灯盏花素对转化生长因子-β2诱导的人晶状体上皮细胞上皮-间质转化的影响  

Effects of breviscapine on epithelial-mesenchymal transition induced by transforming growth factor-β2 in human lens epithelial cells

作　　者：崔琨明[1] 张凤妍[1] 祈颖[1] 王蒙蒙[1] 高航[2] 

机构地区：[1]郑州大学第一附属医院眼科,450052 [2]郑州人民医院眼科,450053

出　　处：《中华实验眼科杂志》2013年第10期930-934,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：基金项目：郑州大学研究生科学研究基金项目（12Y03404）

摘　　要：背景 晶状体上皮细胞（LECs）的上皮-间质转化（EMT）是后发性白内障的主要病理改变,而EMT的主要标志是α-平滑肌肌动蛋白（α-SMA）的表达.研究发现,灯盏花素具有抑制组织纤维化病变进展及EMT的作用,但对LECs的EMT及其增生是否有抑制作用尚不清楚. 目的 探讨灯盏花素对转化生长因子-β2（TGF-β2）诱导的人LECs表达α-SMA和纤维连接蛋白（FN）的影响. 方法 将人LECs HLE-B3细胞株在含质量分数10％胎牛血清的DMEM培养基中进行培养和传代,在培养基中分别加入质量浓度为6.75、12.75、25.00、50.00和100.00 mg/L的灯盏花素作用24、48和72 h,采用细胞计数试剂盒-8（CCK-8）法检测不同质量浓度灯盏花素作用不同时间对HLE-B3细胞增生的抑制率.此外取生长状态良好的HLE-B3细胞以1×106个/孔的密度接种于培养板中,将其分为4个组,无血清培养基培养的HLE-B3为正常对照组；加入10 μg/L TGF-β2处理的HLE-B3为TGF-β2组；10 mg/L灯盏花素处理的HLE-B3为灯盏花素组；用10 mg/L灯盏花素与10 μg/L TGF-β2共同处理的HLE-B3为联合用药组,药物干预72 h后分别用实时荧光定量PCR法及Western blot法检测α-SMA、FN的mRNA及蛋白在HLE-B3中的表达. 结果 随着灯盏花素质量浓度的逐渐增加,对HLE-B3增生的抑制率逐渐增加,差异有统计学意义（F=292.851,P=0.000）；随着灯盏花素作用时间的延长,对HLE-B3增生的抑制率逐渐增加,差异有统计学意义（F=65.037,P=0.000）；HLE-B3培养72 h的半抑制率（IC50）为22 mg/L,故本实验用10 mg/L（≈1/2 IC50）药物质量浓度作用72 h行后续实验.正常对照组HLE-B3可表达一定量的α-SMA及FN,正常对照组、TGF-β2组、灯盏花素组、联合用药组间HLE-B3中α-SMA mRNA及FN mRNA的表达差异均有统计学意义（F=105.490,P=0.000;F=1041.414,P=0.000）,HLE-B3中α-SMA及FN蛋白的表达差异均有统计学意义（F=136.872,P=0.000;F=119.820,P=0.000）.与正常对�Background Epithelial-mesenchymal transition （EMT） is a major event in the pathogenesis of posterior capsular opacification （PCO）,and the expressions of α-smooth muscle actin （α-SMA） is the marker of EMT.Previous studies showed that breviscapine plays an important role in anti-fibrosis and suppression EMT,however,the mechanism of its effect on EMT in LECs is unclear.Objective Present study was to investigate the effect of breviscapine on the expression of α-SMA and fibronectin （FN） in human LECs induced by transforming growth factor-β2 （TGF-β2）.Methods Human LECs strain,HLE-B3,was cultured and passaged in DMEM containing 10% fetal bovine serum.Different concentrations of breviscapine （6.75,12.75,25.00,50.00 and 100.00 mg/L）were added into the medium for 24,48 and 72 hours respectively,and then cell cunting kit-8 （CCK-8） was used to evaluate the half maximal inhibitory concentration （IC50） of breviscapine to HLE-B3.In addition,HLE-B3 was subcultured at the density of 1 × 106/hole and divided into 4 groups.The cells were in free-serum medium as the normal control;the cells were exposed in 10 μg/L TGF-β2 as TGF-β2 group;while in the breviscapine group,10 mg/L of breviscapine was added into the culture medium and another group was the combination of 10 mg/L breviscapine and 10 μg/L of TGF-β2 treatment.Real-time PCR and Western blot were used to detect the mRNA expressions of α-SMA and FN as well as their protein in HLE-B3 72 hours after cultured.Results The inhibitory rate of breviscapine to HLE-B3 proliferation was gradually elevated with the increase of concentration of breviscapine,showing a significant inhibition of the cell proliferation among the different groups and at various time points （F =292.851,P=0.000;F =65.037,P=0.000）.IC50 of HLE-B3 at 72 hours was 22 mg/L,and therefore,in rest of experiment 10 mg/L of breviscapine was used which was 1/2 only half of the IC50.α-SMA and FN were expressed in cultured normal HLE-B3.The expressing level of α-SMA mRNA and F

关 键 词：灯盏花素 晶状体上皮细胞 上皮-间质转化 后发性白内障 

分 类 号：R285[医药卫生—中药学]