中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
7期
605-609
,共5页
解正高%陈放%庄朝荣%王健%华欣%王虹%季河清
解正高%陳放%莊朝榮%王健%華訢%王虹%季河清
해정고%진방%장조영%왕건%화흔%왕홍%계하청
促红细胞生成素%可溶性促红细胞生成素受体%视网膜脱离%光感受器细胞%神经保护作用
促紅細胞生成素%可溶性促紅細胞生成素受體%視網膜脫離%光感受器細胞%神經保護作用
촉홍세포생성소%가용성촉홍세포생성소수체%시망막탈리%광감수기세포%신경보호작용
Erythropoietin%Erythropoietin soluble receptor%Retinal detachment%Photoreceptor%Neuroprotection
背景 促红细胞生成素(EPO)对多种视网膜疾病模型中视网膜神经元具有一定的保护作用,但EPO对视网膜脱离(RD)后光感受器细胞是否具有保护作用尚不清楚.目的 探讨内源性EPO对RD状态下光感受器细胞的保护作用及可能机制.方法 利用视网膜下腔注射质量分数1.4%透明质酸钠建立大鼠RD模型,按每组情况各组玻璃体腔内分别单次注射PBS或不同剂量的外源性可溶性EPO受体(EPOsR),采用计算机产生随机数字法将72只SD大鼠随机平均分为正常对照组、RD组、RD+PBS组、RD+EPOsR 2、20、200ng组.分别于造模后3d和14d用过量麻醉法处死大鼠并获得大鼠视网膜标本,采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测光感受器细胞的凋亡情况,并分别采用Western blot和免疫荧光法检测视网膜中caspase-3的活性,RD造模后14d进行组织病理学检查并测量外核层(ONL)厚度.结果 RD造模后3d,RD组ONL出现凋亡细胞核,玻璃体腔注射EPOsR组光感受器细胞凋亡进一步增加,随着玻璃体腔注射EPOsR的剂量增加,ONL凋亡细胞核有增加趋势.Western blot和免疫荧光检测结果均显示,各组视网膜caspase-3表达的条带灰度值分别为(0.15±0.04)、(0.49±0.03)、(0.50±0.07)、(0.63±0.03)、(0.69±0.04)、(0.83±0.04),各组的总体差异有统计学意义(F=76.016,P=0.000),RD+EPOsR 200ng组的caspase-3活性均强于其他各组,差异均有统计学意义(P＜0.01).RD造模后14d,正常对照组、RD组、RD+PBS组、RD+EPOsR 2、20、200ng组的ONL厚度分别为(47.39±3.39)、(33.96±3.54)、(31.83±5.21)、(31.40±2.63)、(24.99±2.06)、(19.30±3.71)μm,总体差异有统计学意义(F=44.733;P=0.000);EPOsR处理组ONL厚度明显薄于单纯RD组和RD+PBS组,差异均有统计学意义(P＜0.05).结论 RD状态下,EPOsR通过剂量依赖的方式诱导视网膜细胞的凋亡和caspase-3活性增强,而缺氧状态下视网膜神经上皮的内源性EPO表达增强可通过抑制caspase-3活性和抗凋亡作用发挥对光感受器细胞的保护作用.
揹景 促紅細胞生成素(EPO)對多種視網膜疾病模型中視網膜神經元具有一定的保護作用,但EPO對視網膜脫離(RD)後光感受器細胞是否具有保護作用尚不清楚.目的 探討內源性EPO對RD狀態下光感受器細胞的保護作用及可能機製.方法 利用視網膜下腔註射質量分數1.4%透明質痠鈉建立大鼠RD模型,按每組情況各組玻璃體腔內分彆單次註射PBS或不同劑量的外源性可溶性EPO受體(EPOsR),採用計算機產生隨機數字法將72隻SD大鼠隨機平均分為正常對照組、RD組、RD+PBS組、RD+EPOsR 2、20、200ng組.分彆于造模後3d和14d用過量痳醉法處死大鼠併穫得大鼠視網膜標本,採用末耑脫氧覈苷痠轉移酶介導的dUTP缺口末耑標記(TUNEL)法檢測光感受器細胞的凋亡情況,併分彆採用Western blot和免疫熒光法檢測視網膜中caspase-3的活性,RD造模後14d進行組織病理學檢查併測量外覈層(ONL)厚度.結果 RD造模後3d,RD組ONL齣現凋亡細胞覈,玻璃體腔註射EPOsR組光感受器細胞凋亡進一步增加,隨著玻璃體腔註射EPOsR的劑量增加,ONL凋亡細胞覈有增加趨勢.Western blot和免疫熒光檢測結果均顯示,各組視網膜caspase-3錶達的條帶灰度值分彆為(0.15±0.04)、(0.49±0.03)、(0.50±0.07)、(0.63±0.03)、(0.69±0.04)、(0.83±0.04),各組的總體差異有統計學意義(F=76.016,P=0.000),RD+EPOsR 200ng組的caspase-3活性均彊于其他各組,差異均有統計學意義(P＜0.01).RD造模後14d,正常對照組、RD組、RD+PBS組、RD+EPOsR 2、20、200ng組的ONL厚度分彆為(47.39±3.39)、(33.96±3.54)、(31.83±5.21)、(31.40±2.63)、(24.99±2.06)、(19.30±3.71)μm,總體差異有統計學意義(F=44.733;P=0.000);EPOsR處理組ONL厚度明顯薄于單純RD組和RD+PBS組,差異均有統計學意義(P＜0.05).結論 RD狀態下,EPOsR通過劑量依賴的方式誘導視網膜細胞的凋亡和caspase-3活性增彊,而缺氧狀態下視網膜神經上皮的內源性EPO錶達增彊可通過抑製caspase-3活性和抗凋亡作用髮揮對光感受器細胞的保護作用.
