Establishing a primary taste-cell culture system as a basic analysis for new taste engineering

建立原代味觉细胞培养体系作为新味觉工程的基础分析

• 批准号: 13356008
• 负责人: ABE Keiko
• 金额: $ 28.12万
• 依托单位: THE UNIVERSITY OF TOKYO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 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-13356008/
• 关键词: taste bud cell; primary culture; Ca ion; gene introduction; cell adhesion; basement membrane; laminin; integrin; 味覚

We have established an in vitro culture system of taste bud cells from rat circumvallate papillae. The taste bud cells were adhesive and viable for over 3 days when cultured onto Matrigel-coated dishes in medium based on keratinocyte growth medium. The cells retained molecular markers for both the cytoskeleton and intracellular signalling such as cytokeratin 8 and phospholipase Cβ2. In addition, three intracellular signaling molecules, gustducin, phospholipase Cβ2, and inositol 1, 4, 5-trisphosphate receptor type 3, are expressed in the same correlation as those in vivo. We show that an exogenously expressed myc-tagged α1 A-adrenoceptor sorts into the plasma membrane, and transduces a ligand-dependent signal resulting in intracellular [Ca^<2+>] increase in about half of the infected cells.Under high Ca^<2+> condition (about 0.5 mM), the cells were tightly associated with each other and formed packed aggregates. Under low Ca^<2+> condition (below 1.1 mM), the cells were dispersed and had an elongated shape. These two forms were reversible and specifically dependent on Ca^<2+>. The results indicate that extracellular Ca^<2+> regulates cell shape and cell-to-cell adhesion of taste bud cells. We analyzed the subunits of laminin, a major component of the basement membrane, in circumvallate papillae of rat tongue by the method of RT-PCR and immunohistochemistry. As a result, the papillae contain β2 and γ1 as major β and γ subunits, respectively and three integrin β subunit species, β1, β4 and β5.

建立了大鼠轮状乳头味蕾细胞的体外培养体系。当在基于角质形成细胞生长培养基的培养基中培养到基质胶涂覆的培养皿上时，味蕾细胞是粘附的并且存活超过3天。细胞保留了细胞骨架和细胞内信号传导的分子标记物，如细胞角蛋白8和磷脂酶Cβ2。此外，三种细胞内信号分子，味蛋白，磷脂酶Cβ2和肌醇1，4，5-三磷酸受体3型，表达与体内相同的相关性。我们发现，外源性表达的myc标记的α1 A肾上腺素受体进入质膜，并传递配体依赖性信号，导致约一半的感染细胞内[Ca^2+]增加，在高Ca^2+条件下（约0.5 mM），细胞相互紧密结合，形成密集的聚集体。在低Ca^<2+>条件下（低于1.1 mM），细胞分散并具有细长的形状。这两种形式都是可逆的，并且特异性地依赖于Ca^<2+>。结果表明，细胞外Ca^2+调节味蕾细胞的形状和细胞与细胞的粘附。采用RT-PCR和免疫组织化学方法对大鼠舌轮状乳头基底膜的主要成分层粘连蛋白（laminin）的亚单位进行了分析。因此，乳头含有分别作为主要β和γ亚基的β2和γ1以及三种整合素β亚基种类β1、β4和β5。

项目成果

Kishi, M.: "Changes in cell morphology and cell to cell adhesion induced by extracellular Ca2+ in cultured taste bud cells"Biosci.Bitotech.Biochem.. 66. 484-487 (2002)

Kishi, M.：“培养味蕾细胞中胞外 Ca2 诱导的细胞形态和细胞间粘附的变化”Biosci.Bitotech.Biochem.. 66. 484-487 (2002)

Matsumoto, I.: "A comparative study of three cranial sensory ganglia projecting into the oral cavity : in situ hybridization analyzes of neurotrophin receptors and thermosensitive cation channels"Mol. Brain Res.. 93. 105-112 (2001)

Matsumoto，I.：“投射到口腔中的三个颅感觉神经节的比较研究：神经营养蛋白受体和热敏阳离子通道的原位杂交分析”Mol。

Kishi, M.: "Primary culture of rat taste bud cells that retain molecular markers for taste buds and permit functional expression of foreign genes"Neuroscience. 106. 217-225 (2001)

Kishi, M.：“大鼠味蕾细胞的原代培养物保留了味蕾的分子标记并允许外源基因的功能表达”神经科学。

Kishi, M.: "Identification of β and γ subunits of laminins localized in the basement membrane of rat circumvallate papillae"Biosci.Biotech.Biochem.. 67. 1154-1156 (2003)

Kishi, M.：“大鼠周乳头基底膜中层粘连蛋白 β 和 γ 亚基的鉴定”Biosci.Biotech.Biochem.. 67. 1154-1156 (2003)

Okada, S.: "Aquaporin-9 is expressed in a mucus-secreting goblet cell subset in the small intestine"FEBS Lett.. (in press).

Okada, S.：“Aquaporin-9 在小肠分泌粘液的杯状细胞亚群中表达”FEBS Lett..（出版中）。