中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
3期
204-208
,共5页
接合黏附分子-l%紧密连接%角膜%逆转录聚合酶链反应%免疫组织化学
接閤黏附分子-l%緊密連接%角膜%逆轉錄聚閤酶鏈反應%免疫組織化學
접합점부분자-l%긴밀련접%각막%역전록취합매련반응%면역조직화학
Junctional adhesion molecule-1%Tight junction%Cornea%Reverse transcription polymerase chain reaction%hnmunohistochemistry
背景 接合黏附分子-1(JAM-1)是新发现的跨膜蛋白,参与细胞紧密连接的结构组成和功能发挥.在眼组织方面,紧密连接对维持角膜的透明性十分重要,但是目前就JAM-1在角膜紧密连接结构和功能方面的研究较少. 目的 确定JAM-1在大鼠角膜上皮层、基质层和内皮层的构成.方法 选取4只SPF级Wistar大鼠,2只用于JAM-1基因在角膜组织中表达的逆转录聚合酶链反应(RT-PCR)检测,另2只用于免疫组织化学检测.动物过量麻醉处死后获得角膜组织并制备角膜上皮、基质和内皮标本,RT-PCR法检测角膜标本中JAM-1、occludin和claudin-1 mRNA的表达.反应产物行质量分数1.5％琼脂糖凝胶电泳并用凝胶成像系统进行分析.用兔抗鼠JAM-1单克隆抗体对角膜石蜡切片、上皮及内皮铺片行免疫组织化学检测,评估JAM-1蛋白在大鼠角膜组织各层的表达部位和表达强度. 结果 在大鼠角膜各层均可检测到JAM-1、occludin和claudin-1 mRNA的表达,PCR熔解曲线为清晰的单峰.角膜组织各层中JAM-1 mRNA表达水平与occludin mRNA相似,均高于claudin-1 mRNA.3种黏附分子均在上皮层表达最强,角膜基质层表达较弱.免疫组织化学检测显示,JAM-1蛋白在角膜各层均有明确的阳性染色,角膜上皮基底层的表达强于基质层和内皮层.角膜上皮、内皮铺片检测显示,JAM-1蛋白主要表达于上皮细胞和内皮细胞的连接部位,而角膜内皮中JAM-1蛋白的阳性染色广泛而弥散.结论 JAM-1作为细胞连接的构成成分,在角膜上皮层、内皮层和基质层均有表达,但其表达的形态和水平因组织层次的不同而不同.
揹景 接閤黏附分子-1(JAM-1)是新髮現的跨膜蛋白,參與細胞緊密連接的結構組成和功能髮揮.在眼組織方麵,緊密連接對維持角膜的透明性十分重要,但是目前就JAM-1在角膜緊密連接結構和功能方麵的研究較少. 目的 確定JAM-1在大鼠角膜上皮層、基質層和內皮層的構成.方法 選取4隻SPF級Wistar大鼠,2隻用于JAM-1基因在角膜組織中錶達的逆轉錄聚閤酶鏈反應(RT-PCR)檢測,另2隻用于免疫組織化學檢測.動物過量痳醉處死後穫得角膜組織併製備角膜上皮、基質和內皮標本,RT-PCR法檢測角膜標本中JAM-1、occludin和claudin-1 mRNA的錶達.反應產物行質量分數1.5％瓊脂糖凝膠電泳併用凝膠成像繫統進行分析.用兔抗鼠JAM-1單剋隆抗體對角膜石蠟切片、上皮及內皮鋪片行免疫組織化學檢測,評估JAM-1蛋白在大鼠角膜組織各層的錶達部位和錶達彊度. 結果 在大鼠角膜各層均可檢測到JAM-1、occludin和claudin-1 mRNA的錶達,PCR鎔解麯線為清晰的單峰.角膜組織各層中JAM-1 mRNA錶達水平與occludin mRNA相似,均高于claudin-1 mRNA.3種黏附分子均在上皮層錶達最彊,角膜基質層錶達較弱.免疫組織化學檢測顯示,JAM-1蛋白在角膜各層均有明確的暘性染色,角膜上皮基底層的錶達彊于基質層和內皮層.角膜上皮、內皮鋪片檢測顯示,JAM-1蛋白主要錶達于上皮細胞和內皮細胞的連接部位,而角膜內皮中JAM-1蛋白的暘性染色廣汎而瀰散.結論 JAM-1作為細胞連接的構成成分,在角膜上皮層、內皮層和基質層均有錶達,但其錶達的形態和水平因組織層次的不同而不同.
