缓激肽对体外培养兔角膜内皮细胞增殖状况及对闭锁小带蛋白-1与相关性核酸结合蛋白表达的影响  

Effects of bradykinin on the proliferation of rabbit corneal endothelial cells and the expression of tight junction-related proteins ZO-1 and ZONAB

作　　者：贺李娴 邵毅[2] 邹雪香 周双双[1] 周颖[1] 刘二华[1] 谭钢[1] HE Li-Xian;SHAO Yi;ZOU Xue-Xiang;ZHOU Shuang-Shuang;ZHOU Ying;LIU Er-Hua;TAN Gang(Department of Ophthalmology,the First Affiliated Hospital of University of South China,Hengyang 421001,Hunan Province,China;Department of Ophthalmology,the First Affiliated Hospital of Nanchang University,Nanchang 330006,Jiangxi Province,China)

机构地区：[1]南华大学附属第一医院眼科,湖南省衡阳市421001 [2]南昌大学第一附属医院眼科,江西省南昌市330006

出　　处：《眼科新进展》2018年第2期116-120,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81100648;81400372);湖南省教育厅优秀青年基金资助(编号:15B210)~~

摘　　要：目的观察缓激肽(bradykinin,BK)对体外培养家兔角膜内皮细胞增殖及对紧密连接相关蛋白闭锁小带蛋白-1(zonula occludens-1,ZO-1)、闭锁小带蛋白-1相关性核酸结合蛋白(zonula occludens-1-associated nucleic-acid-binding protein,ZONAB)表达的影响,初步探讨BK促进角膜内皮细胞增殖的作用机制。方法选择对数生长期的兔角膜内皮细胞,向培养基中分别加入0.01、0.10、1.00、10.00μmol·L-1BK(BK组),对照组不做加药处理,倒置相差显微镜下观察细胞的形态变化和增殖状况,MTT法检测各组细胞在24 h、48h、72 h、96 h的吸光度(A)值,Western blot检测72 h各组细胞中紧密连接处ZO-1与核内ZONAB蛋白的表达。结果加药72 h,各组细胞融合成片并紧密连成单层,加药96 h后各组细胞生长受限,细胞间隙变大,脱落细胞增多。与对照组相比,除0.01μmol·L-1BK组24 h外,其余浓度BK处理后A值升高、增殖活力增强,差异均有统计学意义(均为P<0.001),并呈现出一定的浓度依赖性,其中1μmol·L-1BK作用最强(P<0.001)。Western blot结果显示BK可促进紧密连接处ZO-1与核内ZONAB蛋白的表达,且呈一定的浓度依赖性。结论 BK体外刺激可促进兔角膜内皮细胞的增殖,具有浓度和时间的依赖性,其作用机制可能与ZO-1与ZONAB介导的信号通路有关。Objective To investigate the effects of bradykinin(BK)on the proliferation of rabbit corneal endothelial cells(RCECs)and the expression of tight junction-related proteins zonula occludens-1(ZO-1)and zonula occludens-1-associated nucleic-acid-binding protein(ZONAB),and to explore the underlying mechanisms of BK on cell proliferation in corneal endothelium.Methods RCECs at logarithmic growth phase were treated with different concentrations of BK(0.01,0.1,1,10μmol·L-1)BK group,with the controls left untreated.Morphological changes of cells in each group were examined under phase-contrast microscope,and MTT assays were used to detect cell proliferation at 24 h,48 h,72 h,96 h after BK treatment.And,at 72 h,the expression levels of ZO-1 and ZONAB protein were determined by Western blot.Results After 72 h of treatment,the cells in each group were fused into pieces and closely linked into a monolayer;but after 96 h,the growth of the cells was restricted,with the intercellular space become larger and the cells exfoliated.Compared with the control group,BK induced a significant increase of absorbance value and cell viability,and the differences were statistically significant(all P<0.001),and the promoting effects showed a concentration-dependent manner,and 1μmol·L-1 BK demonstrated the strongest regulative effect(P<0.001).Western blot results showed that BK upregulated the expression of ZO-1 and ZONAB protein in a concentration-dependent manner.Conclusion BK can stimulate the proliferation of RCECs in a time-and concentration-dependent manner,and the mechanisms are probably associated with ZO-1/ZONAB-mediated signaling pathway.

关 键 词：缓激肽 兔角膜内皮细胞 增殖 ZO-1 ZONAB 

分 类 号：R772.2[医药卫生—眼科]