The Inhibiting Effect Of On Proliferation Of Human Pterygium Fibroblasts

Objective: To investigate the effect of the acetoacetate extract of Vitex negundoseed(Evn-50）inhibitors on proliferation of human pterygium fibroblasts (HPF) inculture and search for a new method to prevent the recurrence after pterygiumsurgery.Methods: The tissues from pterygium were got by operation and cut to clips lessthan1×1×1mm~3. Segments were digested by collagenase type Ⅱ and trypsin. Afterthe primaryculture and subculture, the third or fourth passage of HPF were usedfor immunofluorescence staining with combining with α-SMA morphologicalfeatures, pterygium fibroblasts can be identified. HPF cultured in vitro was treatedWith Evn-50(0.25、0.5、1、2、4μg/ml), and the negative control group without drugs.After48h of culture, cell morphology changes were observed. The expression ofproliferating cell nuclear antigen (Ki-67) in different group was detected byimmunofluorescence method，and the cell cycle distribution was measured by flowcytometic analysis.Result:(1) In vitro culture cells form general for long spindle,oval nuclei islocated in the central cells was examined by inverted phase contrast microscope.The expression of α-SMA in cultured cells in vitro was positive,and pterygiumfibroblasts can be identified.(2) After treated with different doses Evn-50for48h,flow cytometricanalysis indicated that a lot of HPF were induced cell-cycle arrest and stopped inG0/G1phase with the increasing of Evn-50concentration,and cells in S, G2/M phasewere decreased accordingly. The calculation of proliferation rate (S%+G2/M%)showed that the multiplication capacity of HPS were decreased.(3) After treated with different doses Evn-50for48h,the immuno-fluorescence demonstrated that, when the concentration ranged in1-4μg/ml, Evn-50 can inhibit the expression of proliferating cell nuclear antigen (Ki-67) in fibroblast ina concentration-dependent manner (p<0.05).Conclusion: Evn-50can inhibit the proliferation of HPF significantly. Flowcytometic analysis indicated that Evn-50blocked HPF cells in G0/G1phase.