1.The Effect Of ALB1 Gene On Adhesion Of Aspergillus Fumigatus 2.Mechanisms Of Inhibit The Proliferation Of Candida Albicans By Mouse Cornea

PURPOSEExplore the role of ALB1 gene of Aspergillus fumigatus to fungal corneal adherence , To provide information for clarify the interaction Molecular mechanism between fungi and cornea..METHODTo determine the effect of ALB1 gene mutation on Aspergillus fungal corneal adherence, we compare the amount of Aspergillus fumigatus, respectively, using wild-type strain (B5233) and gene mutants strain (â–³alb1) spores adhesion the corneal epithelial cell monolayer model , cornea of eye organ model in vitro, and animal test in vivo, by detecting the amount of adherent spores.1 Use the corneal epithelial cells model to observe the difference between two strains adherenceThe high quality corneal epithelial cells were collected and seeded in 24 well plate, 1×105 cells/hole. The cells were cultured for 6-8 hours and transferred into serum-free medium for overnight and then co-cultured with suspension of fungal spores. The ratio of fungal spores and cells is 10:1. 37℃, saturated humidity and 5% CO₂ incubator for 60min, washed three times with normal saline. Cells stained with calcofluor white stain, photograph with fluorescence microscopy. Plus cell lysis, samples at different dilutions in Sabouraud medium evenly coated plates, 37℃cultured 24h, counting the formation of colonies . The experiment was repeated 3 times.2 Use the eye organ model to observe the difference between the amount of adherence spores of two strains10 eyes of 6-8weeks Balb/c mice were divided into two groups and placed into 96-well plate ,co-cultured with two fungi spores suspension B5233 andâ–³alb1 (concentration of 1×10⁹/ml) respectively. Then, they were placed in 37℃, 5% CO₂ incubator for 30min and washed three times with normal saline. In each group, three eyes were used to detect the colony-forming unit by counting the formation of colonies and two eyes were fixated with formalin, embedded in paraffin and stained glycogen (PAS staining) to observe spores adhesion.3 Compare the difference between two strains, the amount of fungal adhesion with animal experiment10 of Balb/c mice were divided into two groups, n = 5, after Anesthesia the first clean and polish the corneal surface with filter paper, and then fixed a 0.5cm tube on mouse eye , and sew a needle to ensure tube can not fall off. Each tube was added with 20ul 0.5% EDTA and kept on 1h. Then EDTA was removed and two strains of fungi spores suspension (concentration 1×10⁹/ml) 10μl, after 1h role. Removal of the tube, mice were sacrificed, the removal of the eye wash three times with normal saline, in each group, three eyes were used to detect the colony-forming unit, counting the formation of colonies. two eyes fixated with formalin, embedded in paraffin, stained glycogen (PAS staining) observed spores adherence.RESULTS1 The difference between two strains on amount of adherence by corneal epithelial cells adherence assay.Fluorescence micrograph results shows that the adhesion density of B5233 strain was significantly greater than theâ–³alb1 strain. Load of Aspergillus fumigatus detection There were 151635±27330 B5233 spores/hole, (CV=18.0%), 63333±15756â–³alb1 spores/hole (CV=24.9%). Adherence rate of B5233 strain was greater thanâ–³alb1 strain significantly ,(P <0.01).2 The difference of two strains in the amount of corneal adherence on the eye organ model.By the fungal load testing There were1676±313 B5233 spores/cornea, (CV=18.0%), 675±102â–³alb1 spores/cornea (CV=24.9%). Adherence rate of B5233 strain was greater thanâ–³alb1 strain significantly (P <0.01).3 The difference of two strains on the amount of corneal adherence by animal experiments.By the fungal load testing ,There were 680±121 B5233 spores/cornea, (CV=17.8%), 314±45â–³alb1 spores/cornea (CV=14.3%). Adherence rate of B5233 strain was greater thanâ–³alb1 strain significantly (P <0.01).CONCLUSIONALB1 gene mutation enable the fungal adherence decreased on corneal epithelial cells,cornea in vivo and in vitro. Purpose: Mechanisms about Inhibit the proliferation of Candida albicans by mouse cornea Methods: Homogenate supernatant of mouse cornea and cell culture supernatant of HCEC and HTK stimulated by Inactivated fungal spores lysate,co-cultured with Candida albicans individually. Test 600nm optical density (OD) value every 6h, Finally, according to the points OD value drawn a fungal Growth curves. After corneal homogenate with C. albicans co-cultured 2h, 4h, 6h staining with PI, Photograph with the Laser scanning confocal microscope. Observe the proportion of dead fungal (positive of PI stain).Results:1.The influence about corneal homogenate supernatant to candida albicans growth curveCorneal homogenate supernatant were co-cultured with C. albicans 30 hours , after co-cultured 6h,the OD values of experimental group were higher than control group at each time point.2. The influence about HCEC cells culture medium on the growth curve of Candida albicansCollected HCEC cells culture medium after cells stimulated with Inactivated fungal spores lysate, and co-cultured with candida albicans . Fungal growth curve shows that HCEC cells culture medium has no effect on fungal growth。3. The influence about HTK and U937 cells culture medium on the growth curve of Candida albicansCollected HTK and U937 cells culture medium after cells stimulated with Inactivated fungal spores lysate, and co-cultured with candida albicans . Fungal growth curve also shows that HTK and U937 cells co-culture medium has no effect on fungal growth。4. The influence about corneal homogenate supernatant on the survival of Candida albicansAfter corneal homogenates supernatant and Candida albicans co-cultured 2h, 4h, 6h staining with PI. On the three time points, there is no effect about corneal homogenate supernatant can promote or inhibit the death of the spores. But on 4h ,6h points, we acquire corneal homogenates supernatant can promote fungal spore germination.Conclusions: Cornea has a natural defense against fungal ability, and only when this natural defense is destroyed (such as the structural integrity of corneal epithelium is destroyed), FK pathological process to a smooth start.