中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2009年
8期
724-729
,共6页
王佼佼%李绍伟%王宜强%王晔钟%文贤%臧新杰
王佼佼%李紹偉%王宜彊%王曄鐘%文賢%臧新傑
왕교교%리소위%왕의강%왕엽종%문현%장신걸
圆锥角膜%端粒%钙结合蛋白质类%细胞内信号肽和蛋白质类%衰老
圓錐角膜%耑粒%鈣結閤蛋白質類%細胞內信號肽和蛋白質類%衰老
원추각막%단립%개결합단백질류%세포내신호태화단백질류%쇠로
Keratoconus%Telomere%Calcium-binding proteins%Intracellular signaling peptides and proteins%Aging
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.
目的 探討圓錐角膜基質細胞內耑粒長度的變化和圓錐角膜基質內衰老相關β-半乳糖苷酶與衰老標記蛋白30(SMP-30)的錶達,以及其在圓錐角膜髮牛髮展中的臨床意義.方法 實驗研究.收集2006年1月至12月間于山東省眼科研究所青島眼科醫院接受角膜移植治療的圓錐角膜患者(32例)的病變角膜37份、來源于眼庫的正常角膜20份.所收集圓錐角膜患者年齡範圍13～34歲,平均(19±5)歲;正常角膜供體年齡範圍9～25歲,平均(19±4)歲.採用Southern印跡雜交檢測圓錐角膜和正常角膜基質細胞的耑粒長度.以5-溴4-氯-3-吲哚-β-D-半乳糖苷原位染色法染色圓錐角膜和正常角膜基質中的衰老相關β-半乳糖苷酶,應用逆轉錄PCR分彆檢測圓錐角膜和正常角膜基質中的SMP-30.同時對圓錐角膜和正常角膜基質進行組織病理學觀察.應用t檢驗進行統計處理.結果 圓錐角膜基質細胞的耑粒長度為10.29～14.12 kb,平均耑粒長度為(11.54±1.41)kb;正常角膜基質細胞的耑粒長度為12.64～15.32 kb,平均耑粒長度為(13.45±0.99)kb;統計學分析顯示兩者比較差異有統計學意義(t=4.753,P＜0.05).組織化學染色顯示圓錐角膜基質內X-Gal染色町見散在分佈藍色暘性著色,錶明存在衰老相關β-半乳糖苷酶的錶達;而正常角膜基質中X-Gal染色陰性,未見衰老相關-β-半乳糖苷酶錶達.RT-PCR檢測結果 顯示圓錐角膜與正常角膜中均無SMP-30蛋白的錶達.組織學病理學觀察顯示正常角膜基質纖維排列規則緊密,角膜細胞規則地分佈在基質中.而圓錐角膜基質膠原纖維排列呈現疏鬆不規則,細胞分佈較前者散亂且數量減少.結論 與正常角膜基質相比,圓錐角膜基質細胞的耑粒長度有所縮短,基質中衰老相關β-半乳糖苷酶錶達增加.圓錐角膜可能是一種與組織異常老化有關的疾病.
목적 탐토원추각막기질세포내단립장도적변화화원추각막기질내쇠로상관β-반유당감매여쇠로표기단백30(SMP-30)적표체,이급기재원추각막발우발전중적림상의의.방법 실험연구.수집2006년1월지12월간우산동성안과연구소청도안과의원접수각막이식치료적원추각막환자(32례)적병변각막37빈、래원우안고적정상각막20빈.소수집원추각막환자년령범위13～34세,평균(19±5)세;정상각막공체년령범위9～25세,평균(19±4)세.채용Southern인적잡교검측원추각막화정상각막기질세포적단립장도.이5-추4-록-3-신타-β-D-반유당감원위염색법염색원추각막화정상각막기질중적쇠로상관β-반유당감매,응용역전록PCR분별검측원추각막화정상각막기질중적SMP-30.동시대원추각막화정상각막기질진행조직병이학관찰.응용t검험진행통계처리.결과 원추각막기질세포적단립장도위10.29～14.12 kb,평균단립장도위(11.54±1.41)kb;정상각막기질세포적단립장도위12.64～15.32 kb,평균단립장도위(13.45±0.99)kb;통계학분석현시량자비교차이유통계학의의(t=4.753,P＜0.05).조직화학염색현시원추각막기질내X-Gal염색정견산재분포람색양성착색,표명존재쇠로상관β-반유당감매적표체;이정상각막기질중X-Gal염색음성,미견쇠로상관-β-반유당감매표체.RT-PCR검측결과 현시원추각막여정상각막중균무SMP-30단백적표체.조직학병이학관찰현시정상각막기질섬유배렬규칙긴밀,각막세포규칙지분포재기질중.이원추각막기질효원섬유배렬정현소송불규칙,세포분포교전자산란차수량감소.결론 여정상각막기질상비,원추각막기질세포적단립장도유소축단,기질중쇠로상관β-반유당감매표체증가.원추각막가능시일충여조직이상노화유관적질병.
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.
