薬剤スクリーニングの簡素化-ヒト・ヒスチジン脱炭酸酵素

组氨酸脱羧酶（HDC）是哺乳动物细胞中唯一催化组氨酸合成组胺的酶。在造血细胞中，组胺只在嗜碱性细胞和肥大细胞谱系中表达，因此我们对该基因的组织特异性表达机制很感兴趣。我们制备了多种报告基因构建体，并将其转染到不同细胞系中，结果表明该基因上游6kb至下游1kb范围内不存在组织特异性元件。通过基因组DNA印迹，我们在启动子区域发现了组织特异性DNA酶I超敏位点，这表明转录因子很容易接近该区域进行基因表达。此外，在不表达hdc的细胞系中，许多胞嘧啶分子在该启动子区域被甲基化。因此，HDC基因的细胞特异性表达被认为与染色质结构和甲基化模式的差异有关，而不是与反式作用因子有关。我们对小鼠HDC基因也很感兴趣，因为我们可以解决许多在人类身上无法解决的生理和药理问题。首先，我们构建了转录起始位点上游含有1099个碱基的质粒，与荧光素酶基因融合，并将该质粒稳定转染到P815小鼠肥大细胞系中。利用该质粒，我们报道了在同源BDF1小鼠腹腔内诱导P815细胞表达HDC基因的机制[j] .中国生物医学工程学报，2013,28(2):28439-28444。我们正计划用前稳定变换来阐明具有这种诱导性的顺式作用元件。通过将多种药物作用于该细胞系并测定荧光素酶活性，我们可以评估这些药物对HDC基因转激活活性的影响。我们目前正在进行克隆人稳定转化，用于各种药物的活性评估。

Histidine decarboxylase (HDC) is the unique enzyme which catalyze the synthetic reaction of histamine from histidine in mammalian cells. Histamine is expressed exclusively in basophilic and mast cell lineage in hematopoietic cells, therefore we are interested in the mechanism of tissue specific expression of this gene. We prepared various reporter constructs and transfected them into verious cell lines and clarified no tissue specific element exists from 6 Kb upstream to 1 Kb downstream of this gene. We found tissue specific DNase I hypersensitive sites in the promoter region by genomic DNA blotting, suggesting that this region is easily approached by transcription factors for their gene expression. Moreover, many cytosine molecules were methylated in this promoter region in HDC-non-expressing cell lines. Therefore, the cell specific expression of HDC gene is thought to be confirred to the differences of chromatin structure and methylation pattern rather than those of trans-acting factors.We also are interested in mouse HDC gene, because we can approach many physiological and pharmacological problem which could not be approached in human. We, first, constructed a plasmid that contains 1099 bases upstream of the transcription initiation site fused with luciferase gene and transfected this plaamid stably into P815 mouse mast cell line. With this plasmid, we reported the mechanism of how P815 cells was induced to express HDC gene in peritoneal cavity of syngenic BDF1 mouse (J.Biol.Chem.271,28439-28444). We are planning to elucidate the cis-acting element confering this inducibility using former stable transformant. By applying many kinds of medicine to this cell line and measuring the luciferase activity, we can assess the effect of these medicine on the transactivational activity of HDC gene. We are now under cloning of human stable transformant to be used for the assessment of activity of various medicine.