.09)%, (3.66±1.35)% and (2.88±0.66)%, respectively, with no significant difference among them ( F=2.89, P=0.13). Conclusions:FS has no cytotoxicity and has good biocompatibility with HCFs in vitro.
ABSTRACT
Objective To investigate the effects of acute oxidative stress induced by H2O2 on expression of senescence marker protein30 (SMP30) and morphology,survival rate of human lens epithelial cells (HLECs).Methods HLECs were treated with H2O2(0 μmol · L-1,100 μmol · L-1,200 μmol · L-1,300 μmol · L-1) for 24 hours,the acute oxidative stress models were established,the changes of cell morphology was observed,and MTT was used to analyze the cells state,the expressions of SMP30 were measured by Western blot.Results The cell density decreased,morphological changed and viability of cells significant decreased in 100 μmol · L-1 and 200 μmol ·L-1 treated group,the large and round cells appeared,the cell body stretched with unclear boundary.With the H2O2 concentration increased,the viability of cells were gradually decreased in treated group,there were statistical differences compared with 0 μmol · L-1 treated group (all P ＜ 0.05).The relative expression of SMP30 in control group and 100 μmol · L-1 and 200 μmol · L-1 treated group were 0.273 ±0.055,0.464 ± 0.058,0.442 ± 0.050,respectively.There were significant differences between 100 μmol · L-1,200 μmol · L-1 treated group and control group (all P ＜ 0.05),and there was no statistical difference between 100 μmol · L-1 and 200 μmol · L-1 treated group (P ＞ 0.05).Conclusion SMP30 is up-regulated in HLECs under acute oxidative stress induced by H2O2,the cell morphology is changed,the viability of cells is decreased,and SMP30 may be involved in the process of acute oxidative stress in HLECs.
ABSTRACT
Background Senescence marker protein 30 (SMP30) is a new calcium regulatory protein,which plays anti-oxidation,stable calcium and anti-apoptosis roles in vivo.Researches determined that the expression of SMP30 in human cells gradually decreased as ageing.However,the study on the relationship between SMP30 and age-related cataract is rarely.Objective The aim of this study was to investigate the expression of SMP30 in lens epithelial cells (LECs) of different ages of cataract patients.Methods This study was approved by Ethic Commission of the First Affiliated Hospital of Guangxi Medical University.A non-randomized controlled trail was designed.The samples of the anterior capsular membrane of lens were collected during the cataract surgery from the children group (1-18 years),youth group (19-45 years),mid adult group (46-60 years) and elder group (＞60 years) and 12 pieces of capsular membrane for each group.In addition,12 pieces of lens capsular membrane from normal donors aged 19-45 years were obtained as normal control group.The expression of SMP30 in the samples was detected by indirect immunofluorescence method.The average fluorescent values were calculated as absorbance (A) / area.Results SMP30 was expressed in LECs of different groups with the green fluorescence primarily in the cytoplasm.The mean fluorescence intensity was 0.185±0.020,0.181±0.034,0.207±0.018 and 0.126±0.027 in the children group,youth group,mid adult group and elder group,respectively,which were significantly enhanced than 0.087±0.007 of the normal control group(q=3.96,3.82,4.01,3.55,all at P＜0.01).No significant differences in the expression of SMP30 among the children group,youth group and mid adult group (all at P＞0.05).However,the expression of SMP30 in LECs in the elder group was significantly lower than that in the children group,youth group and mid adult group (q =3.42,3.21,3.80,all at P＜ 0.05).Conclusions Expression of SMP30 in LECs dramatically increases in cataract patients,suggesting that SMP30 may be a protective factor for LECs.But SMP30 contents are lower in age-related cataract patients,which may be one of causes of senior cataract.