摘要
目的探讨茄病镰刀菌对人角膜上皮细胞(hCEC)中NOD2表达及下游炎症因子分泌水平的影响。方法将对数生长期的hCEC分为对照组(无真菌刺激)、真菌刺激组(根据加入真菌浓度不同分为10^(3)、10^(4)、10^(5)、10^(6) CFU·mL^(-1)刺激组),真菌刺激组分别刺激hCEC 4 h、8 h、12 h、16 h,RT-qPCR检测各组hCEC中NOD2 mRNA表达;10^(5) CFU·mL^(-1)茄病镰刀菌孢子刺激12 h、24 h、36 h,Western blot检测各组hCEC中NOD2蛋白表达;细胞免疫荧光法检测NOD2分子定位和表达;10^(3)、10^(4)、10^(5)、10^(6) CFU·mL^(-1)茄病镰刀菌孢子刺激hCEC 36 h,ELISA法检测各组hCEC培养上清液中炎症因子白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)蛋白含量。结果随着茄病镰刀菌孢子刺激浓度的升高和刺激时间的延长,hCEC中NOD2 mRNA表达逐渐升高,10^(5) CFU·mL^(-1)茄病镰刀菌孢子刺激8 h,hCEC中NOD2 mRNA相对表达水平达到峰值,差异有统计学意义(P<0.05);10^(5) CFU·mL^(-1)茄病镰刀菌孢子刺激hCEC 12 h、24 h、36 h,hCEC中NOD2蛋白相对表达水平逐渐增多,与对照组相比差异均有统计学意义(均为P<0.05);10^(5) CFU·mL^(-1)茄病镰刀菌孢子刺激24 h后,NOD2主要表达在hCEC细胞质中,其荧光分子表达强度显著增加。随着茄病镰刀菌孢子刺激浓度的增加,hCEC中相关炎症因子的分泌水平增加,10^(4)、10^(5)、10^(6) CFU·mL^(-1)茄病镰刀菌孢子刺激hCEC 36 h,IL-6、IL-8、TNF-α蛋白含量与对照组相比,差异均有统计学意义(均为P<0.05)。结论茄病镰刀菌孢子刺激可上调hCEC中NOD2表达并促进相关炎症因子分泌,NOD2参与了hCEC抗茄病镰刀菌的固有免疫反应。
Objective To investigate the effect of Fusarium solani on the expression of NOD2 and the content of downstream inflammatory factors in human corneal epithelial cells(HCECs).Methods The HCECs in the logarithmic growth phase was divided into a control group(no fungal stimulation)and a fungal stimulation group(according to the concentration of added fungi,there were divided into 10^(3),10^(4),10^(5),10^(6) CFU·mL^(-1)stimulation groups).The fungal stimulation group was stimulated 4 h,8 h,12 h,16 h,separately,and RT-qPCR was used to detect the expression of NOD2 mRNA in HCECs of each group.After HCECs were stimulated by 10^(5) CFU·mL^(-1)spores of Fusarium solani for 12 h,24 h,36 h,Western blot was used to detect the expression of NOD2 protein in each group.Cellular immunofluorescence was applied to detect NOD2 molecular location and its expression.After HCECs were stimulated by 10^(3),10^(4),10^(5),10^(6) CFU·mL^(-1)Fusarium solani spores for 36 h,ELISA was used to detect the content of inflammatory factors,including IL-6,IL-8,and TNF-αin the supernatant of HCEC culture in each group.Results As the stimulation concentration of Fusarium solani spores increased and the stimulation time prolonged,the expression level of NOD2 mRNA in HCECs gradually increased.After HCEC were stimulated by 10^(5) CFU·mL^(-1)Fusarium solani spores for 8 h,the relative expression level of NOD2 mRNA in HCECs reached a peak,and the difference was statistically significant(P<0.05).After 10^(5) CFU·mL^(-1)Fusarium solani spores stimulated HCECs for 12 h,24 h,36 h,the relative expression level of NOD2 protein in HCECs gradually increased,and the difference was statistically significant compared with the control group(all P<0.05).After the spores of 10^(5) CFU·mL^(-1)Fusarium solani stimulated HCECs for 24 h,NOD2 was mainly expressed in the cytoplasm of HCECs,and the expression intensity of its fluorescent molecules increased significantly.As the stimulation concentration of Fusarium solani spores increased,the protein secretion level of related inflammatory factors in HCECs increased,10^(4),10^(5),10^(6) CFU·mL^(-1)Fusarium solani spores stimulated HCECs for 36 h,the differences in IL-6,IL-8,TNF-αprotein secretion levels were statistically significant when compared with the control group(all P<0.05).Conclusion Fusarium solani spore stimulation can up-regulate the expression of NOD2 in HCEC and promote the expression of related inflammatory factors.NOD2 participates in the innate immune response of HCEC against Fusarium solani.
作者
李豪亮
朱然
张希熹
陆志峰
钱晶晶
梁舒
LI Haoliang;ZHU Ran;ZHANG Xixi;LU Zhifeng;QIAN Jingjing;LIANG Shu(Department of Ophthalmology,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China)
出处
《眼科新进展》
CAS
北大核心
2021年第7期633-637,共5页
Recent Advances in Ophthalmology
基金
江苏省卫健委医学科研面上项目(编号H2019083)
南通市科技计划项目(编号MSZ19061)。
关键词
茄病镰刀菌
炎症因子
人角膜上皮细胞
NOD2
Fusarium solani
inflammatory factors
human corneal epithelial cells
NOD2
