丙戊酸钠对体外培养的人晶状体上皮细胞增生的抑制作用

Inhibitory effect of sodium valproate on proliferation of human lens epithelial cells in vitro

目的探讨丙戊酸钠(sodium valproate,VPA)对体外培养的人晶状体上皮细胞(human lens epithelial cells,HLECs)增生和细胞周期的影响。方法采用不同浓度的VPA(0.5mmol·L-1、1mmol·L-1、2mmol·L-1、4mmol·L-1)作用于HLECs,四甲基偶氮唑蓝法(methyl thiazolyl tetrazolium,MTT)检测在不同时间点(24h、48h、72h)VPA对细胞增生的抑制作用;流式细胞仪测定VPA(1mmol·L-1、2mmol·L-1)作用于HLECs48h后细胞周期分布情况;Western blot测定在VPA(1mmol·L-1、2mmol·L-1)作用于HLECs48h后,细胞周期依赖性激酶抑制因子(p21)在蛋白质水平表达量的改变。结果MTT检测显示VPA浓度≥0.5mmol·L-1对体外HLECs增生的抑制作用呈时间和剂量依赖性。VPA作用48h后,各药物浓度组(0.5mmol·L-1、1mmol·L-1、2mmol·L-1、4mmol·L-1)细胞生长抑制率分别为11.05%、21.58%、26.67%和38.25%,流式细胞仪检测显示VPA可阻滞HLECs由G0/G1期向S期转换,使G0/G1期细胞增多,S期细胞减少。经2mmol·L-1VPA作用48h,G0/G1期及S期细胞比例分别为(85.40±3.90)%和(7.35±1.47)%,与阴性对照组[(53.14±1.71)%和(35.31±1.14)%]比较差异具有统计学意义(P<0.05)。Western blot检测发现VPA可促进p21蛋白质水平的表达。VPA处理48h后,各组平均灰度比值分别为0.582±0.082(1mmol·L-1)和0.914±0.113(2mmol·L-1),与阴性对照组(0.303±0.029)比较,差异均有统计学意义(P<0.05)。结论VPA可以通过促进p21表达,使HLECs阻滞于G0/G1期,最终抑制HLECs增生。

Objective To investigate the effect of sodium valproate（VPA） on the proliferation and cell cycle of human lens epithelial cells （HLECs） in vitro. Methods HLECs were treated with VPA in different concentrations （0.5 mmol·L^-1,1 mmol ·L^-1 ,2 mmol ·L^-1 ,4 mmol ·L^-1 ）. Cell proliferation was assessed by MTT assay at different time（24 hours,48 hours,72 hours）. After HLECs were treated with VPA （1 mmol ·L^-1,2 mmol ·L^-1） for 48 hours, cell cycle was analyzed by flow cytometry and the expression of p21 （ cyclin-dependent kinase inhibitor） was detected by Western blot. Resuits MTT staining colorimetry showed that the HLECs proliferation was markedly inhibited and the effects was time and dose dependent when the concentration of VPA was more than 0. 5 mmol ·L^-1. The inhibition rate of cultured lens epithelial cells （LECs） was ll. 05% ,21.58% ,25.57% and 38.25% , respectively,in 0.5 mmol ·L^-11,1 mmol·L^-1,2 mmol·L^-11 and 4 mmol·L^-1 of VPA group after 48 hours treatment（P〈 0.05）. More cultured HLECs were in the G0/G1 phase and fewer in the S phase after treated with VPA by flow cytometry ,which indicated that VPA arrested the transition of HLECs from G0/G1 phase to S phase. The percentage of the LECs in the G0/G1 phase and the S phase at the dosage of 2 mmol ·L^-1 of VPA group was （ 85.40 ± 3.90 ） % and （7.35 ± 1.47 ） % , respectively, after 48 hours treatment, showing statistical significance compared with the control group [ （ 53. 14 ± 1.71 ） % and （ 35.31 ± 1. 14 ） % , P 〈 0.05]. Western blot indicated that VPA can increase the expression of p21. The average grey value of protein band was 0.582 ± 0. 082 （ 1 mmol ·L^-1） ,0. 914 ± 0.113 （2 mmol·L^-1 ） and 0. 303 ± 0. 029 （ control group ）, respectively,indicating statistical significance among groups （P 〈 0.05 ）. Coneluslon VPA arrests HLECs at G0/G1 by increasing the expression of p21 ,which finally inhibits the proliferation of HLECs.