中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
2期
145-150
,共6页
王钧蔚%王林%葛红岩%刘平
王鈞蔚%王林%葛紅巖%劉平
왕균위%왕림%갈홍암%류평
皮质类固醇性白内障%地塞米松%核转录因子κB%核转录因子κB抑制蛋白α%凋亡%人晶状体上皮细胞
皮質類固醇性白內障%地塞米鬆%覈轉錄因子κB%覈轉錄因子κB抑製蛋白α%凋亡%人晶狀體上皮細胞
피질류고순성백내장%지새미송%핵전록인자κB%핵전록인자κB억제단백α%조망%인정상체상피세포
Glucocorticoid cataract%Dexamethasone%Nuclear factor kappa B%Inhibitor kappa B alpha%Apoptosis%Lens epithelial cells
背景 长期全身或眼局部应用糖皮质激素可诱导皮质类固醇性白内障,但其作用机制尚不明确.目的 研究地塞米松作用于人晶状体上皮细胞(LECs)后对LECs中核转录因子KB(NF-κB)/NF-KB抑制蛋白α(IκBα)表达的影响及LECs凋亡的发生情况,了解皮质类同醇性白内障的发病机制.方法取人LECs系(HLE2B3)在含质量分数20%胎牛血清的DMEM中进行培养和传代.将不同浓度的地塞米松(0.01、0.1、1、10、100μmol/L)分别加入DMSO无血清DMEM培养液中作为不同浓度地塞米松组,不含地塞米松的DMSO无血清DMEM培养液培养的LECs作为空白对照组.采用逆转录聚合酶链反应(RT-PCR)、Western blot、流式细胞术等方法,分别在基因水平、蛋白水平检测LECs中NF-κB/IkBα浓度的改变及LECs凋亡率的变化.结果 扩增的基因片段与所设计片段大小一致.地塞米松作用后NF-kB核蛋白在LECs中的表达量随着地塞米松浓度的增加而下降,差异有统计学意义(F＝36.077,P＝0.004);IκBα蛋白的表达随着地塞米松浓度的增加而上升,差异有统计学意义(F＝35.741,P＝0.002).在同浓度地塞米松组,NF-κB核蛋白在LECs中的表达量随作用时间的不同差异有统计学意义(F＝16.606,P＝0.01),与地塞米松作用24h时的表达量比较,36h和48h时的NF-kB核蛋白表达量明显下降,差异有统计学意义(P＝0.002;P＝0.01).与地塞米松作用24h时的表达量比较,36h和48h IκBα蛋白在LECs中的表达量明显增加,差异均有统计学意义(P＝0.014;P＝0.002).流式细胞仪检测显示LECs的凋亡率随着地塞米松浓度的增加明显上升,与空白对照组相比差异有统计学意义(P<0.05).结论 地塞米松通过七调IκBα的表达而过度抑制了NF-κB的活性,并导致LECs凋亡,其作用呈浓度依赖性,这可能是皮质类固醇性白内障发生发展的细胞学和分子生物学机制之一.
揹景 長期全身或眼跼部應用糖皮質激素可誘導皮質類固醇性白內障,但其作用機製尚不明確.目的 研究地塞米鬆作用于人晶狀體上皮細胞(LECs)後對LECs中覈轉錄因子KB(NF-κB)/NF-KB抑製蛋白α(IκBα)錶達的影響及LECs凋亡的髮生情況,瞭解皮質類同醇性白內障的髮病機製.方法取人LECs繫(HLE2B3)在含質量分數20%胎牛血清的DMEM中進行培養和傳代.將不同濃度的地塞米鬆(0.01、0.1、1、10、100μmol/L)分彆加入DMSO無血清DMEM培養液中作為不同濃度地塞米鬆組,不含地塞米鬆的DMSO無血清DMEM培養液培養的LECs作為空白對照組.採用逆轉錄聚閤酶鏈反應(RT-PCR)、Western blot、流式細胞術等方法,分彆在基因水平、蛋白水平檢測LECs中NF-κB/IkBα濃度的改變及LECs凋亡率的變化.結果 擴增的基因片段與所設計片段大小一緻.地塞米鬆作用後NF-kB覈蛋白在LECs中的錶達量隨著地塞米鬆濃度的增加而下降,差異有統計學意義(F＝36.077,P＝0.004);IκBα蛋白的錶達隨著地塞米鬆濃度的增加而上升,差異有統計學意義(F＝35.741,P＝0.002).在同濃度地塞米鬆組,NF-κB覈蛋白在LECs中的錶達量隨作用時間的不同差異有統計學意義(F＝16.606,P＝0.01),與地塞米鬆作用24h時的錶達量比較,36h和48h時的NF-kB覈蛋白錶達量明顯下降,差異有統計學意義(P＝0.002;P＝0.01).與地塞米鬆作用24h時的錶達量比較,36h和48h IκBα蛋白在LECs中的錶達量明顯增加,差異均有統計學意義(P＝0.014;P＝0.002).流式細胞儀檢測顯示LECs的凋亡率隨著地塞米鬆濃度的增加明顯上升,與空白對照組相比差異有統計學意義(P<0.05).結論 地塞米鬆通過七調IκBα的錶達而過度抑製瞭NF-κB的活性,併導緻LECs凋亡,其作用呈濃度依賴性,這可能是皮質類固醇性白內障髮生髮展的細胞學和分子生物學機製之一.
