ABSTRACT
Background Epithelial-mesenchymal transition (EMT) is one of the pathogenesis mechanisms of posterior capule opcification (PCO).Studying EMT is of important significance for the prevention and treatment of PCO.However,EMT model is lack.Objective This study was to establish an in vitro EMT model for the study of human PCO.Methods In vitro mimic cataract enucleation was performed on 40 donor eyes,including anterior capsulorhexis,nucleus hydroexpression,and aspiration of lens fibers.The capsular bag of lens was dissected free during the surgery and pinned flat on a plastic culture dish with DMEM/F12 supplemented containing 10％ fetal bovine serum for 4 weeks.The proliferation of LECs on the capsular bag was observed by phase-contrast and dark-field microscope in 0,3,7,14 and 28 days after culture.The capsular bag tissues were collected in cultured 0,3,7 and 28 days for the preparation of sections and hematoxylin-eosin stain,and the growth and morphology of LECs were examined with optical microscope.The expression and location of α-SMA,E-cadherin and Vimentin were assessed by immunochemistry.The expression levels of α-SMA,E-cadherin and Vimentin mRNA and proteins were detected by using real-time fluorescnce quantitative PCR and Western blot in different time points.Results No LECs were seen on the uncultured capsular bag.LECs appeared in cultured day 3 on the periphery capsular bag and grew toward the center and covered the posterior capsule 7 days after cultured,with a cobblestone-like appearance.Wrinkles occurred and extended gradually along with the enhancement of bag tension.Immunochestry showed that the expression intensity of E-cadherin in the capsular bag gradually weakened,and that of α-SMA or Vimentin was gradually enhanced during the culture duration.The relative expression levels of E-cadherhin mRNA at 0,3,7,14 and 28 days after culture were 3.35±0.13,1.47±0.20,1.13±0.14,1.00±0.85 and 0.23±0.03,and relative expression levels of Vimentin mRNA were 1.00 ± 0.73,1.05 ± 0.14,2.24 ± 0.43,2.84 ± 0.34 and 8.57 ± 0.40,and those of α-SMA mRNA were 1.00 ± 0.06,2.68 ± 0.28,4.24 ± 0.05,2.05 ± 0.90 and 15.30 ± 0.19,showing significant differences among different time points (E-cadherhin mRNA:F =23.430,P =0.000;Vimentin mRNA F =8.915,P =0.002;α-SMA mRNA:F =103.500,P =0.000),with the lowest expression levels in the E-cadherhin mRNA and the highest expression levels in the Vimentin mRNA and α-SMA mRNA at 28 days during the culture period (all at P＜0.01).The gray values of E-cadherin protein expression were 1.443 ± 0.017,1.023 ± 0.003 and 0.568 ± 0.018,and those of Vimentin protein were 0.565±0.012,1.156±0.007 and 1.241±0.009,and those of α-SMA protein were 0.195±0.045,0.693±0.036 and 1.501±0.005 at 0,3 and 28 days,with significnant differences among various time points (E-cadherin:F =2 787.000,P =0.000;Vimentin:F =4 488.000,P =0.000;α-SMA:F =1 173.000,P =0.000).The expression levels were significantly declined in E-cadherhin protein and elevated in Vimentin and α-SMA proteins at 3 and 28 days after culture in comparison with before culture.Conclusions A novel in vitro EMT model of LECs is established in this study.This model can mimic a natural EMT procedure after extracapsular cataract enucleation and therefore is a useful model for the further research of the mechanism and prevention and treatment of PCO.
