中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
4期
313-317
,共5页
丁敏%卢清君%武坤%邓爱军%曾惠阳
丁敏%盧清君%武坤%鄧愛軍%曾惠暘
정민%로청군%무곤%산애군%증혜양
视网膜变性/遗传性%rd小鼠%烟酰胺二磷酸腺苷氧化酶/抑制剂%活性氧%小胶质细胞
視網膜變性/遺傳性%rd小鼠%煙酰胺二燐痠腺苷氧化酶/抑製劑%活性氧%小膠質細胞
시망막변성/유전성%rd소서%연선알이린산선감양화매/억제제%활성양%소효질세포
Retinal degeneration/genetics%rd Mice%Nicotinamide adenine dinucleotide phosphate oxidase/inhibitor%Reactive oxygen species%Microglia
背景 我们先前的研究表明,rd小鼠遗传性视网膜色素变性(RP)过程中小胶质细胞活化与感光细胞的凋亡密切相关.研究显示,小胶质细胞中烟酰胺二磷酸腺苷(NADPH)氧化酶的活化在小胶质细胞活化及神经元损伤中发挥重要作用,但NADPH氧化酶在RP过程中作用机制及其抑制剂的作用有待探讨.目的 探讨rd小鼠发生RP过程中NADPH氧化酶产生活性氧簇(ROS)的活化反应及其抑制剂对感光细胞的保护作用. 方法 按抛掷硬币法将60只SPF级rd小鼠随机分为香荚兰乙酮注射组和PBS对照组,香荚兰乙酮注射组小鼠于出生后9d(P9)腹腔内注射NADPH氧化酶抑制剂香荚兰乙酮10 mg/kg(0.01 ml/kg),每日1次,连续5d(至P13);PBS对照组rd小鼠以同样方式注射等容量的PBS;C57 BL/6N小鼠10只不注射任何药物作为rd小鼠的野生对照鼠.各组小鼠于出生后14 d(P14)处死并制备视网膜冰冻切片,采用二氢乙锭(DHE)荧光染色法检测3个组小鼠视网膜中ROS的表达;采用实时定量PCR(real-time PCR)法测定2个组rd小鼠视网膜感光细胞中视紫红质mRNA的定量表达;采用苏木精-伊红染色法检查2个组rd小鼠视网膜外核层厚度.结果 DHE荧光染色表明,小鼠视网膜中ROS表达呈红色荧光,注药组小鼠视网膜外核层中ROS的红色荧光明显强于C57BL/6N野生鼠,但明显弱于PBS对照组.Real-time PCR检测表明,香荚兰乙酮注射组小鼠感光细胞中视紫红质mRNA相对表达量为4.21 ±0.33,明显低于PBS对照组的0.93±0.24,差异有统计学意义(t=2.360,P=0.000);香荚兰乙酮注射组小鼠视网膜外核层厚度为(35.95±1.63) μm,明显厚于PBS对照组的(23.17±1.38) μm,差异有统计学意义(t=3.850,P=0.016).结论 在rd小鼠视网膜感光细胞变性过程中,NADPH氧化酶生成ROS的活化反应明显增强;香荚兰乙酮能够延缓rd小鼠感光细胞的凋亡过程.
揹景 我們先前的研究錶明,rd小鼠遺傳性視網膜色素變性(RP)過程中小膠質細胞活化與感光細胞的凋亡密切相關.研究顯示,小膠質細胞中煙酰胺二燐痠腺苷(NADPH)氧化酶的活化在小膠質細胞活化及神經元損傷中髮揮重要作用,但NADPH氧化酶在RP過程中作用機製及其抑製劑的作用有待探討.目的 探討rd小鼠髮生RP過程中NADPH氧化酶產生活性氧簇(ROS)的活化反應及其抑製劑對感光細胞的保護作用. 方法 按拋擲硬幣法將60隻SPF級rd小鼠隨機分為香莢蘭乙酮註射組和PBS對照組,香莢蘭乙酮註射組小鼠于齣生後9d(P9)腹腔內註射NADPH氧化酶抑製劑香莢蘭乙酮10 mg/kg(0.01 ml/kg),每日1次,連續5d(至P13);PBS對照組rd小鼠以同樣方式註射等容量的PBS;C57 BL/6N小鼠10隻不註射任何藥物作為rd小鼠的野生對照鼠.各組小鼠于齣生後14 d(P14)處死併製備視網膜冰凍切片,採用二氫乙錠(DHE)熒光染色法檢測3箇組小鼠視網膜中ROS的錶達;採用實時定量PCR(real-time PCR)法測定2箇組rd小鼠視網膜感光細胞中視紫紅質mRNA的定量錶達;採用囌木精-伊紅染色法檢查2箇組rd小鼠視網膜外覈層厚度.結果 DHE熒光染色錶明,小鼠視網膜中ROS錶達呈紅色熒光,註藥組小鼠視網膜外覈層中ROS的紅色熒光明顯彊于C57BL/6N野生鼠,但明顯弱于PBS對照組.Real-time PCR檢測錶明,香莢蘭乙酮註射組小鼠感光細胞中視紫紅質mRNA相對錶達量為4.21 ±0.33,明顯低于PBS對照組的0.93±0.24,差異有統計學意義(t=2.360,P=0.000);香莢蘭乙酮註射組小鼠視網膜外覈層厚度為(35.95±1.63) μm,明顯厚于PBS對照組的(23.17±1.38) μm,差異有統計學意義(t=3.850,P=0.016).結論 在rd小鼠視網膜感光細胞變性過程中,NADPH氧化酶生成ROS的活化反應明顯增彊;香莢蘭乙酮能夠延緩rd小鼠感光細胞的凋亡過程.
