涎腺腺样囊性癌细胞分化行为的实验观察

为探讨涎腺腺样囊性癌（ａｄｅｎｏｉｄｃｙｓｔｉｃｃａｒｃｉｎｏｍａ，ＡＣＣ）中肌上皮细胞的来源和此癌的组织发生，对其细胞的一些分化行为进行了实验观察。用ＡＢＣ免疫组织化学方法，证明了ＡＣＣ－２和ＡＣＣ－３细胞系细胞，在体外能不同程度地表达肌上皮细胞特有的Ｓ－１００蛋白、肌动蛋白（ａｃｔｉｎ）和肌浆球蛋白（ｍｙｏｓｉｎ）成分；加入分化诱导剂ｄＢ－ｃＡＭＰ后，可使上述成分表达增强并使癌胚抗原（ｃａｒｃｉ－ｎｏｅｍｂｒｙｏｎｉｃａｎｔｉｇｅｎ，ＣＥＡ）由阴性转阳性。作者还观察到ＡＣＣ细胞的分化与其增殖速度呈负相关关系；改变细胞生长环境时，部分细胞呈“印戒”样，表明其具有分泌功能；将单个细胞悬浮生长于胶原凝胶为基质的培养基可形成典型的管样结构。作者认为，本研究结果支持ＡＣＣ的组织发生来自闰管细胞的观点，对认识该肿瘤的发生很有参考价值。     

炎症状态下人牙髓神经纤维内Ca^2＋浓度的变化

利用细胞内Ｃａ２＋浓度测定技术，直接测定了牙髓神经纤维内Ｃａ２＋浓度，结果发现：无论是临床炎症性痛过敏牙髓，或是炎症介质组织胺、５－羟色胺处理的牙髓，其神经纤维内Ｃａ２＋浓度均比正常牙髓显著升高。而当降低细胞外液Ｃａ２＋浓度时，虽经炎症介质处理，神经纤维内Ｃａ２＋浓度与正常组无显著差异。说明牙髓神经纤维内Ｃａ２＋升高可能与其炎性痛过敏的发生有关。增加的Ｃａ２＋可能来源于细胞外液的Ｃａ２＋跨膜通道内流     

细胞粘附蛋白在涎腺腺样囊性癌细胞中的表达

目的　探讨具有不同转移能力的涎腺腺样囊性癌（ａｄｅｎｏｉｄ ｃｙｓｔｉｃ ｃａｒｃｉｎｏｍ ａ; Ａ Ｃ Ｃ）细胞系中粘附分子表达的差异。方法　采用免疫组化方法对两株人腺样囊性癌细胞系（ Ａ Ｃ Ｃ２ 和 Ａ Ｃ Ｃ Ｍ）培养细胞及其 Ｓ Ｃ Ｉ Ｄ 小鼠移植瘤模型进行粘附分子 Ｃ Ｄ４４ｓ（标准型）、 Ｃ Ｄ４４ｖ６（变异型）、 Ｅｃａｄｈｅｒｉｎ（ Ｅｃａｄ）、αｃａｔｅｎｉｎ（αｃａｔ）、βｃａｔｅｎｉｎ（βｃａｔ）的检测。结果　（１） Ａ Ｃ Ｃ肺转移灶的肿瘤细胞中粘附分子的表达普遍高于其皮下移植瘤细胞;（２） Ａ Ｃ Ｃ Ｍ 肺转移灶肿瘤生长缓慢,细胞分化相对较好,而其皮下移植瘤和腹腔种植瘤肿块生长迅速,细胞分化差;（３） Ｃ Ｄ４４ｖ６ 在 Ａ Ｃ Ｃ Ｍ 和 Ａ Ｃ Ｃ２ 体外培养细胞中均有弱阳性表达,而在异体移植瘤中包括皮下移植瘤和肺部转移灶均无表达。结论　 Ａ Ｃ Ｃ细胞中 Ｃ Ｄ４４ｓ 的表达受细胞分化程度影响,并影响了肿瘤细胞本身的生长状况; Ｅｃａｄ／ｃａｔ复合体的表达与否或表达强度与肿瘤细胞的分化成正比; Ｃ Ｄ４４ｖ６ 在涎腺 Ａ Ｃ Ｃ中的表达和转移能力可能无关。     

兔腮腺腺细胞的体外培养和功能评价

目的：体外培养获取成活率高且保持功能的兔腮腺腺细胞。方法：采用改良DMEM培养液进行兔腮腺腺细胞的原代及传代培养,观察腮腺腺细胞的生长状况;免疫细胞化学法检测细胞中泛角蛋白及α-淀粉酶蛋白和肌动蛋白的表达情况以鉴定细胞来源;检测α-淀粉酶蛋白在不同代次腺细胞中的表达情况,判断其分泌功能。应用SPSS10.0软件包对数据进行t检验。结果：体外培养的兔腮腺腺细胞具有一定的增殖能力,可传3代,存活4周以上;其特异性抗体泛角蛋白及α-淀粉酶蛋白染色为阳性,肌动蛋白染色阴性;不同代次腺细胞α-淀粉酶的表达强度随传代次数的增加而逐渐减弱,原代培养和传代培养第1代的细胞,α-淀粉酶的表达强度较高且可维持一定水平,两者无显著差异（P〈0.05）;传代培养第2代的细胞,α-淀粉酶含量明显下降,与前者差异显著（P〈0.05）;第3代细胞基本测不到α-淀粉酶的表达。结论：采用改良DMEM培养液体外培养兔腮腺腺细胞,可得到足量的腺细胞,且前2代细胞保持了较强的分泌功能,是选作实验研究的最佳阶段。     

