Ohno Misa, Kida Yuta, Sakaguchi Masayoshi, Sugahara Yasusato, Oyama Fumitaka
Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo, Japan.
BMC Mol Biol. 2014 Oct 8;15:23. doi: 10.1186/1471-2199-15-23.
Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4). Recently, CLPs have attracted considerable attention due to their increased expression in a number of pathological conditions, including asthma, allergies, rheumatoid arthritis and malignant tumors. Although the exact functions of CLPs are largely unknown, the significance of their increased expression levels during pathophysiological states needs to be determined. The quantification of BRP-39, Ym1 and Ym2 is an important step in gaining insight into the in vivo regulation of the CLPs.
We constructed a standard DNA for quantitative real-time PCR (qPCR) by containing three CLPs target fragments and five reference genes cDNA in a one-to-one ratio. We evaluated this system by analyzing the eight target cDNA sequences. Tissue cDNAs obtained by reverse transcription from total RNA from four embryonic stages and eight adult tissues were analyzed using the qPCR system with the standard DNA.
We established a qPCR system detecting CLPs and comparing their expression levels with those of five reference genes using the same scale in mouse tissues. We found that BRP-39 and Ym1 were abundant in the mouse lung, whereas Ym2 mRNA was abundant in the stomach, followed by lung. The expression levels of BRP-39 and Ym1 in the mouse lung were higher than those of two active chitinases and were comparable to glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene which is constitutively expressed in all tissues.
Our results indicate that catalytically inactive BRP-39 and Ym1 are constitutive genes in normal mouse lung.
小鼠和人类会产生几丁质酶样蛋白（CLPs），它们与几丁质酶高度同源，但缺乏几丁质分解活性。小鼠主要表达三种CLPs，包括乳腺退化蛋白-39（BRP-39）[几丁质酶3样-1（Chi3l1）或38 kDa糖蛋白（gp38k）]、Ym1（Chi3l3）和Ym2（Chi3l4）。最近，CLPs因其在包括哮喘、过敏、类风湿性关节炎和恶性肿瘤在内的多种病理状况下表达增加而备受关注。尽管CLPs的确切功能在很大程度上尚不清楚，但需要确定它们在病理生理状态下表达水平升高的意义。对BRP-39、Ym1和Ym2进行定量是深入了解CLPs体内调节的重要一步。
我们通过以一对一比例包含三个CLPs靶片段和五个参考基因cDNA构建了用于定量实时PCR（qPCR）的标准DNA。我们通过分析八个靶cDNA序列对该系统进行了评估。使用该qPCR系统和标准DNA分析了从四个胚胎阶段和八个成年组织的总RNA逆转录获得的组织cDNA。
我们建立了一种qPCR系统，可在小鼠组织中检测CLPs并将其表达水平与五个参考基因的表达水平进行同比例比较。我们发现BRP-39和Ym1在小鼠肺中含量丰富，而Ym2 mRNA在胃中含量丰富，其次是肺。小鼠肺中BRP-39和Ym1的表达水平高于两种活性几丁质酶，并且与甘油醛-3-磷酸脱氢酶相当，甘油醛-3-磷酸脱氢酶是一种在所有组织中组成性表达的管家基因。
我们的结果表明，催化无活性的BRP-39和Ym1是正常小鼠肺中的组成性基因。
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