中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
8期
691-694
,共4页
宋鸿雁%李瑶%卢泽艳%吴刘成%邵义祥
宋鴻雁%李瑤%盧澤豔%吳劉成%邵義祥
송홍안%리요%로택염%오류성%소의상
眼睑/生长和发育%形态发生学%基因突变%血清反应因子%眼部异常/遗传学%角质形成细胞%表型%B6-Co小鼠
眼瞼/生長和髮育%形態髮生學%基因突變%血清反應因子%眼部異常/遺傳學%角質形成細胞%錶型%B6-Co小鼠
안검/생장화발육%형태발생학%기인돌변%혈청반응인자%안부이상/유전학%각질형성세포%표형%B6-Co소서
Eyelids/growth & development%Morphogenesis%Mutation%Serum response factor%Eye abnormality/genetics%Keratinocytes%Phenotype%Mice,B6-Co
背景 C57BL/6角膜混浊表型的突变系(B6-Co)小鼠具有出生眼睑闭合不全(EOB)表型,是研究眼睑发育机制的良好动物模型.探讨血清反应因子(SRF)与B6-Co小鼠EOB表型形成的关系可为人类先天性眼睑发育缺陷产生机制的研究提供理论依据.目的 检测SRF在B6-Co小鼠胚胎眼睑发育关键时期的表达.方法 采用肌内注射戊巴比妥钠安乐死术,分别剖取B6-Co母鼠以及表型正常B6母鼠体内胚胎期(E)16.5 d、E17.5 d和E18.5 d小鼠各9只,分离眼睑组织,分别采用实时定量PCR法和Western blot法检测小鼠眼睑组织中SRF mRNA及其蛋白的相对表达水平.取各胎龄的B6-Co小鼠和B6小鼠制作组织冰冻切片,利用免疫荧光技术检测并比较2种小鼠SRF在眼睑组织中的定位和表达强度.结果 B6-Co小鼠E16.5 d和E17.5 d眼睑组织中SRF mRNA的相对表达水平分别为0.41±0.06和0.24±0.17,明显低于B6小鼠的1.03±0.17和1.01±0.09,差异均有统计学意义(P=0.025、0.017);B6-Co小鼠E16.5 d和E17.5 d眼睑组织中SRF蛋白的表达水平分别为0.08±0.01和0.08±0.01,明显低于B6小鼠的0.12±0.03和0.13 ±0.02,差异均有统计学意义(P=0.036、0.024);而2种小鼠间E18.5 d时眼睑组织中SRF mRNA及其蛋白的表达量差异均无统计学意义(P=0.387、0.774).免疫荧光染色显示,SRF蛋白多表达于B6-Co小鼠和B6小鼠眼睑组织的角质层细胞,但B6-Co小鼠眼睑角质形成细胞中SRF蛋白表达的荧光强度明显弱于B6小鼠.结论 SRF在B6-Co小鼠眼睑组织中的表达量明显下调,SRF可能参与眼睑发育缺陷的发生过程.
揹景 C57BL/6角膜混濁錶型的突變繫(B6-Co)小鼠具有齣生眼瞼閉閤不全(EOB)錶型,是研究眼瞼髮育機製的良好動物模型.探討血清反應因子(SRF)與B6-Co小鼠EOB錶型形成的關繫可為人類先天性眼瞼髮育缺陷產生機製的研究提供理論依據.目的 檢測SRF在B6-Co小鼠胚胎眼瞼髮育關鍵時期的錶達.方法 採用肌內註射戊巴比妥鈉安樂死術,分彆剖取B6-Co母鼠以及錶型正常B6母鼠體內胚胎期(E)16.5 d、E17.5 d和E18.5 d小鼠各9隻,分離眼瞼組織,分彆採用實時定量PCR法和Western blot法檢測小鼠眼瞼組織中SRF mRNA及其蛋白的相對錶達水平.取各胎齡的B6-Co小鼠和B6小鼠製作組織冰凍切片,利用免疫熒光技術檢測併比較2種小鼠SRF在眼瞼組織中的定位和錶達彊度.結果 B6-Co小鼠E16.5 d和E17.5 d眼瞼組織中SRF mRNA的相對錶達水平分彆為0.41±0.06和0.24±0.17,明顯低于B6小鼠的1.03±0.17和1.01±0.09,差異均有統計學意義(P=0.025、0.017);B6-Co小鼠E16.5 d和E17.5 d眼瞼組織中SRF蛋白的錶達水平分彆為0.08±0.01和0.08±0.01,明顯低于B6小鼠的0.12±0.03和0.13 ±0.02,差異均有統計學意義(P=0.036、0.024);而2種小鼠間E18.5 d時眼瞼組織中SRF mRNA及其蛋白的錶達量差異均無統計學意義(P=0.387、0.774).免疫熒光染色顯示,SRF蛋白多錶達于B6-Co小鼠和B6小鼠眼瞼組織的角質層細胞,但B6-Co小鼠眼瞼角質形成細胞中SRF蛋白錶達的熒光彊度明顯弱于B6小鼠.結論 SRF在B6-Co小鼠眼瞼組織中的錶達量明顯下調,SRF可能參與眼瞼髮育缺陷的髮生過程.
