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  

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

作　　者：朱然 梁舒[1] 钱晶晶[1] 李豪亮 张希熹 ZHU Ran;LIANG Shu;QIAN Jingjing;LI Haoliang;ZHANG Xixi(Department of Ophthalmology,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China)

机构地区：[1]南通大学附属医院眼科研究所,江苏省南通市226001

出　　处：《眼科新进展》2021年第6期534-539,共6页Recent Advances in Ophthalmology

基　　金：江苏省卫健委医学科研面上项目(编号H2019083);南通市市级科技计划项目(编号MSZ19054)。

摘　　要：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 mmol·L^(-1)葡萄糖培养基交替培养),根据干预真菌的不同共分9小组(低糖-对照组、高糖-对照组、糖波动-对照组、低糖-茄病镰刀菌组、高糖-茄病镰刀菌组、糖波动-茄病镰刀菌组、低糖-尖端赛多孢子菌组、高糖-尖端赛多孢子菌组、糖波动-尖端赛多孢子菌组)。真菌干预组均加入相应真菌孢子悬液,且孢子与HCEC的比例为10:1,对照组加入等量的无菌生理盐水。观察真菌在培养板上的生长状况,倒置显微镜下观察每组HCEC的形态,荧光白染色法和平板稀释涂布法用于HCEC的黏附真菌计数,利用qRT-PCR检测各组HCEC中MMP-9 mRNA的表达、Western blot检测各组HCEC中MMP-9蛋白的表达。结果倒置显微镜下可见与真菌共培养的HCEC出现不同程度的形变和脱落,尤以高糖-茄病镰刀菌组和糖波动-茄病镰刀菌组最为显著。荧光白染色法和平板稀释涂布法计数结果显示,两种真菌高糖组和糖波动组对HCEC的黏附量均大于低糖组,差异均有统计学意义(均为P<0.05);高糖和糖波动条件下,尖端赛多孢子菌黏附量大于茄病镰刀菌,差异均有统计学意义(均为P<0.05)。qRT-PCR和Western blot检测结果显示,MMP-9 mRNA和蛋白在各组HCEC中均有表达,与低糖组相比,高糖组和糖波动组HCEC中MMP-9 mRNA和蛋白表达均增多;高糖和糖波动条件下,真菌干预组HCEC中MMP-9 mRNA和蛋白表达均高于对照组,差异均有统计学意义(均为P<0.05),而两种真菌干预组之间差异均无统计学意义(均为P>0.05)。结论高糖和糖波动环境能够促进HCEC分泌更多的MMP-9,并增加真菌对HCEC的黏附。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 sp

关 键 词：茄病镰刀菌 尖端赛多孢子菌 人角膜上皮细胞 高糖 糖浓度波动 

分 类 号：R772.2[医药卫生—眼科]