Combretastatin A4 Phosphate对人晶状体上皮细胞增殖及迁移的影响

Effect of Combretastatin A4 Phosphate on Proliferation and Migration of Lens Epithelial Cells

【目的】研究Combretastatin A4 Phosphate(CA4P)对晶状体上皮细胞系SRA01/04增殖及迁移影响,初步探讨其抑制细胞增殖的机制。【方法】以晶状体上皮细胞系SRA01/04为实验对象,设空白对照组及CA4P组,分别以PBS或1μmol/L CA4P处理15 min,于0、24、48、72 h后进行检测。通过WST-1法检测细胞增殖能力;划痕试验检测细胞迁移能力;Annexin V-FITC/PI双染法检测细胞死亡方式;PI染色法检测细胞周期;免疫荧光法检测细胞骨架蛋白(α-微管蛋白、F-肌动蛋白)及细胞形态。【结果】CA4P对细胞增殖的抑制作用呈时间依赖性,24、48、72 h细胞相对增殖活性分别为73%、40%、34%(P<0.05)。空白对照组细胞划痕面见大量细胞覆盖;CA4P组划痕面无细胞覆盖,细胞呈细小圆形,可见部分细胞脱落漂浮。CA4P组24、48、72 h细胞凋亡率为19.7%、43.6%、72.2%,明显高于对照组(1.7%、2.2%、3.6%;P<0.05)。CA4P组各时间点G2/M期分布分别为30.3%、55.4%、70.8%,明显高于对照组(4.4%,9.0%,7.9%;P<0.05)。CA4P组细胞内微管纤维丝状结构消失,可见多核、巨核等有丝分裂障碍表现,但F-肌动蛋白未见明显变化。【结论】1μmol/L CA4P短时间作用于SRA01/04晶状体上皮细胞可明显抑制其增殖和迁移,且作用呈时间依赖性。CA4P抑制细胞增殖可能机制是通过解聚微管蛋白,诱导有丝分裂停止而诱发SRA01/04细胞凋亡。

[Objective ]To study the effect of combretastatin A4 phosphate （CA4P） on proliferation and migration of lens epithelial cells line SRA01 ! 04 and discuss the mechanism on CA4P inhibiting cell proliferation. [Methods] The experimental object was lens epithelial cells line SRA01/04. We divided them into the control group and CA4P group, treating respectively by PBS or lumol/L CA4P for 15 min. The testing time were 0 h, 24 h, 48 h, and 72 h after drug-treating. Cell proliferation capability was detected by WST -1. Cell migration was detected by wound test. Way of cell death was detected by Annexin V-FITC/PI. Cell cycle was detected by PI streaming. Cytoskeleton protein （alpha microtubules protein, F-actin） and cell morphology were detected by immunofluorescence. [Results] Inhibition of cell proliferation by CA4P presented time dependent. After 24 h 48 h, and 72 h treated by CA4P, the cell viability were 73%, 40%, and 34%. Wound test showed a large number of cells covers scratches in the control group. Scratches in CA4P group was smooth. Ceils become tiny and round, falling off the basement to floating. Cell apoptosis in CA4P group were 19.7%, 43.6%, and 72.2% in 24 h, 48 h, and 72 h, respectively. It was conspicuously higher than those in the control group （1.7%, 3.6%, and 2.2%）. G2/M period distribution in CA4P group were 30.3%,55.4%,and 70.8% in each test points respectively, which was significantly higher than those in the control group （4.4% 9.0%, and 7.9%）. Microtubules fiber filamentous structure disappeared in CA4P group. Cells appeared mitotic obstacle performances, such as multi-core and huge nuclear. But F-actin showed no changes. [ Conclusion ] 1 txmol/L CA4P obviously inhibit the proliferation and migration of lens epithelial cells by shortterm incubation. The effect is time dependent. The mechanism of CA4P inhibiting cell proliferation probably relate to depolymerizationmicrotubules protein which induce mitotic catastrophe and apoptosis.