배경 C57BL/6각막혼탁표형적돌변계(B6-Co)소서구유출생안검폐합불전(EOB)표형,시연구안검발육궤제적량호동물모형.탐토혈청반응인자(SRF)여B6-Co소서EOB표형형성적관계가위인류선천성안검발육결함산생궤제적연구제공이론의거.목적 검측SRF재B6-Co소서배태안검발육관건시기적표체.방법 채용기내주사무파비타납안악사술,분별부취B6-Co모서이급표형정상B6모서체내배태기(E)16.5 d、E17.5 d화E18.5 d소서각9지,분리안검조직,분별채용실시정량PCR법화Western blot법검측소서안검조직중SRF mRNA급기단백적상대표체수평.취각태령적B6-Co소서화B6소서제작조직빙동절편,이용면역형광기술검측병비교2충소서SRF재안검조직중적정위화표체강도.결과 B6-Co소서E16.5 d화E17.5 d안검조직중SRF mRNA적상대표체수평분별위0.41±0.06화0.24±0.17,명현저우B6소서적1.03±0.17화1.01±0.09,차이균유통계학의의(P=0.025、0.017);B6-Co소서E16.5 d화E17.5 d안검조직중SRF단백적표체수평분별위0.08±0.01화0.08±0.01,명현저우B6소서적0.12±0.03화0.13 ±0.02,차이균유통계학의의(P=0.036、0.024);이2충소서간E18.5 d시안검조직중SRF mRNA급기단백적표체량차이균무통계학의의(P=0.387、0.774).면역형광염색현시,SRF단백다표체우B6-Co소서화B6소서안검조직적각질층세포,단B6-Co소서안검각질형성세포중SRF단백표체적형광강도명현약우B6소서.결론 SRF재B6-Co소서안검조직중적표체량명현하조,SRF가능삼여안검발육결함적발생과정.
Background Mutant C57BL/6 mouse with corneal opacity (B6-Co) appears eye open at birth (EOB) phenotype,which is a good animal model in the study of developmental mechanism of eyelid.Investigating the relationship between serum response factor (SRF) and EOB phenotype can provide theoretical support for the research on the mechanism of innate defects in eyelid development in humans.Objective This study was to assess the dynamic expressions of SRF in eyelid of embryonic B6-Co mouse.Methods Total RNA was extracted from B6 and B6-Co mice eyelid tissue at embryonic day 16.5 (E16.5 d),E17.5 d and E18.5 d.The relative expression levels of SRF mRNA and protein in the eyelid tissue of B6 and B6-Co embryonic mice were assayed by real-time quantitative PCR and Western blot,respectively.In situ expressions of SRF protein in eyelid of B6-Co mice and B6 mice were detected using immunofluorescence technique.The use and care of the animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals of Nantong University.Results The relative expression levels ofSRF mRNA in the eyelids were 0.41±0.06 and 0.24±0.17 in E16.5 d and E17.5 d of B6-Co mice,showing a significant decline in comparison with 1.03 ±0.17 and 1.01 ±0.09 in the B6 mice (P =0.025,0.017).The expression levels of SRF protein in the eyelids of E16.5 d and E17.5 d B6-Co mice were 0.08±0.01 and 0.08± 0.01,which were significantly lower than 0.12 ±0.03 and 0.13 ± 0.02 of B6 mice (P =0.036,0.024).However,there were no significant differences in the expression levels of SRF mRNA and protein in E18.5 d between the B6-Co mice and B6 mice (P =0.387,0.774).Immunofluorescence assay displayed that SRF was expressed in the keratinocytes of eyelids in both mice,but the fluorescence intensity was weaker in the B6-Co mice.Conclusions SRF probably interrupts the developing process of eyelid in early embryo of B6-Co mice.
