高糖和糖浓度波动对人角膜上皮细胞中MMP-9表达及真菌黏附的影响

目的 探讨不同浓度葡萄糖环境下茄病镰刀菌和尖端赛多孢子菌对人角膜上皮细胞(HCEC)的黏附能力和HCEC中MMP-9表达的影响.方法 将处于对数生长期的HCEC分为3组:低糖组(5.5 mmol·L-1葡萄糖培养基培养)、高糖组(25.0 mmol·L-1葡萄糖培养基培养)和糖波动组(5.5 mmol·L-1和50.0...

Effects of high glucose and fluctuating glucose on the expression of MMP-9 in human corneal epithelium cells and the adhesion of Fungi

Objective　To investigate the adhesion ability of Fusarium solani (F.solani) and Scedosporium apiospermum (S.apiospermum) to human corneal epithelium cells (HCEC) and their effects on the expression of matrix metalloproteinase-9 (MMP-9) in HCEC under different concentrations of glucose.Methods　The cells in logarithmic growth were divided into 3 groups: low glucose group, in which cells were cultured with 5.5 mmol·L^-1 glucose concentration medium, high glucose group, in which cells were cultured with 25 mmol·L^-1 glucose concentration medium and fluctuating glucose group, in which cells were cultured with 5.5 mmol·L^-1 and 50 mmol·L^-1 glucose concentration medium alternately. Then the cells were divided into 9 groups according to different fungal interventions, and they were low glucose-control group, high glucose-control group, fluctuating glucose-control group, low glucose-F.solani intervention group, high glucose-F.solani intervention group, fluctuating glucose-F.solani intervention group, low glucose-S.apiospermum intervention group, high glucose-S.apiospermum intervention group and fluctuating glucose-S.apiospermum intervention group. The fungal intervention groups were treated with corresponding fungal spores suspension and the ratio of spores to HCEC was 10∶1. The control groups were given the sterile normal saline of equal volume. We observed the growth of fungi on the agar plate. The morphology of HCEC in each group was observed by the inverted microscope. Calcofluor white fluorescent staining and spread plate methods were used to count the adherent fungal spores of HCEC. Quantitative real-time PCR was used to determine the relative expression of MMP-9 mRNA and Western blot was used to determine the relative expression of MMP-9 proteins in HCEC. Results　We observed that the HCEC co-cultured with fungi showed varying degrees of deformation and abscission, especially in the high glucose-F.solani intervention group and fluctuating glucose-F.solani intervention group. The results of Calcofluor white fluorescent staining and spread plate methods showed that the adhesion population of fungi to HCEC was higher in high glucose groups and fluctuating glucose groups than that in the low glucose groups (all P<0.05). The adhesion population of S.apiospermum was higher than that of F.solani under the conditions of high glucose and fluctuating glucose, and the differences were statistically significant (all P<0.05). The result of Quantitative real-time PCR and Western blot showed that both MMP-9 mRNA and protein were expressed in HCEC of each group. The expression of MMP-9 mRNA and protein in HCEC was higher in high glucose groups and fluctuating glucose groups than that in low glucose groups. under the conditions of high glucose and fluctuating glucose, the expression levels of MMP-9 mRNA and protein in HCEC of fungal intervention groups was higher than those of the control groups (all P<0.05), but there was no statistically significant difference between the F.solani intervention group and S.apiospermum intervention group (P>0.05).Conclusion　High glucose and fluctuating glucose can promote MMP-9 secretion and increase the fungal adherence to the HCEC.