Development of a new method for detecting gene expression in a single cell and its medical application.

我们已经成功开发了一种新的荧光素酶检测技术，可以在摄影图像上捕捉到转染了含有萤火虫荧光素酶基因的融合基因的色素细胞的表达。由荧光素酶介导的反应发出的光可以通过化学发光测量来量化。利用光计数相机和一种有效的转基因方法，我们能够检测到人黑素细胞中酪氨酸酶基因的启动子活性。此外，我们还取得了以下成果，总结如下。1.人类酪氨酸酶基因有三个顺式调控元件，每个元件都含有一个CATGTG基序的拷贝。我们证明了每个CATGTG基序都是色素细胞特异性转录所必需的。2.在研究巨噬细胞分化过程中血红素加氧酶-1基因表达的调控过程中，我们发现存在一个假定的沉默序列，它抑制了位于人血红素加氧酶-1基因5^1侧翼区域的热休克元件的功能。3.三种类型的一氧化氮(NO)供体均能提高HO-1mRNA的表达水平，提示HO-1和NO合成酶的mRNAs可能存在协同调节。4.与正常脑组织相比，人原发性脑肿瘤组织中血红素氧合酶-1和诱导型一氧化氮合酶mRNAs均有高表达。5.脉络丛存在于脑脊液中，不仅在脑的机械支持中起重要作用，而且在脑内化学平衡中也起着重要作用。我们发现，肾上腺髓质素是一种有效的血管扩张肽，由培养的脉络丛癌细胞产生和分泌，脉络丛癌细胞是一种来自脉络丛上皮的罕见肿瘤。

We have successfully developed a new luciferase assay technology that enables the expression of pigment cells, transfected with the fusion gene containing the firefly luciferase gene, to be captured on photographic images. The light discharged by the luciferase-mediated reaction can be quantified by chemical luminescence measurement. Using the photocounting camera and an efficient transfection method, we were able to detect the promoter activity of the tyrosinase gene in human melanocytes. Furthermore, we have obtained the following results as summarized below. 1. The three cis-regulatory elements of the human tyrosinase gene have been identified, and each of these elements contains one copy of a CATGTG motif. We showed that each CATGTG motif is required for pigment cell-specific transcription. 2. In the course of studying the regulation of the heme oxygenase-1 gene expression during macrophage differentiation, we showed the presence of a putative silencer sequence, repressing a function of the heat shock element, which is located in the 5^1-flanking region of the human heme oxygenase-1 gene. 3. Three types of nitric oxide (NO) donors increased the expression levels of heme oxygenase-1 mRNA,suggesting a possible coordinated regulation of heme oxygenase-1 and NO synthase mRNAs. 4. Both heme oxygenase-1 and inducible NO synthase mRNAs were overexpressed in human primary brain tumors compared to the normal brain tissues. 5. The choroid plexus probuces cerebrospinal fluid which plays an important role not only in the mechanical support of the brain but also in the chemical homeostasis of the brain. We found that adrenomedullin, a potent vasodilator peptide, is produced and secreted by cultured choroid plexus carcinoma cells, a rare neoplasm derived from the epithelium of the choroid plexus.