Anderson P H, O'Loughlin P D, May B K, Morris H A
Hanson Institute, Frome Road, Adelaide, South Australia, 5000, Australia.
J Mol Endocrinol. 2003 Aug;31(1):123-32. doi: 10.1677/jme.0.0310123.
Critical to an understanding of the control of 1,25-dihydroxyvitamin D (1,25D) activity is a molecular appreciation of the regulation of three genes, 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), 25-hydroxyvitamin D-24-hydroxylase (CYP24) and vitamin D receptor (VDR). We now report the sensitivity, reproducibility and accuracy of a real-time reverse transcriptase-polymerase chain reaction protocol (Taqman) for the quantification of mRNA levels for these genes in total RNA extracted from kidney tIssue. The sensitivity of the protocol was at least 150 copies of mRNA per reaction. Reproducibility, expressed as the coefficient of variation, ranged between 14 and 30% at the level of approximately 10(4) copies of mRNA per reaction. Accuracy was estimated at greater than 95% for each of these mRNAs. This protocol allows for the comparison of absolute mRNA levels in extracted total RNA in kidneys from animals fed diets containing different levels of calcium, ranging from 0.05% to 1%. Serum 1,25D levels were decreased when the dietary calcium concentration was increased (P<0.05). The levels of CYP27B1 mRNA were highest in the animals fed the 0.05% calcium diet (P<0.01). Conversely, CYP24 and VDR mRNA levels were highest in the animals fed the 1% calcium diet (P<0.01). Both CYP27B1 and CYP24 mRNA levels were major determinants of serum 1,25D levels when dietary calcium intakes were varied in these adult animals (Multiple R(2)=0.70, P<0.01). No significant relationship was detected between kidney CYP27B1 and serum parathyroid hormone (PTH) suggesting that serum calcium may regulate CYP27B1 mRNA expression directly during normocalcaemia. Low levels of CYP24 mRNA were associated with high PTH levels. These findings suggest that kidney CYP24 activity, possibly regulated by factors such as PTH, acts in concert with kidney CYP27B1 to control serum 1,25D levels.
理解1,25 - 二羟基维生素D（1,25D）活性的调控，关键在于从分子层面认识三个基因的调控，即25 - 羟基维生素D - 1α - 羟化酶（CYP27B1）、25 - 羟基维生素D - 24 - 羟化酶（CYP24）和维生素D受体（VDR）。我们现在报告一种实时逆转录 - 聚合酶链反应方案（Taqman）用于定量从肾脏组织提取的总RNA中这些基因的mRNA水平的灵敏度、可重复性和准确性。该方案的灵敏度为每个反应至少150个mRNA拷贝。以变异系数表示的可重复性在每个反应约10⁴个mRNA拷贝水平时介于14%至30%之间。这些mRNA中每个的准确性估计大于95%。该方案可用于比较喂食含钙量从0.05%至1%不同水平饲料的动物肾脏中提取的总RNA中的绝对mRNA水平。当饮食钙浓度增加时，血清1,25D水平降低（P<0.05）。喂食0.05%钙饲料的动物中CYP27B1 mRNA水平最高（P<0.01）。相反，喂食1%钙饲料的动物中CYP24和VDR mRNA水平最高（P<0.01）。当这些成年动物饮食钙摄入量变化时，CYP27B1和CYP24 mRNA水平都是血清1,25D水平的主要决定因素（复相关系数R² = 0.70，P<0.01）。未检测到肾脏CYP27B1与血清甲状旁腺激素（PTH）之间存在显著关系，这表明在正常血钙水平时血清钙可能直接调节CYP27B1 mRNA表达。低水平的CYP24 mRNA与高PTH水平相关。这些发现表明，可能受PTH等因素调控的肾脏CYP24活性与肾脏CYP27B1协同作用以控制血清1,25D水平。
