Novel Splice Variants of Human Tenascin‐C mRNA Identified in Normal and Bullous Keratopathy Corneas
Novel Splice Variants of Human Tenascin‐C mRNA Identified in Normal and Bullous Keratopathy Corneas
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在正常和大疱性角膜病角膜中鉴定出人腱蛋白-C mRNA 的新型剪接变体
DOI：
10.1097/00003226-199805000-00014
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发表时间：
1998
期刊：
影响因子：
2.8
通讯作者：
A. Ljubimov
中科院分区：
文献类型：
--
作者：
M. Saghizadeh;Htwe L. Khin;M. Bourdon;M. Kenney;A. Ljubimov
目的研究人假晶体/无晶体大泡性角膜病变（PBK/ABK）角膜细胞外基质糖蛋白腱生蛋白C（TN-C）的表达。在此，目的是鉴定正常和PBK/ABK人角膜中的特异性TN-C mRNA剪接变体。方法采用常规和半定量逆转录聚合酶链反应（RT-PCR）方法，以TN-C的可变剪接（插入）和组成型纤连蛋白III型样重复序列为引物。通过RT-PCR产物的克隆和测序或Southern印迹分析鉴定剪接变体。结果角膜TN-CmRNA的大部分种类对应于相对较小的蛋白形式。在正常和PBK/ABK角膜中发现了4种先前未鉴定的TN-C mRNA剪接变体，其含有插入重复序列A1+A2+B+D、A1+A2+D、A1+B+D或A1+D。先前在小鼠和大鼠中描述的具有插入重复A1+A2或A1的变体也在人角膜中鉴定。半定量RT-PCR结果显示，与正常角膜相比，新的TN-C mRNA变体在PBK/ABK中显著升高。结论TN-C蛋白在PBK/ABK中表达，而在正常角膜中不表达，但在正常角膜和病变角膜中均含有15种TN-C亚型的mRNA。PBK/ABK角膜有六个相对较小的TN-C mRNA变体，包括五个新的水平升高。这些特异性亚型可能对角膜细胞的粘附和迁移产生不利影响，从而导致PBK/ABK的恶化。
Purpose Pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas accumulate an extracellular matrix glycoprotein tenascin-C (TN-C), an important modulator of cell adhesion and migration. Here, the purpose was to identify specific TN-C mRNA splice variants in normal and PBK/ABK human corneas. Methods Conventional and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) with primers to alternatively spliced (insertional) and constitutive fibronectin type III-like repeats of TN-C was used. Splice variants were identified by cloning and sequencing of RT-PCR products or by Southern blot analysis. Results The majority of corneal TN-C mRNA species corresponded to relatively small forms of the protein. Four previously unidentified TN-C mRNA splice variants were found in normal and PBK/ABK corneas that contained insertional repeats A1+A2+B+D, A1+A2+D, A1+B+D, or A1+D. Variants with insertional repeats A1+A2 or A1, previously described in mouse and rat, were also identified in human corneas. Semiquantitative RT-PCR showed that novel TN-C mRNA variants were dramatically elevated in PBK/ABK compared to normal corneas. Conclusion TN-C protein was found in PBK/ABK but not in normal corneas; however, both normal and diseased corneas contained mRNA for 15 different TN-C isoforms. PBK/ABK corneas had elevated levels of six relatively small TN-C mRNA variants including five novel ones. These specific isoforms may adversely affect adhesion and migration of corneal cells thus contributing to the exacerbation of PBK/ABK.