葡萄籽原花青素提取物对牛晶状体上皮细胞增殖及细胞周期的影响

Effects of grape seed proanthocyanidin extract on the growth and cell cycle of bovine lens epithelial cells in vitro

目的观察葡萄籽原花青素提取物(grape seed proanthocyanidin extract,GSPE)对牛晶状体上皮细胞(bovine lens epithelial cells,BLECs)的增殖及细胞周期的影响,为后发性白内障的药物治疗提供新的研究方向。方法首先进行BLECs的原代及传代培养,取第2～第3代的BLECs,分别采用MTT法、流式细胞术及免疫组化SP染色法进行实验,研究不同浓度及作用时间的GSPE对体外培养的BLECs的增殖及细胞周期的影响。结果GSPE能明显抑制BLECs的增殖,并呈现剂量和时间依赖性。在作用24h后,随着GSPE浓度的增高(20μg/ml,60μg/ml,100μg/ml),A值逐渐变小(0.343±0.079,0.252±0.029,0.137±0.011),抑制作用逐渐增强(P<0.01)。100μg/ml GSPE作用BLECs 24h、48h、72h后,A值分别为0.137±0.011、0.096±0.025、0.055±0.008,差异有非常显著的统计学意义(P<0.01)。流式细胞术结果显示:经过GSPE处理,BLECs的G0/G1期细胞数量增加,S期、G2/M期明显减少。100μg/ml GSPE作用24h后,G0/G1期细胞为83.67%±5.63%,阴性对照组为59.13%±5.71%,差异有统计学意义(P<0.05)。免疫组化结果:GSPE剂量组中p21蛋白的表达增加,阳性细胞率从28.75%±1.25%增加到86.73%±5.11%,与阴性对照组7.73%±1.75%相比,差异有非常显著的统计学意义(P<0.01)。结论GSPE通过上调p21蛋白的表达诱导细胞周期停滞于G0/G1期,能有效地抑制牛晶状体上皮细胞增殖,可能成为后发性白内障的药物治疗研究的新方向。

Objective To observe the effects of grape seed proanthocyanidin extract （GSPE） on the growth and cell cycle of bovine lens epithelial cells （BLECs） in vitro in an attempt to find a drug that can prevent the effects of cataracts. Methods BLECs were first cultured in vitro and then different concentrations of GSPE were added to the second ithelial cell cultures. The effects BLECs in vitro were examined by and third generations of the ep- of GSPE on the proliferation of the MTY method. Cell cycle dis- tribution was determined with flow cytometry （FCM） and immunohis- tochemistry staining. The SP method was used to detect the effect of GSPE on the expression of p21. Results With GSPE treatment, the proliferation of BLECs was significantly inhibited in both a dose-and time-dependent manner. Following GSPE （20 p.g/ml, 60 ~g/ml, 100 Ixg/ml） treatments for 24 h, the A values were 0.343± 0.079, 0.252±0.029 and 0.137±0.011, respectively （P〈0.01）. Moreover, the A values of 100μg/ml GSPE were 0.137±0.011, 0.096± 0.025 and 0.055±0.008 at 24 h, 48 h and 72 h after GSPE treatment, respectively （P〈0.01）. An increase in counts for the G0/G1 phase cell population in the GSPE-treated group was indicated by flow cytometry analysis, while there was a decrease in the S phase and G：/M phase. The percentage of cells in the GdG~ phase was 83.67%＋_5.63% at a 100 Ixg/ml dose for 24 h, which was signifi- cantly different compared to the negative control group （59.13%-＋ 5.71%, P〈O.05）. The results of immunohistochemistry demonstrate that the expression of p21 protein increased in the GSPE-treated group, and the positive rate of cells in the GSPE-treated group in- creased from 28.75%±1.25% to 86.73%＋5.11% as compared to the negative control group （7.73%±1.75%, P〈0.01）. Conclusion GSPE can suppress the proliferation of BLECs by up-regulating the expression of p21 and inducing G0/G1 phase arrest. GSPE may be a new agent for post-cataract drug therapy.