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

我们建立了大鼠周围乳头味蕾细胞的体外培养体系。味蕾细胞在以角化细胞生长培养基为基础的基质涂布培养皿中培养，具有粘附性，存活时间超过3天。这些细胞保留了细胞骨架和细胞内信号的分子标记，如细胞角蛋白8和磷脂酶Cβ2。此外，三种细胞内信号分子gustducin，磷脂酶c - β2和肌醇1,4,5 -三磷酸受体3型表达与体内相同。我们发现外源性表达的myc标记的α1 a -肾上腺素能受体分选到质膜上，并转导一个配体依赖的信号，导致大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，形成致密的聚集体。低Ca^<2+>条件下（低于1.1 mM），细胞呈分散状，呈细长状；这两种形式是可逆的，并且特别依赖于Ca^<2+>。结果表明，细胞外Ca^<2+>调节味蕾细胞的细胞形状和细胞间粘附。采用RT-PCR和免疫组织化学方法，对大鼠舌周乳头基底膜的主要成分层粘连蛋白亚基进行了分析。结果表明，这些乳头分别含有β2和γ1作为主要的β亚基，以及β1、β4和β5三个整合素β亚基。

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.