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

我们建立了大鼠环状乳头味蕾细胞的体外培养体系。在以角质形成细胞为基础的培养液中，味蕾细胞在Matrigel涂层培养皿上贴壁存活3d以上。此外，细胞内信号分子Gustducin、磷脂酶Cβ2和1，4，5-三磷酸肌醇受体3型的表达与体内相同。我们发现，外源表达的Myc标记的α1 A-肾上腺素受体进入质膜，并传递配体依赖的信号，导致大约一半感染细胞内[Ca^&lt；2+&gt；]的增加。在高Ca^&lt；2+&gt；条件下(约0.5 mm)，细胞彼此紧密联系并形成紧密的聚集体。在低钙条件下(<1.1 mm)，细胞分散，呈拉长状。这两种形式是可逆的，并特异性地依赖于钙离子。结果表明，细胞外Ca~(2+)~(2+)对味蕾细胞形态和细胞间黏附有调节作用。用RT-PCR和免疫组织化学方法分析了大鼠舌环乳头层粘连蛋白的亚基组成。结果表明，乳突中分别含有β2和γ1作为主要的β和γ亚基，以及3种整合素β亚基β1、β4和β5。

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.