Feng Yi, Feng Yumei, Zhu Xudong, Dang Ying, Ma Qingjun
Genetic Engineering Lab, Beijing Institute of Biotechnology, Beijing, Peoples Republic of China.
Mol Vis. 2004 Nov 8;10:845-50.
Aldehyde dehydrogenase 3A1 (ALDH3A1) is the most abundant soluble protein component in the mouse cornea, produced mainly by corneal epithelial cells. High levels of ALDH3A1 in cornea contribute to maintenance of a stable an d transparent corneal structure. Alkali burn is a common damage to the corneal surface, which produces an alkaline hydrolysis of matrix proteins and induces an inflammatory reaction. Our study was intended to detect changes in ALDH3A1 expression after corneal alkaline burn.
To address this issue we employed RTQ-PCR to monitor the transcriptional change of ALDH3A1 after alkali burn. We used zymography to test enzyme activity changes of ALDH3A1 in the alkali burn cornea; And SDS-PAGE and mass spectrometry technology were used to verify protein content changes and to identify ALDH3A1 protein.
Using zymography, ALDH3A1 enzymic activity was observed to decrease immediately after corneal alkali burn and the levels recovered following healing. Proteins extracted from alkali burned corneas, when run on SDS-PAGE, showed the same sized band (about 54 kDa, which is the molecular weight of ALDH3A1) but in much smaller quantity, compared to normal corneas. This result was further verified by mass spectrometry fingerprinting of the in-gel lysis product. An immediate decrease of ALDH3A1 transcription after alkali burning of the cornea was also found using RTQ-PCR. This level of transcription was gradually restored during healing.
Alkali burn of the corneal surface caused a rapid decrease of ALDH3A1 in the corneal at both the RNA and protein levels, which leads to the loses of the protective component of the corneal surface and makes it vulnerable to further damage. The ALDH3A1 level in the cornea gradually recovered during the healing process. Use of an anti-oxidation reagent as a treatment ingredient for alkali burn of the corneal surface could compensate for the decrease of anti-oxidation protection potential caused by ALDH3A1 loss.
醛脱氢酶3A1（ALDH3A1）是小鼠角膜中含量最丰富的可溶性蛋白质成分，主要由角膜上皮细胞产生。角膜中高水平的ALDH3A1有助于维持稳定且透明的角膜结构。碱烧伤是角膜表面常见的损伤，会导致基质蛋白发生碱性水解并引发炎症反应。我们的研究旨在检测角膜碱烧伤后ALDH3A1表达的变化。
为解决此问题，我们采用实时定量聚合酶链反应（RTQ-PCR）监测碱烧伤后ALDH3A1的转录变化。我们使用酶谱法检测碱烧伤角膜中ALDH3A1的酶活性变化；并采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和质谱技术验证蛋白质含量变化并鉴定ALDH3A1蛋白。
使用酶谱法观察到，角膜碱烧伤后ALDH3A1酶活性立即降低，愈合后水平恢复。与正常角膜相比，从碱烧伤角膜中提取的蛋白质在SDS-PAGE上显示出相同大小的条带（约54 kDa，即ALDH3A1的分子量），但数量少得多。凝胶内裂解产物的质谱指纹图谱进一步验证了这一结果。使用RTQ-PCR还发现角膜碱烧伤后ALDH3A1转录立即下降。这种转录水平在愈合过程中逐渐恢复。
角膜表面碱烧伤导致角膜中ALDH3A1在RNA和蛋白质水平均迅速下降，这导致角膜表面保护成分丧失，使其更容易受到进一步损伤。角膜中的ALDH3A1水平在愈合过程中逐渐恢复。使用抗氧化剂作为角膜表面碱烧伤的治疗成分可以弥补因ALDH3A1丧失而导致的抗氧化保护潜力的下降。