배경 아문선전적연구표명,rd소서유전성시망막색소변성(RP)과정중소효질세포활화여감광세포적조망밀절상관.연구현시,소효질세포중연선알이린산선감(NADPH)양화매적활화재소효질세포활화급신경원손상중발휘중요작용,단NADPH양화매재RP과정중작용궤제급기억제제적작용유대탐토.목적 탐토rd소서발생RP과정중NADPH양화매산생활성양족(ROS)적활화반응급기억제제대감광세포적보호작용. 방법 안포척경폐법장60지SPF급rd소서수궤분위향협란을동주사조화PBS대조조,향협란을동주사조소서우출생후9d(P9)복강내주사NADPH양화매억제제향협란을동10 mg/kg(0.01 ml/kg),매일1차,련속5d(지P13);PBS대조조rd소서이동양방식주사등용량적PBS;C57 BL/6N소서10지불주사임하약물작위rd소서적야생대조서.각조소서우출생후14 d(P14)처사병제비시망막빙동절편,채용이경을정(DHE)형광염색법검측3개조소서시망막중ROS적표체;채용실시정량PCR(real-time PCR)법측정2개조rd소서시망막감광세포중시자홍질mRNA적정량표체;채용소목정-이홍염색법검사2개조rd소서시망막외핵층후도.결과 DHE형광염색표명,소서시망막중ROS표체정홍색형광,주약조소서시망막외핵층중ROS적홍색형광명현강우C57BL/6N야생서,단명현약우PBS대조조.Real-time PCR검측표명,향협란을동주사조소서감광세포중시자홍질mRNA상대표체량위4.21 ±0.33,명현저우PBS대조조적0.93±0.24,차이유통계학의의(t=2.360,P=0.000);향협란을동주사조소서시망막외핵층후도위(35.95±1.63) μm,명현후우PBS대조조적(23.17±1.38) μm,차이유통계학의의(t=3.850,P=0.016).결론 재rd소서시망막감광세포변성과정중,NADPH양화매생성ROS적활화반응명현증강;향협란을동능구연완rd소서감광세포적조망과정.
Background Our previous study demonstrated that microglial activation is closely associated with photoreceptor apoptosis in rd mice.Recent studies on central nervous system (CNS) showed that activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the microglia activation and neural cell death.However,the mechanism of NADPH oxidase during the retinal degeneration and the effect of NADPH oxidase inhibitor on photoreceptor apoptosis are concerned.Objective The aim of this study was to further explore the production of reactive oxygen species (ROS) by NADPH oxidase in the retinal degenerative process of rd mice and protection of NADPH oxidase inhibitor on photoreceptors.Methods Sixty rd mice at postnatal day 9 (P9) were randomized into the experimental group and the control group by throwing coins method.Apocynin,a NADPH oxidase inhibitor,was intraperitoneally injected in the dose of 10 mg/kg (0.01 ml/kg) once daily for 5 days (P13) in the experimental group,and the equal amount of PBS was used in the same way in the control group,and 10 C57BL/6N mice without injection of any drugs served as the wild type mice group.All the mice were sacrificed in P14 for the preparation of retinal sections.The expression of ROS in the retina was detected by dihydroethidium (DHE) fluorescence staining.Expression level of rhodopsin mRNA in the photoreceptor of the mice was determined by real-time PCR,and the thickness of retinal outer nuclear layer (ONL) in the mice of the experimental group and the control group was measured using hematoxylin & eosin staining.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology.Results DHE staining showed that the ROS presented with the red fluorescence in the mouse retinas.In the rd mice of the experimental group,the ROS fluorescence intensity was dramatically enhanced in comparison with C57BL/6N mice,but weakened in comparison with the rd mice of the control group.Real-time PCR revealed that the relative expressing level of rhodopsin mRNA in the photoreceptor was (4.21±0.33) in the experimental group and (0.93±0.24) in the control group,showing a significant difference between them (t =2.360,P =0.000).The thickness value of retinal ONL was (35.95±1.63)μm in the mice of the experimental group,which was significantly higher than that in the mice of the control group ([23.17±1.38] μm) (t=3.850,P=0.016).Conclusions In the retinal degeneration of rd mice,activation of NADPH oxidase increases the ROS production.Apocynin can slow the apoptosis procedure of photoreceptor cells of rd mice.
