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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.
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Objectives@#To explore the expression regulation of type 1 and type 2 (Th1 and Th2) cytokines from serum of coal miners and the evaluation in surveillance of coal workers' pneumoconiosis, 630 coal miners were studied.@*Methods@#A total of 90 male patients diagnosed as coal workers' pneumoconiosis (CWP) in a institute for occupational health and 19 male workers newly diagnosed as CWP patients was chosen as CWP group with simple random sampling method from a coal mine group from January 2013 to December in 2015. 180 male coal miners with abnormal but not diagnosed as CWP were selected as CWP suspected group with simple random sampling methods, meanwhile 180 male coal miners with normal chest X-ray photograph was as dust-exposed group by 1∶1 matched as age. And 161 healthy males accepted pre-employed examination were selected as control group, CWP suspected group, dust-exposed group and control group called as non-CWP group. According to screening test and diagnosis test, the basic information and occupational history of all subjects were collected, and cytokines including IL-1β, IL-8, IFN-γ, IL-6 and IL-10 of serum were detected. Receiver operator characteristic (ROC) curve was used to determine the optimal cutoff value of each cytokine. Area under curve (AUC), the validity and reliability were calculated and judged.@*Results@#The average age of control group, dust-exposed group, CWP suspected group and CWP group were (27.4±5.0) , (43.4±10.7) , (48.2±6.2) , (64.7±7.0) years old, respectively. The median level of IL-1β, IL-8, IFN-γ and IL-6 in cases group (1 638.30, 2 099.49, 815.18,140.32 pg/ml) were higher than that of non-cases group (1 445.57, 1 402.26, 736.38, 95.73 pg/ml) (P<0.05) . The level of IL-8 (1 503.99 pg/ml) in CWP suspected group was higher than that of control group (1 295.67 pg/ml) and dust-exposed group (1 376.94 pg/ml) , but the level of IL-10 (654.08 pg/ml) was lower than that of control group (596.64 pg/ml) . The ratio of IFN-γ/IL-6 ranged from 5 to 8, and the ratio in CWP group (5.87) was lower than that of non-CWP group (7.61) . The IL-6 and IL-8 among the subjects of dust-exposed group in terms of the age distribution of among had reached statistical significance. According to ROC, the cutoff value of IL-1β, IL-6, IL-8, IL-10 and INF-γ reached 1 582.65, 116.53, 1 791.54, 581.08 and 792.69 pg/ml, respectively. The AUC was 0.668, 0.895, 0.859, 0.716 and 0.637, respectively. It was found that IL-6 and IL-8 could be used as biomarkers in detecting CWP, the sensitivity and specificity was 82.6% and 84.6%, 78.0% and 84.8%, respectively; Youden's index was 0.674 and 0.628 and the consistency rate was 84.3% and 83.7%, while Kappa value was 0.55 and 0.52.@*Conclusion@#There was Type 1 and type 2 cytokine dysregulation in CWP patients. IL-6 and IL-8 can be used as effective biomarkers to forecast lung injury before X-ray changes.
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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.
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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.
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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.
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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.
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In the treatment of 80 cases of the patients with deficient and cold pattern in the spleen and stomach by external application of herbal paste on the acupoints, the results showed remarkable effect in 52 cases, improvement in 24 cases, failure in 4 cases and the total effective rate in 95.0%. Remarkable therapeutic effects were achieved in three different diseases of same pattern. External application of herbal paste on the acupoints is definitely effective for stomachache in deficient and cold pattern of the spleen and stomach.
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Combined with the clinical practice of Xi'an Eye Bank for over four decades,this article focuses on the ethical issues related to the practice of eye bank,such as voluntary cornea donation free of charge,the relationship between donors and recipients,the choice of operation indication and application of heterogeneous cornea,in order to promote the all-round development of eye bank system.