维拉帕米对体外人晶状体上皮细胞增殖和黏附的抑制作用

Inhibitory effect of verapamil on proliferation and adhersion of human lens epithelial cells in vitro

目的：研究钙离子拮抗剂维拉帕米（verapamil,Ver）对体外培养的人晶状体上皮细胞（human lens epithelial cell,HLEC）增殖、凋亡和黏附的影响。方法：组织块法培养人晶状体上皮细胞，用Ver处理第二代人晶状体上皮细胞。MTT法观察不同浓度的Ver对HLEC增殖和黏附的影响，AO/EB双染法观察细胞形态，Annexin V-FITC/PI双染法观察细胞凋亡情况，流式细胞术（FCM）检测Ver对HLEC周期的影响。结果：组织块贴壁4~6d可见HLEC从组织块边缘长出，15~20d接近融合。随着Ver药物浓度的增加，HELC增殖率减少，以72h最为明显。FCM检测细胞周期变化时发现，G1期细胞在10,40g/L Ver（78.1±0.2，83.2±0.6）较正常组（72.0±0.9）增加（P〈0.05）；S期的细胞在10,40g/L Ver（10.2±0.1,8.5±0.3）较正常组（16.9±0.3）明显减少（P〈005）。形态学观察发现，HELC在40g/L Ver作用48h后发生了一系列形态改变，包括细胞核固缩、染色质边集等凋亡改变。10，20，40，80g/L的Ver可以诱导HLEC发生凋亡，且随着浓度的升高凋亡率显著升高。用药24h后HELC的贴壁量为对照组的73.2%。结论：Ver能改变细胞周期而抑制HLEC增殖、诱导细胞凋亡，并且抑制HLEC的黏附。

AIM：To investigate the effect of the calcium channel blocker verapamil （Ver） on the proliferation, apoptosis and adhersion of the human lens epithelial cell （HLEC） in vitro. METHODS： Primary lens epithelial cells were cultured by the tissue explants adherent method. The second generations of lens epithelial cells were treated with Ver, the effect of proliferation and adhersion were examined with MTT. Cell morphological changes were examined by using AO/EB staining. Staining with Annexin V-FITC/PI was used to detect the apoptotic cells. The effect of Ver on HLEC cycle was analyzed by flow cytometer （FCM）. RESULTS：HLEC grew out of the edge of tissue pieces after incubation for 4 to 6 days, they reached confluence after 15 to 20 days. HELC proliferation decreased with the increase of the concentration of Ver. The result of flow cytometer showed that the percentage of HLEC in G1 phase in drug group （78.1±0.2,83.2±0.6）compared with control group （72.0±0.9） was increased, HLEC in S phase in drug group （10.2±0.1,8.5±0.3） decreased in comparison with control group （16.9±0.3, P〈0.05）.Morphological observation demonstrated that treatment of HLEC with 40g/L Ver for 48 hours caused the typical morphological charateristics of apoptosis, including nuclear condensation and chromatin margination. The amount of adhered cells in drug group was 73.2% compared with control group. CONCLUSION：Ver could alter the cycle of HLEC and effectively inhibit the HLEC proliferation. It could induce apoptosis of HLEC and inhibit the adhersion of HLEC.