title: [The effects of chloride channel inhibitor on bFGF-induced proliferation of lens epithelial cells].
abstract: OBJECTIVE
  To study the effect of chloride channel on proliferation induced by basic-fibroblast growth factor (bFGF) in human lens epithelial cells HLE B-3 (LEC).
  
  
  METHODS
  HLE B-3 cells at Logarithmic growth phase were incubated in the 6 or 96 well plate overnight and the proliferation was induced by 10 µg/L bFGF. The cells were divided into bFGF group treated with bFGF and the blank control group with the regular cells culture. LEC were treated with different concentrations of chloride channel inhibitors: 5-nitro-2-[3-phenylpropylamino] benzoic acid (NPPB) at 50, 100, 150 µmol/L and 4 , 4'-2 diisothiocyanostilbene-2, 2' -disulfonic acid (DIDS) at 10, 50, 100 µmol/L with bFGF or without bFGF in 10% serum for 24, 48, 72 hours. The cell viability and the drug toxicity were detected by CCK-8 colorimetric assay. The expression of proliferating cell nuclear antigen (Ki-67) of LEC treated with NPPB or DIDS were observed by immunocytochemistry staining assay. The phase change of cell cycle was examined by flow cytometry. Each experiment group and control group were compared using two-way ANOVA, further pairwise comparisons using LSD-t test or by the Independent-Samples t test.
  
  
  RESULTS
  10 µg/L bFGF induced LEC proliferation and the values of A stage were gradually declined with the increase of chloride channel inhibitors' concentrations and time at 24 hours, 48 hours and 72 hours (bFGF+NPPB group: Ftime = 305.28, Fconcentrations = 18.76, P = 0.000;bFGF+ DIDS group:Ftime = 94.44, Fconcentrations = 24.42, P = 0.000). Different concentrations of chloride channel inhibitor reduced the values of a stage with 10 µg/L bFGF in a dose- and time-dependent manner. The expression of Ki-67 was significantly lowered compared with the bFGF group (bFGF+NPPB group: 18.32% ± 1.23%, F = 580.3, P = 0.000;bFGF+DIDS group: 11.21% ± 1.02%, F = 507.4, P = 0.000), when 150 µmol/L NPPB and 100 µmol/L DIDS with 10 µg/L bFGF were added at 48 hours. After 150 µmol/L NPPB and 100 µmol/L DIDS treatment at the 48th hour, the rate of G1 stage was significantly increased (F = 390.754, P = 0.000), where as that of S stage decreased significantly (F = 166.240, P = 0.000).
  
  
  CONCLUSIONS
  These results indicate that chloride channel inhibitors play an important role in modulating the proliferation cycle of LEC treated with bFGF by limiting the cell at cycle S/G1 stage from dividing into S stage.
authors: Department of Ophthalmology,Union Hospital of Fujian Medical University, Fuzhou 350001, China.
authors: Min-hua
authors: MH
authors: Zhang
authors: Department of Ophthalmology,Union Hospital of Fujian Medical University, Fuzhou 350001, China.
authors: Xiao-dong
authors: XD
authors: Zhuang
authors: Department of Ophthalmology,Union Hospital of Fujian Medical University, Fuzhou 350001, China. Email: wjn55@sina.com.
authors: Jing-ning
authors: JN
authors: Weng
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D004847:Epithelial Cells / Q000187:drug effects*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology; D009579:Nitrobenzoates / Q000494:pharmacology*
chemical_list: D018118:Chloride Channels; D009579:Nitrobenzoates; D016222:Fibroblast Growth Factor 2; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid