CAJ | 학술논문

背景临床研究表明多数角膜营养不良的发病与转化生长因子β诱导(TGFBI)基因突变有关,但其发病的分子机制尚不完全清楚. 目的研究TGFBI基因在人角膜组织及体外培养的角膜上皮细胞和基质细胞中的表达,为进一步研究角膜营养不良的发病机制奠定基础. 方法对人角膜上皮细胞和基质细胞进行培养和传代,应用逆转录聚合酶链反应( RT-PCR)法检测TGFBI mRNA在人角膜组织及细胞中的表达,将供体角膜组织制成石蜡包埋切片,利用免疫组织化学法检测TGFBI蛋白在角膜组织、人角膜上皮细胞和角膜基质细胞中的表达,采用免疫荧光技术检测TGFBI蛋白在细胞爬片的表达. 结果 RT-PCR检测显示人角膜组织和基质细胞中在1274 bp处可见TGFBI mRNA的清晰条带,而角膜上皮细胞中亦有TGFBImRNA表达.免疫组织化学检测显示,TGFBI蛋白在人角膜组织中基质细胞的细胞质中呈阳性表达,但人角膜上皮细胞中未见TGFBI蛋白表达.免疫荧光检测技术显示,人角膜基质细胞胞质中TGFBI蛋白呈红色荧光,而角膜上皮细胞未见TGFBI蛋白表达. 结论 TGFBI主要在人角膜基质层表达,而在上皮层几乎不表达,有助于进一步研究TGFBI在角膜营养不良发病机制中的作用.
배경 림상연구표명다수각막영양불량적발병여전화생장인자β유도(TGFBI)기인돌변유관,단기발병적분자궤제상불완전청초. 목적 연구TGFBI기인재인각막조직급체외배양적각막상피세포화기질세포중적표체,위진일보연구각막영양불량적발병궤제전정기출. 방법 대인각막상피세포화기질세포진행배양화전대,응용역전록취합매련반응( RT-PCR)법검측TGFBI mRNA재인각막조직급세포중적표체,장공체각막조직제성석사포매절편,이용면역조직화학법검측TGFBI단백재각막조직、인각막상피세포화각막기질세포중적표체,채용면역형광기술검측TGFBI단백재세포파편적표체. 결과 RT-PCR검측현시인각막조직화기질세포중재1274 bp처가견TGFBI mRNA적청석조대,이각막상피세포중역유TGFBImRNA표체.면역조직화학검측현시,TGFBI단백재인각막조직중기질세포적세포질중정양성표체,단인각막상피세포중미견TGFBI단백표체.면역형광검측기술현시,인각막기질세포포질중TGFBI단백정홍색형광,이각막상피세포미견TGFBI단백표체. 결론 TGFBI주요재인각막기질층표체,이재상피층궤호불표체,유조우진일보연구TGFBI재각막영양불량발병궤제중적작용.
Background Clinical studies indicated that the pathogenesis of most corneal dystrophy is associated with the mutation of the transforming growth factor beta-induced (TGFBI) gene.However,the molecular mechanism of mutated TGFBI gene in corneal dystrophy is unclear. Objective The present study was to investigate the expression of the TGFBI gene in human corneal tissue and cells in vitro. Methods Human corneal epithelial cells and keratocytes were cultured and passaged,and donor corneal tissue was obtained for the section preparation.RT-PCR was used to detect the expression of TGFBI mRNA in human corneal tissue and cells.Immunofluorescence was used to test the expression of the TGFBI protein in the human corneal tissue,and immunohistochemistry was used to test the expression of the TGFBI protein in human corneal epithelial cells and corneal stromal cells. Results RT-PCR analysis showed that TGFBI mRNA could be detected as a 1274 bp band in human corneal tissue and corneal stromal cells,but no TGFBI mRNA was observed in corneal epithelial cells.Immunofluorescence assay revealed that corneal stromal cells were positive ly expressed for the TGFBI protein,but the corneal epithelial cells did not express the TGFBI protein.Immunohistochemistry indicated that the expression of TGFBI was detected the red fluoressence in the cytoplasm of corneal stromal cells;however,no positive response was found in corneal epithelial cells. Conclusions The expression of the TGFBI gene occurs in human corneal stromal cells but not in the corneal epithelial cells.This result might be of helpful for studying the function and role of TGFBI gene in pathogenesis of corneal dystrophy.