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

采用real - time PCR方法定量比较Caco-2细胞与人十二指肠肠细胞中转运蛋白mrna和一种与口服吸收相关的代谢酶的表达水平。在Caco-2细胞的培养和传代过程中，也评估了这些蛋白mRNA表达水平的变化。此外，我们还研究了MDR1基因外显子26 C3435T突变对人十二指肠肠细胞MDR1 mRNA表达水平的影响。研究结果如下：1。人肠细胞中转运体mrna （MDR1、MRP1和MRP2）和代谢酶（CYP3A）的相对浓度分别比Caco-2细胞高约12倍、3倍、7倍和8000倍。5个Caco-2细胞系中有3个未检测到CYP3A mrna。Caco-2细胞中MDR1、MRP1和CYP3A mRNA水平与正常结直肠组织和结直肠腺癌细胞相当。在Caco-2细胞中检测到MRP2 mRNA，而在正常结直肠组织和结直肠腺癌中几乎检测不到MRP2 mRNA。突变型等位基因纯合子（T/T）的受试者十二指肠MDR1 mRNA水平高于野生型等位基因纯合子（C/C）的受试者。MDR1和CYP3A4 mRNA表达水平在十二指肠活检中也有很好的相关性（r=0.797； P<0.01）。综上所述，Caco-2细胞株口腔吸收相关蛋白的基因表达谱更接近于人正常结直肠组织和结直肠腺癌，而不是人十二指肠肠细胞。MDR1基因外显子26的突变体C3435T与十二指肠中较高水平的MDR1- mrna相关。在携带MDR1基因突变体C3435T的受试者中，可以观察到CYP3A4底物的血浆浓度较低。

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.