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ClィイD1-ィエD1 Channel Gene Expression in Guinea-pig Gastric Parietal Cells

CliiD1-ieD1 通道在豚鼠胃壁细胞中的基因表达

基本信息

批准号：
10670466
负责人：
UEDA Shunji
金额：
$ 2.11万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

We applied whole-cell patch clamp to single parietal cells isolated from the guinea-pig then found the ETb-receptor coupled Maxi-ClィイD1-ィエD1 and pH-sensitive Mini-ClィイD1-ィエD1 channels on the parietal cells. In the 10ィイD1thィエD1 year, we detected the expression of ClィイD1-ィエD1 channel mRNA by the method of RT-PCR at the whole gastric epithelial cell level. Then, we made a cDNA library from guinea-pig gastric epithelial cells and determined the cDNA sequence of guinea-pig CLC-2 ClィイD1-ィエD1 channel. We analyzed the ClィイD1-ィエD1 current using whole-cell patch clamp in the CLC-2 gene expression cell system of Xenopus oocytes and CHO cells, but could not detect the channel current previously founded. To analyze the ClィイD1-ィエD1 channel gene expression in parietal level, we amplified the CFTR, CLC-2, 3, 4 mRNA by RT-PCR method using the purified guinea-pig parietal cells. But we could not detect any single band. In 11ィイD1thィエD1 year, we further applied whole-cell patch clamp and single cell mRNA analysis by RT-PCR method to single parietal cell. Single parietal cell did not show any ClィイD1-ィエD1 channel gene expression though G3PDH did the expression in single intestinal cell as positive control. These results did not come from the technical failure. Recent our intestinal work showed that discrepancy between electrophysiological and molecular biological results depended on the difference of cellular maturation. Therefore, we concluded that parietal cells have several grade of maturation and this result induced the discrepancies of these works.

我们将全细胞膜片钳应用于从豚鼠分离的单个壁细胞，然后在壁细胞上发现了与 Maxi-CliiD1-ィエD1 和 pH 敏感的 Mini-CliiD1-ィD1 通道偶联的 ETb 受体。第10ィイD1thエD1年，我们采用RT-PCR方法在全胃上皮细胞水平检测ClィイD1-ィD1通道mRNA的表达。然后，我们构建了豚鼠胃上皮细胞的cDNA文库，并确定了豚鼠CLC-2 ClィイD1-ィエD1通道的cDNA序列。我们利用全细胞膜片钳分析了爪蟾卵母细胞和CHO细胞的CLC-2基因表达细胞系统中的ClィイD1-ィD1电流，但无法检测到先前建立的通道电流。为了分析壁层水平的ClィイD1-ィD1通道基因表达，我们使用纯化的豚鼠壁细胞通过RT-PCR方法扩增了CFTR、CLC-2、3、4 mRNA。但我们无法检测到任何单个带。 11ィイD1thエD1年，我们进一步将全细胞膜片钳和RT-PCR方法的单细胞mRNA分析应用于单个壁细胞。尽管G3PDH在作为阳性对照的单个肠细胞中表达，但单个壁细胞未显示任何CliiD1-ィD1通道基因表达。这些结果并非来自技术故障。最近我们的肠道工作表明，电生理学和分子生物学结果之间的差异取决于细胞成熟的差异。因此，我们得出结论，壁细胞有几个成熟级别，这一结果导致了这些工作的差异。