Faculty of Dentistry, McGill University, Montreal, Canada.
Tissue Eng Part A. 2011 May;17(9-10):1229-38. doi: 10.1089/ten.TEA.2010.0297. Epub 2011 Feb 8.
Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to <5% of huSG cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.
目前，对于由干燥综合征或头颈部癌症放疗引起的功能性唾液腺腺泡不可逆转丧失的患者，尚无有效的治疗方法。组织工程人工唾液腺将有助于这些患者。该设备的移植物细胞除了具有分泌液的性质外，还必须建立紧密连接。本研究分析了在 Matrigel 上培养的人唾液腺 (huSG) 的移植物来源。从腮腺和颌下腺获得细胞，体外扩增，然后分别种植在涂有 Matrigel（2 mg/mL）或未涂覆的培养皿上。采用免疫组织化学、透射电子显微镜、实时定量聚合酶链反应、Western blot 和跨上皮电阻进行分析。在 Matrigel 上，huSG 细胞通过形成三维腺泡样单位（在接种后 24 小时内）以及单层细胞来采用腺泡表型。在未涂覆的表面（塑料）上，huSG 细胞仅形成单层导管细胞。这两种培养条件都允许 huSG 细胞表达紧密连接蛋白（claudin-1、-2、-3、-4；occludin；JAM-A；和 ZO-1）和足够的跨上皮电阻。重要的是，与塑料上的 huSG 细胞相比，99%的 Matrigel 上的 huSG 细胞表达α-淀粉酶和水通道蛋白 Aquaporin-5，而只有<5%的 huSG 细胞表达α-淀粉酶和水通道蛋白 Aquaporin-5。透射电子显微镜证实了具有许多分泌颗粒的腺泡表型。Matrigel 将α-淀粉酶的分泌量增加到培养基中的两到五倍，下调了某些唾液基因，并调节了腺泡蛋白的翻译。这种无血清的三维体外细胞培养方法允许 huSG 细胞组织和分化为具有腺泡表型的唾液细胞。
