Cultivation and identification of keratoconus associated-fibroblasts / 中华实验眼科杂志

Objective To culture and identify corneal fibroblasts from human keratoconus patients (HKCs).Methods HKCs and corneal fibroblasts from human healthy controls (HCFs) were cultured by tissue block adherence method.Cellular morphology and ultrastructure were observed by inverted phase contrast microscope and electron microscopy respectively.Cell viability was detected by cell counting kit-8 (CCK-8) assay.α-Smooth muscle actin (αt-SMA),collagen type 1 alpha 1 (COL1A1) and collagen type 3 alpha 1 (COL3A1) protein expression levels were detected by Western blot.This study protocol was approved by Ethic Committee of Xi'an No.1 Hospital (No.1504).Results Compared with HCFs,HKCs showed several distinguishing properties.First of all,its growth speed was faster,with collagen fibers decreased and attenuated.At the same time,mitochondrion swelled and mitochondrial cristae disappeared.Additionally,Golgi apparatus presented significant expansion and endoplasmic reticulum displayed severe swelling.There were statistically significant differences in A values between the two kinds of corneal fibroblasts at different time points after culture (Fgroup =5 023.13,P＜0.01;Ftime =38 518.16,P＜0.01),the A value of HKCs was significantly higher than that of HCFs at the same time point,and the difference was statistically significant (all at P＜0.01).The relative expression of α-SMA,COL3A1 and COL1 A1 was 120.00±5.77,158.33 ±4.41 and 88.33± 1.67,respectively in HKCs,the relative expression of α-SMA,COL3A1 and COL1 A1 was 100.00±0.00,100.00±0.00 and 100.00±0.00,respectively in HCFs,the relative expressions of α-SMA and COL3A1 were significantly increased in HKCs than those in HCFs,the relative expression of COL1 A1 was significantly decreased in HKCs than that in HCFs,with significant differences between them (t =-3.46,P ＜ 0.05;t =-13.23,P ＜ 0.01;t =7.00,P＜0.05).Conclusions HKCs are cultured and identied,which is suitable for establishing in vitro cell model of keratoconus.

Research on the Cytotoxicity of the Heterogeneous Acellular Corneal Stromal Leaching Solution on the CHL Cells / 现代检验医学杂志

Objective To select the largest non-toxic leaching solution concentration through the experimental observation of the cytotoxicity of the ostrich acellular corneal stromal leaching solution to the Chinese hamster lung fibroblasts cells(CHL) for the further chromosome distortion experiment.Methods The leaching solution made from the ostrich acellular corneal stromal material was diluted with concentrate of 1 ∶ 2,1 ∶ 4 and the original concentration were used to culture with the CHL cells,the negative and positive control were also set up at the same time,to evaluate the impact on cell growth after 24 hour by MTT colorimetric method.Results The leaching solution diluted with 1∶4 was non-toxic,and could promote the growth of the cells.Conclusion Combined with the results of classification and cell morphological features,this cytotoxicity test can be used to screen the best benchmark non-toxic concentrations for the chromosome aberration test of the CHL cells.

Management of fungal corneal ulcer by corneal collagen cross-linking technique / 中华实验眼科杂志

Background Fungal corneal ulcer is a visual-threatening eye disease,and drug therapy has a limiting efficacy.Corneal transplantation or eye enucleation sometimes is necessary to the severe patients.Corneal collagen cross-linking (CXL) is an effective method for some corneal diseases,but the study on CXL for fungal corneal ulcer is lack.Objective This study was to evaluate the clinical effectiveness and safety CXL for fungal corneal ulcer.Methods Fifteen 8-week-old healthy New Zealand white rabbits were used in this study and other 5 rabbits served as normal controls.Fungal corneal ulcer models were established in the right eyes of other 10 rabbits by infecting sickle bacteria liquid after corneal scratching and removing corneal epithelium,then decellularized ostrich corneal patch covered the defected cornea.The models were randomly divided into the non-treatment group and the CXL treatment group.Corneal lesions were examined under the slit lamp microscope every day,and cornea was pictured by laser scanning confocal microscope on the 3rd,7th,14th,21st and 28th day individually after CXL.All rabbits were sacrificed and corneal tissues were obtained 4 weeks after treatment,and the collagen fiber diameter and fibrocytes were observed under the scanning electron microscope.Results Fungal corneal ulcer models were successfully established by corneal scratching and decellularized ostrich cornea covering.The gray ulcer lesions and hypbae like bean pod were seen by slit lamp microscope and laser scanning confocal microscope 3 days after modeling.Corneal ulcer deepened and expanded 1 week later,and there were a large number of spore and hyphae criss-crossing as short rod in shallow stroma.Inflammatory cells were observed in corneal endothelial cells and ocular anterior chamber.In the CXL treatment group,the range of corneal epithelial deficiency was less than that in the nontreatment group on the 3rd,7th,14th,and 21st (all at P＜ 0.05).The diameters of collagen fibers were (24.6± 1.8) nm,(24.9 ± 1.9) nm and (43.0 ± 7.4) nm in the normal control group,non-treatment group and CXL treatment group,showing a significant difference among the 3 groups (F =27.05,P =0.00),and the collagen diameters were thicker in the CXL treatment group than those in the normal control group and non-treatment group (t =5.40,-5.30,both at P＜0.05),and fibrocytes were seen among the collagen fibers.No significant difference was found in the collagen diameters between the non-treatment group and normal control group,and the fibrocytes were less in the non-treatment group.Conclusions CXL therapy can treat fungal corneal ulcer by enhancing collagen,promoting fibrocytes proliferation,suppressing fungus and inflammatory response and accelerating tissue repair.

