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1.The Effect of ALB1 Gene on Adhesion of Aspergillus Fumigatus 2.Mechanisms of Inhibit the Proliferation of Candida Albicans by Mouse Cornea

Author: XiHaiJie
Tutor: WangYiQiang
School: Jinan University
Course: Ophthalmology
Keywords: Of the A1b1 gene △ alb1 B5233 Adhere to Cornea Candida albicans Suppression OD values
CLC: R378
Type: Master's thesis
Year: 2011
Downloads: 22
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Abstract

Objective To explore the role Aspergillus mold the A1b1 of gene in fungal corneal adhesion, to provide information to clarify the molecular mechanisms of fungal corneal interactions. The method of use of the wild-type strain of Aspergillus fumigatus (B5233) gene mutant (△ alb1) of spore adhesion monolayer of corneal epithelial cells and corneal organ in vitro and in vivo animal models, by detecting cells and corneal number of adhered spores, determine the A1b1 gene mutations Aspergillus fumigatus corneal adhesion. 1 compare these two strains of corneal epithelial cells in monolayer adhesion amount of the good growth of corneal epithelial cells (HCEC) were seeded on 24-well plates, 1 × 10 5 cells per well and incubated for 6-8 hrs cells attached wall after serum-free culture medium overnight. Fungal spore suspension was added, the amount of fungal spores, and the proportion of cell volume 10:1,37 ° C, saturated humidity, 5% CO2 incubator and incubated for 60min, washed three times with physiological saline, the hole plus fluorescent white staining 30s fluorescence photomicrograph . The coupled cell lysates epithelial cells samples at different dilutions uniform coating in Sabouraud medium plates, 37 ° C cultured for 24 hours, counting the colonies formed. The experiment was repeated three times. 5. Observation of two strains of the difference of the amount of adhesion of the eye organ models in mice Balb / c mice after treatment to remove the eye, placed in 96-well plates. 10 eyes were divided into two groups, the two sets of holes respectively join B5233 and △ alb1 the fungal spore suspension (concentration of 1 × 10 9CFU/ml), and placed in 37 ° C, 5% CO2 incubator incubated for 30 min. Remove the eye, washed three times with saline, three in each eye detect bacteria carrying amount; another two eye neutral formalin-fixed, paraffin-embedded, sliced ??glycogen staining (PAS staining) observed spore adhesion. The experiment was repeated five times. (3) the difference between in vivo experiments comparing the two bacteria adhesion 8-10 weeks Balb / c mice were divided into two groups of five each. Were anesthetized with filter paper to wipe the surface of the cornea, then about 0.5cm long infusion tube sets in the mouse eye, suture fixation. Tube plus 0.5? The TA 20 ul role 1h. Pipette go EDTA, the two groups were added to two strains of fungal spore suspension (concentration of 1 × 10 9CFU/mL) 10 ul role 1h, remove the casing, cervical dislocation, the mice were killed, the removal of the eye with physiological brine three times. Three in each eye is used to set the amount of bacteria detection, counting the colonies formed; 2 eye fixed with neutral formalin, embedded in paraffin, glycogen staining (PAS staining) observed spore adhesion. The experiment was repeated two more times. Results 1. Corneal epithelial cell adhesion the assay two strains adhesion amount of the difference between a fluorescence microscope images visible B5233 strain adhesion density was significantly higher than the △ alb1 strain. Fungal load testing, the B5233 strain group adhesion number 151635 ± 27330 cells / well (CV = 18.0%), the number of △ of alb 1 strain group adhesion of 63333 ± 15756 cells / well (CV = 24.9%), the B5233 strain adhesion rate is greater than the the △ alb1 strains. And the difference between the two groups was statistically significant (P lt; 0.01). (2) the eye organ model to detect the amount of corneal adhesion of the two strains difference fungal load detection, the amount of adhesion of the two strains were 1676 ± 313 cells / organs, 675 ± 102 / organ. The B5233 strain adhesion rate is greater than the △ alb1 strain, and the difference between the two groups was statistically significant (P lt; 0.01). Animal experiments comparing two strains of corneal adhesion fungal load detection, the number of adhesion B5233 strain group was 680 ± 121 / (CV = 17.8%) of the cornea, △ of alb 1 strain group the number of adhesion of 314 ± 45 / (CV = 14.3%) of the cornea. The B5233 strain adhesion rate is greater than the △ alb1 strain. And the difference between the two groups was statistically significant (P lt; 0.01). Concluding the A1b1 gene mutations in the fungal spores decreased adhesion of corneal epithelial cells and in vivo and in vitro corneal. Purpose of murine corneal mechanism inhibit the growth of Candida albicans mouse corneal homogenate supernatant and inactivation of fungal spores Lysates stimulation of human corneal epithelial cells, human corneal stromal cell culture supernatant of the culture of Candida albicans, respectively, every 6h measured 600nm optical density (OD) values, the final fungal growth curves plotted according to the OD value of each point measured. The corneal homogenate Candida albicans Total culture 2h, 4h, 6h after PI staining, laser scanning confocal microscope photography to observe the death fungi (PI staining positive) ratio. Results: 1. Corneal homogenate supernatant of Candida albicans growth curve of the cornea homogenate supernatant with Candida albicans co-cultured for 30 hours, the experimental group at each time point after 6h OD values ??were higher . HCEC cells 2.HCEC cell culture supernatant of Candida albicans growth curve of the cell culture supernatant collected plus inactivated broth stimulate co-cultured with Candida albicans. Fungal growth curves show that HCEC culture supernatant no effect on the growth of fungi. 3.HTK and U937 cell culture supernatant of Candida albicans growth curve of co-culture system HTK and U937 cells were stimulated with inactivated fungal lysates collected culture supernatant and Candida albicans were cultured for 30 hours fungal growth curve of the experimental group also showed that the bacterium growth has no effect. Cornea homogenate supernatant of Candida albicans survival of corneal homogenate with Candida albicans were cultured for 2 h, 4H, 6 h after PI staining, three time points showed no corneal lysate promote or inhibit spore death role, but in the role of 4h, 6h when visible corneal lysate for fungal spore germination and sprouting. The conclusion has a natural defense capability, fungal corneal only when this natural defense (such as the structural integrity of the corneal epithelium is damaged), fungi may have on the cornea colonization only possible fungal keratitis.

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