배경 장기전신혹안국부응용당피질격소가유도피질류고순성백내장,단기작용궤제상불명학.목적 연구지새미송작용우인정상체상피세포(LECs)후대LECs중핵전록인자KB(NF-κB)/NF-KB억제단백α(IκBα)표체적영향급LECs조망적발생정황,료해피질류동순성백내장적발병궤제.방법취인LECs계(HLE2B3)재함질량분수20%태우혈청적DMEM중진행배양화전대.장불동농도적지새미송(0.01、0.1、1、10、100μmol/L)분별가입DMSO무혈청DMEM배양액중작위불동농도지새미송조,불함지새미송적DMSO무혈청DMEM배양액배양적LECs작위공백대조조.채용역전록취합매련반응(RT-PCR)、Western blot、류식세포술등방법,분별재기인수평、단백수평검측LECs중NF-κB/IkBα농도적개변급LECs조망솔적변화.결과 확증적기인편단여소설계편단대소일치.지새미송작용후NF-kB핵단백재LECs중적표체량수착지새미송농도적증가이하강,차이유통계학의의(F＝36.077,P＝0.004);IκBα단백적표체수착지새미송농도적증가이상승,차이유통계학의의(F＝35.741,P＝0.002).재동농도지새미송조,NF-κB핵단백재LECs중적표체량수작용시간적불동차이유통계학의의(F＝16.606,P＝0.01),여지새미송작용24h시적표체량비교,36h화48h시적NF-kB핵단백표체량명현하강,차이유통계학의의(P＝0.002;P＝0.01).여지새미송작용24h시적표체량비교,36h화48h IκBα단백재LECs중적표체량명현증가,차이균유통계학의의(P＝0.014;P＝0.002).류식세포의검측현시LECs적조망솔수착지새미송농도적증가명현상승,여공백대조조상비차이유통계학의의(P<0.05).결론 지새미송통과칠조IκBα적표체이과도억제료NF-κB적활성,병도치LECs조망,기작용정농도의뢰성,저가능시피질류고순성백내장발생발전적세포학화분자생물학궤제지일.
Background Researches demonstrated that the long-term application of glucocorticoids can induce cataract. However, its molecular mechanism is unclear. Objective Present study was to investigate the effects of dexamethasone on the regulation of nuclear factor kappa B( NF-κB)/ inhibitor kappa B alpha( IκBα) line on human lens epithelial cells (LECs) and the LECs apoptosis. Methods Human LECs line(HLE2B3) were cultured and passaged in DMEM containing 20% fetal bovine serum and treated by different concentrations of dexamethasone(0. 01,0. 1,1,10,100 μmol/L) for 24,36 and 48 hours respectively. The LECs cultured in free-serum DMEM without dexamethasone were as blank control group. The expressions of IκBo: in the LECs were examined by reverse transcription polymerase chain reaction ( RT-PCR) and Western blot, and the expressions of NF-κB neucleoprotein in LECs were detected by Western blot after exposure to dexamethasone. The apoptosis rate of LECs was determined by flow cytometer. Results Agarose gel electrophoresis showed that the amplified gene fragment was coincident to designed one. The expressing level of NF-κB neucleoprotein in LECs was significantly lowed with the increase of dexamethasone concentration ( F = 36. 077 , P = 0. 004 ) , and that of IkBo: was evidently ascended ( F = 35. 741 ,P = 0. 002). In the same concentration of dexamethasone group,the expression of NF-κB in LECs showed the considerable alteration in different duration after treated of dexamethasone with the lowest expressing level in 36 hours, and significant differences were found in the expressing level between 24 hours and 36 hours ( P = 0. 002) and between 24 hours and 48 hours (P = 0. 01). The differences of expression of IκBá in LECs appeared the same pattern to NF-κB neucleoprotein. Flow cytometry showed that the apoptosis rate of LECs was obviously enhanced after action of dexamethasone in a dose-dependent manner, showing a significant difference among different groups ( F = 73. 261, P = 0.001). Conclusion It is implied that dexamethasone results in the pathogenesis and development of glucocorticoid cataract by up-regulating the expression of IκBα in LECs and suppressing the activity of NF-κB and herein induce the apoptosis of LECs at concentration-and time-dependent manner. This might be one of cellular and biological mechanisms of glucocorticoid cataract formation.
