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Real time PCR analysis of transporter and metabolism enzyme expression level in human alimentary canal epidermic cell

人消化道表皮细胞转运蛋白和代谢酶表达水平的实时PCR分析

基本信息

批准号：
13672387
负责人：
SAKAEDA Toshiyuki
金额：
$ 0.9万
依托单位：
KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672387/
关键词：
Caco-2 Human duodenal enterocytes Real time PCR mRNA MDR1 MRP1 CYP3A ヒト十二指腸上皮細胞

项目摘要

The expression levels of mRNAs for transporters and a metabolic enzyme related to oral absorption in Caco-2 cells were quantitatively compared with those in human duodenal enterocytes using real time PCR method. Alteration in mRNA expression levels of these proteins was also evaluated in Caco-2 cells during culture and passage. Furthermore, the effect of the C3435T mutation in exon 26 of MDR1 gene on the expression levels of MDR1 mRNA was evaluated in human duodenal enterocytes.The study results are shown in the following.1. Relative concentrations of mRNAs for transporters (MDR1, MRP1 and MRP2) and a metabolic enzyme (CYP3A) in human enterocytes were about 12-, 3-, 7- and 8000-fold higher than in Caco-2 cells, respectively. Three of five Caco-2 cell lines had no detectable CYP3A mRNA.2. MDR1, MRP1 and CYP3A mRNA levels in Caco-2 cells were comparable with those in normal colorectal tissues and colorectal adenocarcinomas. MRP2 mRNA was detected in Caco-2 cells, whereas those in normal colorectal tissues and colorectal adenocarcinomas were barely detectable.3. The duodenal mRNA level of MDR1 in the subject with the homozygote of the mutant allele (T/T) was higher than that in those with homozygote of wild-type allele (C/C). A good correlation (r=0.797 ; P<0.01) was also observed between the mRNA expression levels of MDR1 and CYP3A4 in the individual duodenal biopsies.In conclusion, Caco-2 cell lines showed the gene expression profiles of oral absorption related proteins closer to those of human normal colorectal tissue and colorectal adenocarcinoma, rather than human duodenal enterocytes. The mutant C3435T at exon 26 of the MDR1 gene associated with the higher level of MDR1-mRNA in the duodenum. Lower plasma concentrations of the substrates for CYP3A4 may be observed in subjects harboring the mutant C3435T of the MDR1 gene.

采用实时定量聚合酶链式反应技术，定量比较Caco-2细胞和人十二指肠肠上皮细胞转运蛋白和代谢酶基因的表达水平。在Caco-2细胞的培养和传代过程中，也评估了这些蛋白的mRNA表达水平的变化。此外，我们还研究了mdr1基因26号外显子C3435T突变对人十二指肠肠上皮细胞mdr1基因表达水平的影响。人肠上皮细胞转运蛋白(MDR1、MRP1和MRP2)和代谢酶(CYP3A)的相对浓度分别是Caco-2细胞的12倍、3倍、7倍和8000倍。5个Caco-2细胞系中有3个没有检测到CYP3A mRNA2。Caco-2细胞中mdr1、MRP1和CYP3a基因的表达水平与正常大肠组织和结直肠腺癌中的mdr1、MRP1和CYP3a的表达水平相近。MRP2基因在Caco-2细胞中表达，而在正常大肠组织和结直肠腺癌组织中几乎检测不到。突变等位基因纯合子(T/T)患者十二指肠MDR1mRNA水平高于野生型等位基因纯合子患者(C/C)。十二指肠活检组织中多药耐药基因mdr1和细胞色素P3A4mRNA的表达水平之间也存在良好的相关性(r=0.797；P&lt0.01)。结论：Caco-2细胞株的口腔吸收相关蛋白的基因表达谱更接近于人正常大肠组织和结直肠癌组织，而不是人十二指肠肠细胞。Mdr1基因第26外显子C3435T突变与十二指肠组织中mdr1-mRNAs水平升高有关。在携带MDR1基因突变C3435T的受试者中，可以观察到较低的CYP3A4底物的血浆浓度。