A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules
A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules
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DOI：
10.1007/s00441-005-1076-x
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发表时间：
2005-06-01
影响因子：
3.6
通讯作者：
Sugiya, H
中科院分区：
文献类型：
--
作者：
Fujita-Yoshigaki, J;Tagashira, A;Sugiya, H
外分泌腺泡细胞，如腮腺细胞，在细胞系或原代培养中难以维持其功能。由于这个原因，外分泌细胞功能的分子研究是不令人满意的。为了研究腮腺腺泡细胞功能维持的机制，我们试图建立一个原代培养和外源基因转染的系统。用酶消化法从大鼠腮腺分离腺泡细胞，并在含大鼠血清的培养基中培养。大多数培养细胞具有含有淀粉酶的分泌颗粒，表明它们来自腺泡细胞，尽管它们散布在培养皿表面并形成丝状伪足。培养的细胞保留颗粒和释放淀粉酶的能力，响应β-肾上腺素能和胆碱能激动剂，甚至48小时后分散。但分散后24 ~ 48 h，细胞内淀粉酶总量迅速下降。这些结果表明，在体外培养过程中，淀粉酶的合成比胞吐机制受到更大的损害。将与增强型绿色荧光蛋白融合的VAMP 2基因转染到分散的腺泡细胞中，并且VAMP 2蛋白表达并定位于含淀粉酶的颗粒，如通常所见的内源性VAMP 2蛋白。这表明产生了新的颗粒，并且蛋白质分选是功能性的。用此方法培养的细胞至少能维持48 h的功能。它们可用于检查外源基因对腮腺腺泡细胞功能的影响，如调节胞吐作用和分泌颗粒的成熟。
Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to beta-adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.