Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.

Measurement of retinal thickness in macular region of high myopic eyes using spectral domain OCT

AIM: To investigate the changes of retinal thickness in macula of high myopic eyes using spectral domain optical coherence tomography(OCT). ·METHODS: Middle-aged and young myopic patients were divided into three groups according to their refractive error/axial length: low and medium myopia group(LMMG),high myopia group(HMG) and super high myopia group(SHMG). Cirrus HD-OCT was used to evaluate total average macular thickness,central subfield thickness,inner/outer macular thickness and macular volume. The differences among experimental groups were analyzed by one-factor analysis of variance. Associations between macular thickness and refractive error/axial length were analyzed by Pearson correlation analysis. ·RESULTS: There was no significant difference in age among the three groups(P =0.2789). The mean refraction error in the LMMG,HMG,and SHMG groups was-2.49 ± 1.38D,-8.53 ±1.95D and-13.88 ±1.76D,respectively(P 【 0.001). The central subfield thickness of three groups was 244.56 ±12.19μm,254.33 ±11.61μm and 261.75 ± 11.83μm,respectively,and there were statistically significance between random two groups. The total average macular thickness,inner/outer macular thickness,and macular volume decreased with increased myopia/axial length. Average foveal thickness had negative correlations with refractive error(P 【0.001),and positive correlations with axial length. The inferior and temporal inner macular thickness,all the quadrants of outer ring,total average macular thickness and macular volume featured positive correlations with refractive error,and negative correlations with axial length. Average foveal thickness,superior and temporal innermacular thicknesses,and temporal outer macular thickness was lower in females compared to males. ·CONCLUSION: With an increase in myopia degree/axial length,the average foveal thickness increased and the inner/outer macular thickness decreased. Females featured thicker average foveal thickness,and thinner macular thickness compared to males.

Phylogenetic incongruence in Cymbidium orchids

Cymbidium,which includes approximately 80 species,is one of the most ornamental and cultivated orchid genera.However,a lack of markers and sparse sampling have posed great challenges to resolving the phylogenetic relationships within the genus.In the present study,we reconstructed the phylogenetic relationships by utilizing one nuclear DNA(nrITS)and seven plastid genes(rbcL,trnS,trnG,matK,trnL,psbA,and atpI)from 70 species(varieties)in Cymbidium.We also examined the occurrence of phylogenetic conflict between nuclear(nrITS)and plastid loci and investigated how phylogenetic conflict bears on taxonomic classification within the genus.We found that phylogenetic conflict and low support values may be explained by hybridization and a lack of informative characteristics.Our results do not support previous classification of the subgenera and sections within Cymbidium.Discordance between gene trees and network analysis indicate that reticulate evolution occurred in the genus Cymbidium.Overall,our study indicates that Cymbidium has undergone a complex evolution.

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

AIM：To study the inhibition of nuclear factor kappa-B p65（NF-κB p65）antisense oligodeoxynucleotide（ASODN）on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2（TGF-β2）.·M ETHODS：NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide（MSODN）were designed and synthesized.Human lens epithelial cell line（HLE B-3）cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in dulbecco’s modified eagle medium（DMEM）.T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent（Hi Per Fect）for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction（RT-PCR）,and the expression ofα-smooth muscle actin（α-SMA）protein was assayed with ELISA.·RESULTS：With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant（〈0.05）.NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A＋T group was not statistically significant（〉0.05）,but the difference among A＋T group and other groups was statistically significant（〈0.05）.·CONCLUSION：NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Interactions of thymic stromal lymphopoietin with interleukin-4 in adaptive immunity during Aspergillus fumigatus keratitis

AIM:To investigate the potential interactions of thymic stromal lymphopoietin(TSLP)with interleukin-4(IL-4)in adaptive immunity during fungal keratitis(FK).METHODS:An FK mouse model was induced with Aspergillus fumigatus(AF)hyphal infection.Mice were divided into several groups:untreated,phosphate buffer saline(PBS),infected with AF,and pretreated with a scrambled siRNA,a TSLP-specific siRNA(TSLP siRNA),murine recombinant TSLP(rTSLP),immunoglobulin G(IgG),murine recombinant IFN(rIFN-γ),murine recombinant IL-4(rI L-4),rIL-13,murine recombinant IL-17A(rIL-17A),and murine recombinant IL-17F(rIL-17F)groups.Quantitative realtime reverse transcription-polymerase chain reaction(qRTPCR)and enzyme-linked immunosorbent assay(ELISA)or Western blot were performed to determine mRNA and protein levels in the inflamed cornea.Cytokine locations were observed by immunofluoresence staining after AF hyphal infection.RESULTS:Compared to those in the untreated group,TSLP and T helper type 1(Th1)cytokine levels in the AF group were upregulated at 24 h post infection(hpi),and those of T helper type 2(Th2)and T helper type 17(Th17)cytokines were increased at 5 d post infection(dpi).Th2 cytokine levels were decreased in the TSLP siRNA-pretreated group and increased in the rTSLP-pretreated group compared with the AF group.The TSLP level was increased in the rIL-4-pretreated group,but there were no significant changes among the other groups.Immunofluorescence staining showed cytokine locations after AF hyphal infection.CONCLUSION:TSLP induces a Th2 immune response and promots Th2 T cell differentiation in vivo.IL-4 promotes TSLP secretion.Therefore,TSLP with IL-4 regulates adaptive immunity in FK.

