Bordukalo-Niksic Tatjana, Cicin-Sain Lipa, Jernej Branimir
Laboratory for Neurochemistry and Molecular Neurobiology, Rudjer Boskovic Institute, Bijenicka 54, HR-10000 Zagreb, Croatia.
Neurosci Lett. 2004 Oct 7;369(1):44-9. doi: 10.1016/j.neulet.2004.07.039.
By selective breeding, two sublines of rats, termed Wistar-Zagreb 5HT rats, with constitutionally high or low values of platelet serotonin (5HT) level and activity of platelet serotonin transporter (5HTt) have been developed. Previous studies demonstrated significant differences between the sublines in the expression of platelet 5HTt at the level of both mRNA and protein. Neurochemical and behavioural studies demonstrated differences in functional activity of brain 5HTt indicating that, similarly to platelets, differences in mRNA level might be expected in brains of selected animals. In this work, semi-quantitative RT-PCR method for measuring the 5HTt expression in rat tissues was described and then used to quantify the 5HTt mRNA in brains and platelets of animals from high-5HT and low-5HT sublines. Three different housekeeping genes: GAPDH, beta-actin and cyclophylin B, were used as internal standards to normalise 5HTt signals. Significant differences in platelet 5HTt mRNA between the sublines were confirmed, as contrasted to only a tendency toward higher 5HTt mRNA levels in midbrain of animals from the high-5HT subline. Results indicate differences in transcriptional regulation of central and peripheral 5HT transporters, suggesting that homeostatic control in the brain counteract more efficiently the selection pressure than in the periphery.
通过选择性育种，已培育出两个大鼠亚系，称为Wistar - 萨格勒布5羟色胺（5HT）大鼠，其血小板5羟色胺（5HT）水平和血小板5羟色胺转运体（5HTt）活性在体质上呈现高值或低值。先前的研究表明，这两个亚系在血小板5HTt的mRNA和蛋白质水平表达上存在显著差异。神经化学和行为学研究表明，脑5HTt的功能活性存在差异，这表明与血小板类似，在所选动物的大脑中可能预期mRNA水平也存在差异。在这项工作中，描述了用于测量大鼠组织中5HTt表达的半定量逆转录 - 聚合酶链反应（RT - PCR）方法，然后用于定量高5HT和低5HT亚系动物大脑和血小板中的5HTt mRNA。使用三种不同的管家基因：甘油醛 - 3 - 磷酸脱氢酶（GAPDH）、β - 肌动蛋白和亲环蛋白B，作为内标来标准化5HTt信号。证实了亚系之间血小板5HTt mRNA存在显著差异，与之形成对比的是，仅在高5HT亚系动物的中脑中存在5HTt mRNA水平升高的趋势。结果表明中枢和外周5HT转运体的转录调控存在差异，这表明大脑中的稳态控制比外周更有效地抵消了选择压力。