배경 촉홍세포생성소(EPO)대다충시망막질병모형중시망막신경원구유일정적보호작용,단EPO대시망막탈리(RD)후광감수기세포시부구유보호작용상불청초.목적 탐토내원성EPO대RD상태하광감수기세포적보호작용급가능궤제.방법 이용시망막하강주사질량분수1.4%투명질산납건립대서RD모형,안매조정황각조파리체강내분별단차주사PBS혹불동제량적외원성가용성EPO수체(EPOsR),채용계산궤산생수궤수자법장72지SD대서수궤평균분위정상대조조、RD조、RD+PBS조、RD+EPOsR 2、20、200ng조.분별우조모후3d화14d용과량마취법처사대서병획득대서시망막표본,채용말단탈양핵감산전이매개도적dUTP결구말단표기(TUNEL)법검측광감수기세포적조망정황,병분별채용Western blot화면역형광법검측시망막중caspase-3적활성,RD조모후14d진행조직병이학검사병측량외핵층(ONL)후도.결과 RD조모후3d,RD조ONL출현조망세포핵,파리체강주사EPOsR조광감수기세포조망진일보증가,수착파리체강주사EPOsR적제량증가,ONL조망세포핵유증가추세.Western blot화면역형광검측결과균현시,각조시망막caspase-3표체적조대회도치분별위(0.15±0.04)、(0.49±0.03)、(0.50±0.07)、(0.63±0.03)、(0.69±0.04)、(0.83±0.04),각조적총체차이유통계학의의(F=76.016,P=0.000),RD+EPOsR 200ng조적caspase-3활성균강우기타각조,차이균유통계학의의(P＜0.01).RD조모후14d,정상대조조、RD조、RD+PBS조、RD+EPOsR 2、20、200ng조적ONL후도분별위(47.39±3.39)、(33.96±3.54)、(31.83±5.21)、(31.40±2.63)、(24.99±2.06)、(19.30±3.71)μm,총체차이유통계학의의(F=44.733;P=0.000);EPOsR처리조ONL후도명현박우단순RD조화RD+PBS조,차이균유통계학의의(P＜0.05).결론 RD상태하,EPOsR통과제량의뢰적방식유도시망막세포적조망화caspase-3활성증강,이결양상태하시망막신경상피적내원성EPO표체증강가통과억제caspase-3활성화항조망작용발휘대광감수기세포적보호작용.
Background Erythropoietin (EPO) has a protective effect on retinal neurons in many retinal diseases,but regarding the effect of EPO on apoptosis of retinal photoreceptor cells in retinal detachment (RD) is uncompletely clear.Objective This study was to investigate the protective effect of endogenous EPO on photoreceptors in a rat model of RD and explore its possible mechanism.Methods Seventy-two Sprague- Dawley (SD) rats were randomly assigned to control group,RD group,RD+PBS group,RD+erythropoietin soluble receptor (EPOsR) 2, 20, 200ng groups with 12 rats for each group.1.4% hyaluronic acid was slowly injected into the subretinal space to induce RD in rats,and PBS or 2,20 or 200ng EPOsR was then injected into the vitreous space.On day 3 after RD,apoptotic photoreceptors were detected using transferase-mediated dUTP nickend labeling (TUNEL),and caspase-3 activity was assessed by Western-blot and immunofluorescence staining.On day 14 after RD,retinal histopathologic examination was carried out and outer nuclear layer (ONL) thickness was measured under the light microscope.The use of animals complied with the Statement of Association for Research in Vision and Ophthalmology. Results Apoptotic photoreceptors were seen in ONL of rats of the RD group.Apoptotic photoreceptors were gradually increased with the elevation of EPOsR dose in the vitreous cavity.Western blot and immunofluorescence consistently showed that the gray scale of caspase-3 activity was 0.15±0.04,0.49±0.03,0.50±0.07,0.63±0.03,0.69±0.04 and 0.83±0.04 in the normal group,RD group,RD +PBS group,RD+EPOsR 2,20,200ng groups respectively with statistically significant differences (F=76.016;P=0.000),and caspase-3 activity was considerably stronger in the RD+EPOsR 200ng group than the other groups (P＜0.01).On day 14 after RD,the ONL thicknesses in the normal control group,RD group,RD+PBS group,RD+EPOsR 2,20,200ng groups were (47.39±3.39)μm,(33.96±3.54)μm,(31.83±5.21)μm,(31.40±2.63)μm,(24.99±2.06)μm and (19.30±3.71)μm,showing significant differences among these groups (F=44.733,P=0.000).ONL thicknesses the groups treated with different doses of EPOsR were markedly thinner than that of the RD group and RD +PBS group (P＜0.01).Conclusion EPOsR induces apoptosis of retinal cells and enhances the activity of caspase-3 in a dose-dependent manner.Endogenous EPO can protect photoreceptors against anoxia-mediated damage in RD eyes through decreasing caspase-3 activity and inhibiting apoptosis.