General anesthesia versus local anesthesia for penetrating keratoplasty: a prospective study

AIM:To examine which anesthesia general or local is more effective for penetrating keratoplasty(PKP).METHODS:Patients with indications for PKP(n=141)were enrolled in a prospective study and randomly divided into general anesthesia group(group A,70 eyes)and local anesthesia group(group B,71 eyes).Patients received optical PKP(group A1,30 eyes;group B1,30eyes)or therapeutic PKP(group A2,40 eyes;group B2,41 eyes).Measurement of anterior chamber treatment time(T)for PKP patients and the ratio(R)of the area of the pupils to that of recipient graft region.T and R values,as well as perioperative and postoperative complications,were compared between groups A and B using t-test orχ2test.RESULTS:Patients were followed for 2wk after PKP.T was(13.45±8.64)min for group A and(7.36±5.24)min for group B,a statistically significant difference(P【0.001).The R value for group A was stable during the operation,while for PKP patients in group B the value initially increased then gradually decreased to normal after suturing.In group B,extrusion of intraocular contents occurred in 5 eyes,and iridal prolapse occured in 11cases;no perioperative complications occurred in group A.Relapse rate for fungal keratitis was 13.04%in group B and 0%in group A.CONCLUSION:Under general anesthesia,pupils remaine stable during PKP and perioperative complications are averted.General anesthesia gives more time to treat pathological changes in the anterior chamber and treatment success rate is higher.

Pharmacokinetic Studies of Factor VIII in Chinese Boys with Severe Hemophilia A： A Single-Center Study

Background： Although much attention has been paid to the pharmacokinetics （PKs） of different factor VIII （FVIlI） concentrates in persons with hemophilia A （HA）, limited information is available in young boys with severe HA. In this study, we aimed to assess the PK parameters of FVIII products in boys with severe HA in China. Methods： A total of 36 boys （plasma-derived [pd]-FVIII, n= 15; recombinant [r] FVIII, n = 21） were enrolled between January 2015 and May 2016 in Beijing Children＇s Hospital. PK characteristics of FVIII products were studied according to a reduced 4-sampling time point design （1 h, 9 h, 24 h, and 48 h postinfusion）. Results： The mean FVIII half-life （t1/2） was 10.99 ± 3.45 h （range 5.52-20.02 h）, the mean in vivo recovery （IVR） was 2.01 ± 0.42 IU/dl per IU/kg （range 1.24-3.02 IU/dl per IU/kg） and mean clearance （CL） of FVIII is 4.34 ± 1.58 ml·kg^-1·h ^-1 （range 2.29-7.90 ml·kg^-1·h·^-1）. We also analyzed the influence of several parameters that potentially modulate FVIII PK. The age was closely associated with FVIII half-life （R^2 = 0.32, P 〈 0.01 ）. The t1/2 of FVIII increased by 0.59 h per year. Besides age, yon Willebrand factor antigen （VWF：Ag） also was associated with FVIII half-life （R^2 = 0.52, P 〈 0.01）. Patients with blood Group O had a shorter FVIII halt-life than patients with non-O blood group （9.40 ± 0.68 h vs. 12.3 ± 0.79 h, t = 2.70, P = 0.01）. The FVIII IVR correlated with age （R^2 =0.21, P 〈 0.01） and VWF：Ag level （R^2 = 0.28, P 〈 0.01 ）. CL rates were taster in young patients and in those with low-VWF：Ag levels. CL rates of FVIII are higher in blood Group O versus non-blood Group O persons （5.02 ± 0.38 vs. 4.00 ± 0.32 ml·kg^-1·h^-1 , t = 2.53, P = 0.02）. Conclusions： Chinese boys with severe HA have similar PK values to other ethnic groups and large differences in FVIII PK between individual patients. Age, blood group, and VWF：Ag levels are important determining factors for FVlll CL.