圆锥角膜基质细胞端粒长度与衰老相关标记物的变化

目的 探讨圆锥角膜基质细胞内端粒长度的变化和圆锥角膜基质内衰老相关β-半乳糖苷酶与衰老标记蛋白30(SMP-30)的表达,以及其在圆锥角膜发牛发展中的临床意义.方法 实验研究.收集2006年1月至12月间于山东省眼科研究所青岛眼科医院接受角膜移植治疗的圆锥角膜患者(32例)的病变角膜37份、来源于眼库的正常角膜20份.所收集圆锥角膜患者年龄范围13～34岁,平均(19±5)岁;正常角膜供体年龄范围9～25岁,平均(19±4)岁.采用Southern印迹杂交检测圆锥角膜和正常角膜基质细胞的端粒长度.以5-溴4-氯-3-吲哚-β-D-半乳糖苷原位染色法染色圆锥角膜和正常角膜基质中的衰老相关β-半乳糖苷酶,应用逆转录PCR分别检测圆锥角膜和正常角膜基质中的SMP-30.同时对圆锥角膜和正常角膜基质进行组织病理学观察.应用t检验进行统计处理.结果 圆锥角膜基质细胞的端粒长度为10.29～14.12 kb,平均端粒长度为(11.54±1.41)kb;正常角膜基质细胞的端粒长度为12.64～15.32 kb,平均端粒长度为(13.45±0.99)kb;统计学分析显示两者比较差异有统计学意义(t=4.753,P＜0.05).组织化学染色显示圆锥角膜基质内X-Gal染色町见散在分布蓝色阳性着色,表明存在衰老相关β-半乳糖苷酶的表达;而正常角膜基质中X-Gal染色阴性,未见衰老相关-β-半乳糖苷酶表达.RT-PCR检测结果 显示圆锥角膜与正常角膜中均无SMP-30蛋白的表达.组织学病理学观察显示正常角膜基质纤维排列规则紧密,角膜细胞规则地分布在基质中.而圆锥角膜基质胶原纤维排列呈现疏松不规则,细胞分布较前者散乱且数量减少.结论 与正常角膜基质相比,圆锥角膜基质细胞的端粒长度有所缩短,基质中衰老相关β-半乳糖苷酶表达增加.圆锥角膜可能是一种与组织异常老化有关的疾病.

Correlations of telomere length changing and pathogeny of keratoconus

Objective To study telomere length,senescence-associated-beta-galactosidase(SAbeta-galactosidase) and senescence marker prote-30 (SMP-30)in the stromal cells of keratoconus or normal corneas respectively,aiming finding the association of these indexes with the phenotype of keratoconus.Methods Experiment research.37 keratoconus lesions corneas were removed from 32 keratoconus patients who were operated in Shangdong Eye lnstitute between January 2006 and December 2006, and 20 normal corneas were collected from eye bank.The keratoconus corneas ages were from 13 to 34 years mean ages (19±5) years] and the control group consists of 20 normal corneas donor ages from 9 to 30 years mean ages (19±4) years ].And there was no statistical diffefence of ages between keratoconus and normal comeas.Southern blot method was utilized to detect telomere length of genomic DNA.SA-betagalactosidase was detected by 5-bromo-4-ehloro-3-indolyl-β-D-galactopyranoside (X-Gal) staining method respectively in keratoconus and normal corneas.Isolated mRNA from keratoconus and normal corneas were reverse-transcribed to cDNA and SMP-30 was detected using PCR with specific primers (sense:5' ccg tgg atg cot ttg act at 3';anti-sense:5' caa ctt cat gc tgct ttg ga 3').To compare normal corneas and keratoconus corneas by histopathological study.Statistical analysis by t test.Results The telomere lenth in stromal cells in keratoconus corneas were from 10.29 to 14.12 kb,mean (11.54±1.41) kb,while that of normal comeas were from 12.64 to 15.32 kb,mean (13.45±0.99) kb, The difference of telomere length in stromal cells of keratoconus and normal coroeas reached a statistical significant level (t=4.753,P＜0.05).That means the telomere length of keratoconus stroma was shorter than that of normal corneal stroma.Light microscopy revealed that collagen fibers in keratoconus corneal stroma were aranged in an irregular manner.Cells density in keratoconus stroma appeared lower than in normal ones but the decrease wag not significant.The staining of SA-beta-gacltosidase in the keratoconus section wos evident,but there was no staining in the normal corneas.SMP-30 was not detectable with RT-PCR method in either keratoconus or normal corneas.Conclusion Telomeres in the keratoconus stromas manifest higber SA-beta-galactosidase than control, implying that improper senescence might be involved in pathogenesis of keratoconus.