배경 접합점부분자-1(JAM-1)시신발현적과막단백,삼여세포긴밀련접적결구조성화공능발휘.재안조직방면,긴밀련접대유지각막적투명성십분중요,단시목전취JAM-1재각막긴밀련접결구화공능방면적연구교소. 목적 학정JAM-1재대서각막상피층、기질층화내피층적구성.방법 선취4지SPF급Wistar대서,2지용우JAM-1기인재각막조직중표체적역전록취합매련반응(RT-PCR)검측,령2지용우면역조직화학검측.동물과량마취처사후획득각막조직병제비각막상피、기질화내피표본,RT-PCR법검측각막표본중JAM-1、occludin화claudin-1 mRNA적표체.반응산물행질량분수1.5％경지당응효전영병용응효성상계통진행분석.용토항서JAM-1단극륭항체대각막석사절편、상피급내피포편행면역조직화학검측,평고JAM-1단백재대서각막조직각층적표체부위화표체강도. 결과 재대서각막각층균가검측도JAM-1、occludin화claudin-1 mRNA적표체,PCR용해곡선위청석적단봉.각막조직각층중JAM-1 mRNA표체수평여occludin mRNA상사,균고우claudin-1 mRNA.3충점부분자균재상피층표체최강,각막기질층표체교약.면역조직화학검측현시,JAM-1단백재각막각층균유명학적양성염색,각막상피기저층적표체강우기질층화내피층.각막상피、내피포편검측현시,JAM-1단백주요표체우상피세포화내피세포적련접부위,이각막내피중JAM-1단백적양성염색엄범이미산.결론 JAM-1작위세포련접적구성성분,재각막상피층、내피층화기질층균유표체,단기표체적형태화수평인조직층차적불동이불동.
Background Junctional adhesion molecule-1 (JAM-1) is intercellular transmembrane protein newly discovered and associated with the tight junction.Tight junction plays an important role in keeping the transparency of cornea,but there are few studies about JAM-1 in cornea tight junction. Objective This study was to determine the expression of JAM-1 in corneal epithelium,stroma,endothelium,and locate the distribution of JAM-1.Methods The corneas of two SFP Wistar rats were obtained,and the samples of epithelial lamella,corneal stroma and endothelium with Descemet membrane were prepared respectively for the detection of the expression of JAM-1 mRNA using reverse transcription polymerase chain reaction(RT-PCR).Primers were designed according to the genes as RGD web provided,and the objective genes were JAM-1,occludin and claudin-1.Products of PCR were examined by 1.5％ agarose gel electrophoresis and assayed with GelDoc-lt UVP Imaging System.The corneal paraffin sections and stretched preparations of epithelium and endothelium of corneal sections from other two SFP Wistar rats were prepared for the examination of JAM-1 protein expression and location by immunohistochemistry.The use of experimental animals followed the Statement of ARVO. Results JAM-1,occludin and claudin-1 mRNA were expressed in rat cornea epithelium,stroma and endothelium.PCR melting curves showed the limpid unimedality.The expression level of JAM-1 mRNA was similar to occludin mRNA and higher than that of claudin-1 with the highest level in the epithelium layer.Immunohistochemistry assay revealed that there was definite JAM-1 antibody staining in the cornea epithelium,stroma and endothelium layers.However,the basal layer of corneal epithelium presented with the strongest staining in comparison with stromal and endothelial layers.Stretched preparations of corneal epithelium and endothelium showed that JAM-1 protein appeared at the junction site of epithelial cells and endothelial cells.The basal layer of the corneal epithelium showed the strongest response,and the staining of corneal endothelium was extensive and diffuse. Conclusions JAM-1 is a composition of intercellular tight junction which expresses in cornea epithelium,endothelium and stroma.However,its appearance and level vary from different corneal layers.
