年龄相关性白内障晶状体上皮细胞中hTERT的活性表达(1)
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[摘要] 目的:探讨hTERT在年龄相关性白内障晶状体上皮细胞(lens epithelial cells,LEC)中的活性表达及在白内障形成中的作用。方法:对40例年龄相关性白内障患者和10例正常LEC中的hTERT进行原位杂交技术检测,并进行比较。结果:年龄相关性白内障LEC中hTERT的阳性表达率明显增高,与正常晶状体组相比,有显著性差异(P<0.01)。结论:LEC中hTERT活性的变化与年龄相关性白内障的形成密切相关,随着年龄的增长,hTERT的表达活性升高,使LEC发生衰老,从而形成白内障。
[关键词] 端粒酶逆转录酶;晶状体上皮细胞;年龄相关性白内障
[中图分类号] R776.1[文献标识码]A [文章编号]1673-7210(2010)10(a)-019-03
Expression of human telomerase reverse transcriptase in lens epithelial cells of age-related cataract
TAO Dan,WANG Xuemei, LEI Huo
(Department of Ophthalmology, the First People's Hospital of Yunnan Province &the Affiliated Kunhua Hospital of Kunming Medical College, Kunming 650031, China)
[Abstract] Objective: To investigate the expression of human telomerase reverse transcriptase (hTERT) in the lens epithelial cells(LEC)of age-related cataract and its effect on age-related cataract formation. Methods: In situ hybridization was used to detect the mRNA expression of hTERT in 40 LEC specimen of age-related cataract, 10 LEC specimen from normal persons cataracts. Results: The positive rate of hTERT in age-related cataract LEC was significantly higher with the moderate and strong staining intensity in comparison with traumatic LEC (P<0.01). Conclusion: Over expression of hTERT in age-related cataract LEC is related to cataract. With age the increase of telomerase activity is the mechanism of age-related cataract.
[Key words] hTERT; LEC; Age-related cataract
年龄相关性白内障 (age-related cataract,ARC)是最常见的白内障类型,其患病率随年龄的增长而明显增高。据不完全统计在我国50~60岁年龄相关性白内障发病率为60%~70%,70岁以上的达80%,80岁以上的老年人几乎达100%。年龄相关性白内障的病因复杂,其发病机制迄今为止尚未完全达成共识。本研究采用原位杂交的方法检测人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)在人LEC中的活性表达,探讨其在年龄相关性白内障发病机制中的作用,为进一步阐明其发病机制提供实验依据。
1 材料与方法
1.1 材料
年龄相关性白内障组晶状体上皮细胞取自2009年3~5月份在我院行白内障超声乳化术的病人40例,其中,男27例,女13例;年龄55~80岁,平均69.8岁。术前裂隙灯显微镜排外其他眼部疾病及全身性疾病。正常人组晶状体上皮细胞取自透明晶状体白内障摘除术的患者10例,其中,男6例,女4例;年龄18~30岁,平均24岁。二者均在术中环形撕囊取前囊膜。
1.2 试剂
兔抗TERT多克隆抗体(BA0564)及人TERT原位杂交检测试剂盒(MK1158-h)均购自武汉博士德生物工程有限公司。 1.3 实验方法
1.3.1 组片制作晶体前囊膜取出后,用BSS液冲洗黏附的晶状体皮质,用4%多聚甲醛固定,石蜡包埋,组织均为4 μm连续切片,其中一张行原位杂交研究,6张行免疫组化研究,贴附于多聚赖氨酸处理的载玻片上,放至烤箱60℃烤6 h,取出待用。
1.3.2 hTERTmRNA表达的检测石蜡切片常规脱蜡;3%H2O2 室温10 min;滴加3%柠檬酸新鲜稀释的胃蛋白酶,室温消化10 min;1%多聚甲醛/0.1M PBS室温固定10 min;每张切片加预杂交液20 μl,恒温箱37℃ 2 h;每张切片加杂交液20 μl,用原位杂交专用盖玻片盖在切片上,恒温箱37℃过夜;滴加封闭液,37℃ 30 min;滴加生物素化鼠抗地高辛 ......
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