Accumulation of mitochondrial DNA damage in keratoconus corneas
Accumulation of mitochondrial DNA damage in keratoconus corneas
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DOI：
10.1167/iovs.04-1395
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发表时间：
2005-04-01
影响因子：
4.4
通讯作者：
Kenney, MC
中科院分区：
文献类型：
--
作者：
Atilano, SR;Coskun, P;Kenney, MC
目的。目的：探讨角膜圆锥（KC）角膜是否存在比正常角膜更多的线粒体DNA损伤。正常角膜33例，KC角膜34例。对线粒体编码的细胞色素c氧化酶（复合体IV）亚基1 （CO-I）和孔蛋白进行免疫组化。分离总DNA，采用长延伸聚合酶链反应（LX-PCR）或短延伸聚合酶链反应（SX-PCR）扩增mtDNA基因组。用限制性内切酶切取LX-PCR mtDNA，确定全长mtDNA扩增子。采用Southern blot法检测SX-PCR mtDNA。采用多肽核酸定向夹持PCR对T414G突变进行分析。Real-time PCR检测mtDNA与细胞核(n) dna的比值。KC角膜在角膜变薄区域CO-I降低。限制性内切酶酶切的LX-PCR mtDNA显示出预期大小的条带，但PstI在一些KC角膜中显示了两条额外的条带（2/18）。通过LX-PCR （7.4 +/- 3.8 vs. 4.3 +/- 2.7, P < 0.04）和SX-PCR (5.5 +/- 0.55 vs. 2.4 +/- 2.0, P < 0.006)，与正常角膜相比，KC角膜的小条带数量增加（代表mtDNA缺失/突变）。SX-PCR产物的Southern blot分析证实了它们的mtDNA来源。在KC和正常角膜中均未检测到T414G突变。KC角膜mtdna / ndna比值低于正常角膜（26%,P < 0.7）。KC角膜表现出比正常角膜更多的mtDNA损伤。先前报道的氧化应激增加和mtDNA完整性改变可能相互关联，并且可能在KC发病机制中起重要作用。
PURPOSE. To determine whether keratoconus ( KC) corneas have more mitochondrial (mt) DNA damage than do normal corneas.METHODS. Thirty-three normal corneas and 34 KC corneas were studied. Immunohistochemistry for mitochondria-encoded cytochrome c oxidase ( complex IV) subunit 1 (CO-I) and porins was performed. Total DNA was isolated and mtDNA genome amplified by either long-extension -polymerase chain reaction (LX-PCR) or short-extension-PCR (SX-PCR). LX-PCR mtDNA was digested with restriction enzymes to confirm full-length mtDNA amplicon. SX-PCR mtDNA was probed by Southern blot analysis. The T414G mutation was analyzed by peptide nucleic acid directed clamping PCR. Real-time PCR measured the ratio of mtDNA to nuclear (n) DNA.RESULTS. KC corneas had decreased CO-I in areas of corneal thinning. LX-PCR mtDNA digested with restriction enzymes showed expected size bands except for PstI, which showed two additional bands in some KC corneas (2/18). By both LX-PCR ( 7.4 +/- 3.8 vs. 4.3 +/- 2.7, P < 0.04) and SX-PCR (5.5 +/- 0.55 vs. 2.4 +/- 2.0, P < 0.006), KC corneas had an increased number of smaller-sized bands ( representing mtDNA deletions/ mutations) compared with normal corneas. Southern blot analysis of SX-PCR products confirmed their mtDNA origin. The T414G mutation was not detected in either KC or normal corneas. KC corneas showed a trend of lower mtDNA-to-nDNA ratio (26%, P < 0.7) than did normal corneas.CONCLUSIONS. KC corneas exhibit more mtDNA damage than do normal corneas. The previously reported increased oxidative stress and altered integrity of mtDNA may be related to each other and may be important in KC pathogenesis.