Determination of mRNA Expression of Human UDP-Glucuronosyltransferases and Application for Localization in Various Human Tissues by Real-Time Reverse Transcriptase-Polymerase Chain Reaction
Determination of mRNA Expression of Human UDP-Glucuronosyltransferases and Application for Localization in Various Human Tissues by Real-Time Reverse Transcriptase-Polymerase Chain Reaction
复制标题
DOI：
10.1124/dmd.108.023598
复制
发表时间：
2009-01-01
影响因子：
3.9
通讯作者：
Nakajin, Shizuo
中科院分区：
文献类型：
--
作者：
Ohno, Shuji;Nakajin, Shizuo
采用实时反转录聚合酶链反应（PCR）定量方法测定了15个具有催化活性的人udp -葡萄糖醛基转移酶（UGT）亚型（1A1、1A3、1A4、1A5、1A6、1A7、1A8、1A9、1A10、2B4、2B7、2B10、2B11、2B15和2B17）。为不同的人ugt开发了特异性引物进行鉴别测定。在食道中检测到来自1A7异构体的cDNA，在小肠中检测到1A8和1A10异构体，通过PCR至少在肝脏中检测到所有其他异构体。在所有情况下，琼脂糖凝胶上预期大小的单个条带被证实与预测的UGT同工型序列相对应。每个校正曲线在PCR交叉点与校正拷贝数之间呈线性关系。相关系数大于0.9957，重现性好。这种穷举测量方法应用于23种人体组织类型的UGT表达。UGT主要在消化系统和肝脏表达。我们惊奇地发现，UGT2B4和UGT2B15在肝脏中的表达量极高，分别是UGT2B7在肝脏中的表达量的8.98倍和4.38倍。此外，尽管表达水平较低，但几种UGT异构体在类固醇生成组织中也有表达，如乳腺、前列腺、心脏和肾上腺。因此，这种定量方法可以为多种ugt代谢药物的疗效或药代动力学特性提供有价值的信息。
An exhaustive real-time reverse transcriptase-polymerase chain reaction (PCR) quantification method was used to determine 15 of the catalytically active human UDP-glucuronosyltransferase (UGT) isoforms (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17). The specific primers for respective human UGTs were developed for differential determination. The cDNA derived from the 1A7 isoform was detected in the esophagus, the 1A8 and 1A10 isoforms were detected in the small intestine, and all other isoforms were detected in at least the liver by PCR. In all cases, single bands of the expected size on the agarose gel were confirmed to correspond with the predicted UGT isoform sequences. Each calibration curve showed linearity between the PCR crossing point and the calibrator copy number. The correlation coefficients were greater than 0.9957 with high reproducibility. This exhaustive measurement method was applied to UGT expression in 23 human tissue types. UGT was mostly expressed in the alimentary system and liver. We were surprised to find that extremely high expression in the liver was found for UGT2B4 and UGT2B15, which had, respectively, 8.98 and 4.38 times greater expression than UGT2B7 in the liver. In addition, even though expressed at low levels, several UGT isoforms were expressed in steroidogenic tissues, such as the breast, prostate, heart, and adrenal. Therefore, this quantification method may provide valuable information about the medical efficacy or pharmacokinetic characteristics of a wide variety of UGT-metabolized drugs.