ABSTRACT
Background Endophthalmitis is a serious complication of intraocular surgery.Conventional identification methods for bacteria are becterial culture and smear method,but these laboratory tests spend a long time and have low positive rates.16S rDNA is the bacterial chromosome encoding ribosomal RNA sequences,and it is determined that 16S rDNA sequencing has a high specificity for the identification of bacteria.Objective This study was to identify the infectious bacteria from aqueous humor or vitreous body in the eyes with endophthalmitis by 16S rDNA sequencing technique,and to investigate the diagnosis efficency of 16S rDNA sequencing technique on bacterial endophthalmitis.Methods Anterior chamber fluid (0.1-0.2 ml) or vitreous humor (0.5-1.0 ml) specimens were collected from 5 eyes of 5 patients with endophthalmitis in Qingdao Eye Hospital from June to December 2015 and used for high throughput sequencing,bacterial culture and smear,respectively.Bacteria DNA was extracted from the specimen with D3096-01 trace DNA kit for the amplification of V3-V4 region of 16S rRNA gene and sequencing of hypervariable region of all microbes in the samples by MiSeq Illumina Sequencing Platform.Then the bioinformatic analysis including analysis of taxonomy,abundance and alpha diversity were performed.Nucleasefree water of 50 μl in the centrifuge tube was used as control.Results Five aqueous humor or vitreous body samples were collected,and the positive results were exhibited by smear examination,with the Gram positive bacilli in the trumatic endophthalmitis eye and Gram negative bacilli in the filtering bleb infectious endophthalmitis eye,and all culture results were negative.16S rDNA squencing showed the positive outcomes in all the 5 samples.The high abundent nacteral genuses were Staphylococcus (65.28％),Streptococcus (18.90％) and Pseudomonas (12.76％) in the trumatic endophthalmitis eye;the major components of sample were Pseudomonas (53.68％),Acinetobacter (8.62％) and Limnobacter (5.96％) in the eye with acute endophthalmitis occurring at 2 days following cataract surgery;the major components in the filtering bleb infectious endophthalmitis eye were Moraxella (88.89％) and Pseudomonas (9.52％);the Pseudomonas was major components in the later-onset endophthalmitis eye (84.63％) and the eye with acute endophthalmitis occurring at 1 day after cataract surgery (97.89％).Conclusions A distinct advantage is found in 16S rDNA sequencing technique for the indentification of the pathogenic bacteria in endophthalmitis eyes due to its high positive rate in comparison with bacterial culture and smear method.
ABSTRACT
Objective To study the expression and clinical significance of serum levels of transforming growth factor-31 (TGF-β1) and tumor necrosis factor-alpha(TNF-α) in patients with hypertensive disorder complicating pregnancy(HDCP).Methods The serum levels of TGF-β1 and TNF-αin 50 cases with HDCP and 30 cases of normal third trimester pregnant women(control group) were detected by ELISA.50 cases with HDCP were divided into gestational hypertension group,mild preeclampsia group and severe preeclampsia group according to the severity of HDCP.Results The serum levels of TGF-β1 and TNF-α in HDCP patients were significantly higher than the control group (t =13.283,13.607,all P ＜ 0.05).The serum levels of TGF-β1 and TNF-α gradually increased with the aggravating of HDCP disease,the serum levels of TGF-β1 and TNF-αwere significantly different among the three subgroups of HDCP(P ＜ 0.05).The correlation analysis showed that the serum levels of TGF-β1,TNF-α were positively correlated with the severity of HDCP (r =0.575,0.512,all P ＜ 0.05),there was significantly positive correlation between the serum TGF-β1 and TNF-α(r =0.515,P ＜0.05).Conclusion The serum levels of TGF-β1 and TNF-α were high in the HDCP patients,the high levels of TGF-β1 and TNF-α can reflect the changes and development of HDCP disease.The abnormal levels of TGF-β1 and TNF-α may be associate with dysfunction of trophoblast cell.
ABSTRACT
Objective To discuss treatment effect of ritodrine and ambroxol on acceleration of fetal lung maturation,reduction of asphyxia of premature infants in premature women,and nursing. Methods 218 pregnant woman with premature delivery were divided into the treatment group (110 cases) and the control group ( 108 cases)stochastically.The treatment group adopted ritodrine and ambroxol,the control group adopted routine method.The curative effect was compared between two groups. Results The prolonged pregnancy time,rate of term labor,rate of newborn body weight ＞2500 g,incidence rate of puerperal infection,rate of neonatal pneumonia in the treatment group were better than those of the control group. Conclusions Appli cation of ritodrine in treating premature delivery proved to be safe and effective.For premature infants ＜ 34 weeks,prenatal use of ambroxol may promote the fetal lung maturity and reducecomplication rate of mothers and infants.
