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目的 探讨白细胞抗原相关酪氨酸磷酸酶 (LAR)在人圆锥角膜组织中的表达及其临床意义。方法 收集 2001年 12月至 2002年 3月于北京同仁眼科中心行角膜移植手术的患者术中取下的角膜片,分离 15例人圆锥角膜、7例正常角膜、6例大泡性角膜病变、3例无新生血管性角膜白斑和 2例角膜营养不良标本的总RNA,进行逆转录聚合酶链反应 (RT PCR)扩增,产物进行琼脂糖凝胶电泳分析。收集 2002年 7月至 12月于北京同仁眼科中心行角膜移植手术的患者术中取下的角膜片,提取 6例圆锥角膜、3例正常角膜、3例大泡性角膜病变、3例无新生血管性角膜白斑和 2例角膜营养不良样本总蛋白,进行Western印迹法,用增强的化学发光法显迹。对LAR的RNA和蛋白的显迹条带分别采用相对定量RT PCR和Western印迹法分析。结果 使用等量总RNA进行RT PCR后,圆锥角膜样本LAR扩增产物检测到强信号条带,正常和其他角膜疾病样本只检测到弱信号或检测不到信号。圆锥角膜的LAR扩增产物测序发现其mRNA有外显子 13的删除。使用等量总蛋白进行Western印迹法分析,用LAR抗体检测发现:圆锥角膜样本有清晰的信号条带,正常和其他角膜疾病样本仅有弱信号或检测不到信号。结论 LAR的mRNA和蛋白表达量在圆锥角膜中明显增强,而在正常角膜、大泡性角膜病变、无新生
Objective To investigate the expression of leukocyte antigen-associated tyrosine phosphatase (LAR) in human keratoconus and its clinical significance. Methods Fifteen patients with corneal keratitis, seven normal corneas, six cases of bullous keratopathy were collected from patients undergoing corneal transplantation in Beijing Tongren Eye Center from December 2001 to March 2002. Total RNA was obtained from 3 cases of neovascular corneal leukoplakia and 2 cases of corneal malnutrition. RT - PCR was used to amplify the products. The products were analyzed by agarose gel electrophoresis. Corneal slices removed from patients undergoing corneal transplantation operation at Beijing Tongren Eye Center from July to December 2002 were collected, and 6 cases of keratoconus, 3 cases of corneal diseases, 3 cases of bullous keratopathy and 3 cases of no newborn Vasculogenic leukoplakia and 2 cases of corneal dystrophy samples were subjected to Western blotting and visualized by enhanced chemiluminescence. The bands of RNA and protein of LAR were analyzed by relative quantitative RT-PCR and Western blot, respectively. Results After RT PCR was carried out with the same amount of total RNA, strong signal bands were detected in LAR amplification products of keratoconus samples. Only weak or undetectable signals were detected in normal and other corneal disease samples. The LAR amplification product of the keratoconus was sequenced and found to have exon 13 deletion. Western blot analysis using equal amounts of total protein revealed that the keratoconus samples had a clear signal band detected by LAR antibody and only weak or undetectable signals for normal and other corneal disease samples. Conclusion LAR mRNA and protein expression in keratoconus was significantly enhanced, while in normal corneal, bullous keratopathy, no newborn