Ⅰ型前弹力层角膜营养不良患者角膜与正常角膜蛋白的差异表达

Differences of protein expression between normal corneas and corneas of patients with corneal dystrophy of Bowman layer type 1

目的探讨中国人I型前弹力层角膜营养不良（corneal dystrophy of Bowman layertype1,CDB1）患者角膜与正常角膜的蛋白差异表达。方法取1个CDB1家系3例患者外周血,提取基因组DNA,PCR扩增TGFBI基因第4、11、12、14外显子片段并进行直接测序,证实其突变位点。取板层或穿透性角膜移植术后的病变角膜,部分标本行HE染色、阿辛蓝、PAS、刚果红、Masson三色染色后光学显微镜观察,剩余标本提取组织蛋白后进行双向凝胶电泳。以3例正常角膜组织为对照。结果 3例CDB1患者均发现TGFBI基因R124L突变。HE染色证实病变主要累及前弹力层,PAS、刚果红、Masson三色染色阳性,阿辛蓝染色阴性。在PI4-7,相对分子质量7×103~30×103之间病变与正常角膜蛋白表达存在27个差异点。结论中国人CDB1患者以R124L基因突变为主。CDB1角膜异常沉着性质为细胞外淀粉样纤维蛋白沉着。在PI4-7,相对分子质量7×103~30×103之间的差异蛋白可能在CDB1发病过程中起着重要作用。

Objective To investigate the differences of protein expression between corneas of patients with corneal dystrophy of Bowman layer type 1（CDB1）and normal corneas in Chinese.Methods Peripheral blood was obtained from 3 patients of CDB1 family.DNA was extract,and the 4th,11th,12th and 14th exons of the TGFBI gene were amplified by polymerase chain reaction and were sequenced directly.Mutable sites were confirmed.Pathological corneas were obtained from these 3 patients after lamellar keratoplasty or penetrating keratoplasty.A part of specimen was observed by HE staining,alcian blue,Periodic Acid-Schiff,Congo red staining and Masson trichrome staining.Proteins of left tissues were extracted to perform two-dimensional electrophoresis.Three normal corneas were observed at the same time as control group.Results All of these 3 patients were found with R124L mutations of TGFBI gene.HE staining confirmed that the lesion of CDB1 mostly involved in the Bowman layer.PAS,Congo red and Masson trichrome staining was positive.With 4 to 7 isoelectric points and molecular weight from 7×103 to 30×103,there were 27 different protein points between the abnormal and normal corneas.Conclusions R124L mutation is the mutational hot spot of TGFBI gene in Chinese CDB1 patients.The deposits of CDB1 are amyloid fibers out of the corneal cells.The different protein points from 4 to 7 isoelectric points and with molecular weight from 7×103 to 30×103 may be associated with the pathogenesis of CDB1.