RANTES与SPARC基因在人角膜移植排斥反应中的表达

Expression of RANTES and SPARC gene in human corneal transplant rejection

目的 应用分子生物学技术检测角膜上皮中激活正常T细胞表达和分泌的调节物 (RANTES)和酸性富含胱氨酸分泌型蛋白 (SPARC)基因表达 ,以期为角膜移植排斥反应的早期诊断提供实验依据。方法 随机选择行穿透性角膜移植 (PK)手术患者 30例(30眼 )。其中男 2 1例 2 1眼 ,女 9例 9眼 ;年龄 6～ 78岁 ,平均 5 3.2岁 ;分 3组 ,并以行准分子激光屈光性角膜手术 (PRK)患者的正常角膜上皮作为对照。应用环钻取组Ⅰ和Ⅱ角膜上皮。应用RNA提取、RT PCR、Northern印迹杂交、组织病理、激光密度扫描半定量等技术 ,对比观察排斥、非排斥离体角膜片上皮及正常、PK术后在体角膜上皮中RANTES和SPARC基因的表达。与此同时应用裂隙灯、共聚焦显微镜 (CMTF)检查患者角膜有无排斥反应的体征。结果 正常角膜SPARC呈现低表达 ,未见RANTES表达。SPARC在3组表达率分别为 10 0 %、90 %、10 0 % ,3组比较无显著性差异 (P >0 .0 5 )。RANTES在组Ⅰ的离体片上皮中呈高表达 80 % ;在组Ⅱ 10例患者中均未见表达 ;在组Ⅲ角膜上皮样本中 ,有 6 0 %呈现高表达。这说明RANTES的表达与排斥的发生呈正相关。在对组Ⅲ患者进行裂隙灯、CMTF检查的同时进行RANTES基因检测 ,阳性率分别为 30 %、5 0 %、6 0 % ,3组比较有一定的差异性 (P <0 0 5 )

Objective To access RANTES (regulated on activation, normal T cell expressed and secreted) and SPARC (secreted protein, acidic and rich in cysteine) gene expression as an diagnostic criteria for early rejection in human corneal transplantation. Methods Thirty patients (30 eyes) included 21 male and 9 female, aging from 6 to 78 years with mean 53.2 years. They were divided into three groups. Another 10 patients underwent PRK were used as control. Corneal epithelial cells were collected from the triphined corneal button in groups Ⅰand Ⅱ. Both RATENS and SPARC gene expression in corneal epithelial cells were studied by molecular biological techniques including RT-PCR for mRNA in total extracted RNA. The cDNA was semi-quantified by laser densitometry, and Northern blotting. In addition, the patients were examined by slit-lamp and confocal microscopy through focusing (CMTF) for detecting signs of allograft rejection.Results There were no RANTES gene expression and minimal expression of SPARC in normal cornea epithelium;over expression of RANTES gene was detected in rejected allograft corneal epithelial cells, but not detected in isograft. Over expression of RANTES gene was correlated with clinical signs of corneal rejection. Over expression of RANTES genes was found in 8 of 10 patients (80%) in groupⅠas indicated in increasing mRNA level. Over expression of SPARC gene was found to be 100%, 90% and 100% in groupsⅠ,Ⅱand Ⅲrespectively. Positive for slit-lamp microscopic and CMFT findings and over expression of RANTES were found to be 30%, 50% and 60% respectively; that was highly statistically significant (P<0.05).Conclusion RANTES over expression appear to be a monitorial parameter to detect early rejection of corneal rejection, its clinical significance remains for further study.