Millar T J, Herok G, Koutavas H, Martin D K, Anderton P J
Co-operative Research Centre for Eye Research and Technology, University of New South Wales, Australia.
Tissue Cell. 1996 Jun;28(3):301-12. doi: 10.1016/s0040-8166(96)80017-6.
The purpose of this study was to establish conditions for isolation and long term culture of acinar cells from the Harderian gland, and superior and inferior lacrimal glands of the rabbit and to compare the in vitro growth patterns of cultured cells from these glands. In order to determine the predominant cell type in the cultures, cells and tissue sections were stained using a variety of antibodies to cytokeratins, smooth muscle actin, and neuron specific enolase. Similarly, PAS and alcian blue histochemistry were used to test for the presence of mucins. The glands were excised and cells isolated using enzymatic digestion and then established in long term culture. Different media and substrata were trialed for suitability. When cultured on uncoated Costar plastic in DMEM/10%FBS, the pattern of cell growth was similar for all glands with distinct phases involving aggregation and migration out from the aggregates before cells died between 20 to 30 days. Immunohistochemical staining indicated that the cultures were of acinar cells with a small percentage of ductal cells. The acinar cells of the lacrimal glands in situ and in vitro stained with antibody MNF116 directed against cytokeratins 5, 6, 8 and 17 but did not stain for antibodies to cytokeratin 18. The reverse staining pattern was true for the Harderian gland. Sections from the white lobe of the Harderian gland showed islets of serous secreting cells which showed positive staining when MNF116 was used. In situ, PAS positive cells were found in a small number of demilunes in the superior and inferior lacrimal glands and also in cells of the intercalated ducts. Surprisingly, in culture nearly all cells, including those isolated form the Harderian gland became PAS positive. In this study we have demonstrated that acinar cells from the Harderian and lacrimal glands of rabbit can be isolated and maintained in culture for 20 to 30 days, and that despite dramatic morphological changes, these cells retain their distinctive phenotype as indicated by antibody staining to specific cellular structural proteins such as cytokeratins and actin. However, the cultured cells also begin to produce mucins as indicated by PAS staining.
本研究的目的是建立从兔的哈德氏腺、上下泪腺分离腺泡细胞并进行长期培养的条件，并比较这些腺体培养细胞的体外生长模式。为了确定培养物中主要的细胞类型，使用多种针对细胞角蛋白、平滑肌肌动蛋白和神经元特异性烯醇化酶的抗体对细胞和组织切片进行染色。同样，采用PAS和阿尔辛蓝组织化学方法检测黏蛋白的存在。切除腺体，通过酶消化分离细胞，然后进行长期培养。对不同的培养基和底物进行了适用性试验。当在未包被的Costar塑料上于DMEM/10%胎牛血清中培养时，所有腺体的细胞生长模式相似，具有明显的阶段，包括聚集以及从聚集体中迁移出来，然后细胞在20至30天之间死亡。免疫组织化学染色表明，培养物为腺泡细胞，含有少量导管细胞。泪腺的原位和体外腺泡细胞用针对细胞角蛋白5、6、8和17的抗体MNF116染色，但对细胞角蛋白18的抗体不染色。哈德氏腺的染色模式则相反。哈德氏腺白叶的切片显示浆液分泌细胞岛，使用MNF116时呈阳性染色。在原位，上下泪腺的少数半月形结构以及闰管细胞中发现PAS阳性细胞。令人惊讶的是，在培养中，几乎所有细胞，包括从哈德氏腺分离的细胞都变成了PAS阳性。在本研究中，我们证明了兔的哈德氏腺和泪腺的腺泡细胞可以分离并在培养中维持20至30天，并且尽管形态发生了显著变化，但这些细胞通过对细胞角蛋白和肌动蛋白等特定细胞结构蛋白的抗体染色，保留了其独特的表型。然而，PAS染色表明培养的细胞也开始产生黏蛋白。
