地塞米松和RU-486作用人晶状体上皮细胞后糖皮质激素受体和Caspase-3的表达变化

Expressional changes of glucocorticoid receptor and Caspase-3 in human lens epithelial cells after exposed to dexamethasone and RU-486

目的研究在地塞米松和糖皮质激素受体抑制剂RU-486作用下,人晶状体上皮细胞(human lens epithelial cells,HLECs)中糖皮质激素受体(glucocorticoid receptor,GR)和Caspase-3的表达变化。方法用0.1μmol/L、0.5μmol/L、1μmol/L、5μmol/L地塞米松作用人晶状体上皮细胞24 h、32 h、40 h和48 h或与RU-486共作用4个时间段后,糖皮质激素反应元件(glucocorticoid response element,GRE)和GR-alpha基因的表达变化用转染和实时定量PCR法测定。细胞的增殖情况用MTT法测定。Caspase-3的表达情况用Caspase-3活性试剂盒测定。凋亡细胞率和GR蛋白的表达情况用流式细胞术(flow cytometry,FCM)和间接免疫荧光法测定。结果 0.1μmol/L浓度地塞米松作用HLECs 4个时间段后可以导致GRE-荧光素酶,GR-alpha基因,GR-蛋白和Caspase-3活性的升高,而在4个浓度地塞米松作用24 h后可以导致GRE-荧光素酶的,GR-alpha基因的升高,作用40 h后可导致GR-蛋白的升高。结论GRs与HLECs的增殖力有密切关系;RU486可以全部或部分地抑制GR的表达。虽然RU486可以提高Caspase-3的表达,但可能抑制由GR介导的其它凋亡蛋白的表达,因此它可以提高HLECs的增殖率并且无明显的凋亡细胞率的升高。

Objective To identify the expressional changes of glucocorticoid receptor and Caspase-3 in human lens epithelial cells （HLECs） after being exposed to dexamethasone and glueocorticoid receptor antagonist RU-486. Methods Human lens epithelial cells were treated with 0. 1 μmol/L, 0. 5 μmol/L, 1 μmol/L, 5 μmol/L dex or cotreated with R486 for 24 h, 32 h, 40 and 48 h. then the expressional changes of glucocorticoid response element （GRE） and GR-alpha gene were detected with transfection and real time PCR. The proliferation of condition of cells were detected with MTY assay. The expressional condition of Caspase-3 were detected with Caspase-3 activity detection kit. Apoptotic ceils ratios and the expressional condition of GR protein were detected with flow cytometry （FCM） and indirect immunofluorescence. Restilts 0. 1μmol/L Dex treated HLECs for 4 periods resulted in increased expression of GRE luciferases, GR-alpha gene, GR protein and Caspase-3 activity. The expression of GRE-lucif- erase and GR-alpha gene were increased when HLECs were treated with 4 concentrations of Dex for 24 h however the expression of GR-protein were increased when HLECs were treated with 4 concentrations of Dex for 40 h. Conclusion GRs have closely relationship with HLECs prolif- eration activity and RU486 could wholly or partially inhibit the expression of GR. Although RU486 could increase the expression of Caspase-3, maybe it could inhibit the expression of other apoptotic proteins mediated with GR. Overall, RU486 could increase the proliferation ratios of HLECs and there is lao obvious increase of apoptotic cells ratios.