Effect of ostrich acellular heterogeneous corneal stroma ectopic transplantation on T lymphocyte subsets in peripheral blood of BALB/c mice / 中华实验眼科杂志

Background Ostrich acellular corneal stroma possesses a similar constitution to human corneal stroma,so it is expected to become one of ideal biological corneal carriers.Objective This study was to investigate the immunogenicity of acellular stroma carrier of ostrich cornea and offer the information for the development of industrialization and clinical use of acellular stroma carrier of ostrich cornea.Methods Twenty fresh ostrich eyeballs and 20 porcine eyeballs were collected.Acellular corneal stroma carriers of ostriches and pigs were prepared using low temperature freezing joint enzyme digestion method and desiccant dehydration method and sterilized by cobalt-60 irradiation.The corneal stroma carriers were preserved using drying and dehydration method.Forty-five male BALB/c mice were randomly divided into the sham operation group,ostrich acellular corneal stroma group and porcine acellular corneal stroma group.Acellular corneal stroma carriers of ostriches and pigs(wet weight after rewatering was 10 mg/piece) were subcutaneously implanted to the back of BALB/c mice,respectively.Wound healing and inflammatory response on the operative site were observed,and phenotype and activating rate of CD4+,CD8+ and CD25+in peripheral blood of mice were dynamically detected 7,14 and 28 days after ectopically implantation of heterogeneous corneal stroma by immunofluorescence labeling and flow cytometry analysis.Results No swelling and exudation were seen in the skin of operative site of the mice with a good healing of wound after surgery.There were no significant differences in the activating rates of CD4+,CD8+ and CD25+ cells in the peripheral blood of mice among the sham operation group,the porcine acellular corneal stroma group and ostrich acellular corneal stroma group in the three time points after surgery(CD4+:F=0.74,P=0.50;F=0.39,P=0.05;F=3.46,P=0.58.CD8+:F=1.75,P=0.21 ;F=1.14,P=0.35;F=0.78,P=0.48.CD25+:F=0.52,P=0.61 ;F=3.53,P=0.62;F=2.42,P=0.13).Conclusions The ostrich acellular heterogeneous corneal stroma carrier possesses low immunogenicity.It is inferred that ostrich acellular corneal stroma carrier can be used in heterogeneous corneal transplantation.

Cytotoxicity of dehydrated ostrich acellular corneal stroma as a carrier material / 中国组织工程研究

BACKGROUND:Previous studies from Shaanxi Institute of Ophthalmology have shown that ostrich cornea has the advantages to be developed into the alternatives of human corneal material. OBJECTIVE:To determine the potential toxic effects of ostrich corneal stromal scaffold on cel s. METHODS:Cel culture methods were used to culture L-929 cel s in the extracts of ostrich acel ular corneal stroma which was dried and dehydrated. 3-(4,5)-Dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay was used to evaluate the growth and proliferation of cel s after cultured for 1, 2 and 3 days. RESULTS AND CONCLUSION:After the cel s were cultured in the extracts of ostrich acel ular corneal stroma subjected to dryness and dehydration for 1, 3 and 5 days, and the toxicity level of cultured cel s was graded as level 1. The cytotoxicity test was conducted according to the“National Standard of the People's Republic of China GB/T16886.5-2003”. After cultured in the extracts of ostrich acel ular corneal stroma, a smal number of cel s were round in shape and loosely adherent without intracytoplasmic granules, and cel lysis could be observed occasional y. The results of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay showed that the ostrich acel ular corneal stromal scaffold which was dried and dehydrated had level 1 of cytotoxicity and could be considered as a qualified material.

Etiological identification of 50 isolates from fungal corneal ulceration by semi-nested PCR amplification of ITS2 region / 中华检验医学杂志

Objective To identify the pathogens of 50 cases of fungal corneal ulceration by using semi-nested PCR amplification of ITS2 region.Methods Fifty isolates of fungal corneal ulceration and 3standard fungal strains cultures were collected and their DNAs were extracted.Their ITS2 regions were amplified by semi-nested PCR and sequenced.The results were compared with the nucleotide sequences in the NCBI GenBank.The pathogens of the fungi were identified and their distribution were analysed.Results The sequences results of the 3 standard fungal strains were consistent with the information in the GenBank.The pathological microorganisms of 50 cases of fungal corneal ulceration were:24 Fusarium (48％),including 17 Fusarium solani,6 Fusarium oxysporum and 1 Fusarium verticillioide; 10 Aspergillius (20％),including 5 Aspergillius flavus,3 Aspergillius sydowii and 2 Aspergillius nidulans; 6 Penicillium (12％),including 2 Penicillium citreo-viride,2 Penicillium multicolor and 2 Penicillium oxalicum ;5 Candida (10％),including 3 Candida albicans and 2 Candida parapsilosis; 3 Cladosporium (6％),including 2 Cladosporium herbarum and 1 case of Cladosporium cladosporioides ; 1 case of Neurospora crassa (2％) ;1 Alternaria alternata(2％).Conclusion Semi-nested PCR amplification of ITS2 region was proved to be a fast,simple and accurate method to identify pathogens of fungal corneal ulceration,and may be useful for personalized treatment and epidemiological investigation of local fungi.