关于唾液分泌机制的研究

应用全细胞膜片钳技术（ｐａｔｃｈ—ｃｌａｍｐｗｈｏｌｅｃｅｌｌｒｅｃｏｒｄｉｎｇ）对即刻分离的颌下腺腺泡细胞进行膜电流的测量。实验中发现乙酰胆碱、ＧＴＰ—ｒ—ｓ以及肌醇１．４．５－三磷酸二者均可引起性质相同的、振动性的、内向性的Ｃｌ－离子电流。由于Ｃａ２＋离子螯合剂能够消除三者引起的电流反应，因此这种振动性的Ｃｌ－离子电流是细胞内游离Ｃａ（２＋）离子浓度振动性变化的表示。即乙酰胆碱通过ＧＴＰ结合蛋白的激活，ＩＰ３的产生，引起Ｃａ（２＋）振动。     

口腔癌肿瘤浸润淋巴细胞对人舌癌裸鼠移植瘤的生长抑制作用

目的探讨在体外具有强大细胞毒活性的口腔癌浸润淋巴细胞在体内的抑瘤效果以及化疗药物环磷酰胺（ｃｙｃｌｏｐｈｏｓｐｈａｍｉｄｅ，Ｃｙ）与肿瘤浸润淋巴细胞（ｔｕｍｏｒｉｎｆｉｌｔｒａｔｉｏｎｌｙｍｐｈｏｃｙｔｅ，ＴＩＬ）联合应用治疗口腔癌的可能性。方法将舌鳞癌细胞系Ｔｃａ８１１３注入裸鼠背部皮下建立移植瘤模型，取培养４周的ＴＩＬ联合低剂量Ｃｙ局部注射，观察肿瘤体积变化及肿瘤生长情况。结果①ＴＩＬ＋ｒＩＬ２和ＴＩＬ＋ｒＩＬ２＋Ｃｙ组在３周内抑瘤率较高（分别为：８６１％±０４％和９７７％±０６％），３周后抑瘤作用减弱；②ＴＩＬ＋ｒＩＬ２＋Ｃｙ组抑瘤率较ＴＩＬ＋ｒＩＬ２组高（第８周抑瘤率分别为：２００％±１４％和７５５％±２５％，Ｐ＜００１）。结论口腔癌ＴＩＬ在应用后３周内具较强抑瘤作用，３周后减弱；联合应用Ｃｙ可获得更佳抑瘤效果     

氢氧化钙激动人牙髓细胞内钙释放及其通道的研究

目的：检测Ｃａ（ＯＨ）２激动体外培养的人牙髓细胞内Ｃａ＾２＋的释放和钙释放通道。方法：在ＲＰＭＩ１６４０培养液中分别加入Ｃａ（ＯＨ）２、Ｃａ（ＯＨ）２和肝素、Ｃａ（ＯＨ）２和普鲁卡因,培养第６代人牙髓细胞,用Ｆｌｕｏ－３荧光探针负载后,再次用Ｃａ（ＯＨ）２作用于牙髓细胞,用激光共聚焦显微镜检测胞内Ｃａ＾２＋。结果：含Ｃａ（ＯＨ）２、Ｃａ（ＯＨ）２和普鲁卡因的培养液培养的细胞内Ｃａ＾２＋浓度升高;含     

E-cadher in-caten in复合体在涎腺腺样囊性癌中的表达

目的　研究粘附分子 Ｅｃａｄｈｅｒｉｎｃａｔｅｎｉｎ（ Ｅｃａｄｃａｔ）复合体在涎腺腺样囊性癌（ａｄｅｎｏｉｄｃｙｓｔｉｃ ｃａｒｃｉｎｏｍ ａ; Ａ Ｃ Ｃ）中的表达,探讨 Ｅｃａｄｃａｔ 复合体与 Ａ Ｃ Ｃ分化、侵袭和转移之间的关系及其临床意义。方法　采用免疫组化技术检测５０ 例涎腺 Ａ Ｃ Ｃ标本和１０ 例正常涎腺组织中 Ｅｃａｄｈｅｒｉｎ（ Ｅｃａｄ）及其相关蛋白αｃａｔｅｎｉｎ（αｃａｔ）、βｃａｔｅｎｉｎ（βｃａｔ）的表达。结果　５０ 例病例中 Ｅｃａｄ、αｃａｔ 和βｃａｔ共同表达者有２９ 例（５８％ ）,但在１８ 例有神经血管侵犯者,其原发灶 Ｅｃａｄｃａｔ复合体成份共同表达仅有７ 例（３９％ ）。结论　正常涎腺上皮组织中 Ｅｃａｄｃａｔ复合体成份呈强烈而稳定的表达,而在涎腺 Ａ Ｃ Ｃ 组织中其表达显著下调; Ｅｃａｄ／ｃａｔ复合体的表达水平与 Ａ Ｃ Ｃ神经血管侵犯呈负相关,但与病理分型无关; Ｅｃａｄ／ｃａｔ复合体的表达下降可能和涎腺组织恶变有关。     

耐长春新碱人舌鳞癌细胞系Tca8113/V100的研究

目的建立耐长春新碱（Ｖｉｎｃｒｉｓｔｉｎｅ，ＶＣＲ）人舌鳞癌细胞系。方法采用间歇性加药，逐步提高培养基中ＶＣＲ的浓度，连续体外诱导，培养，建立了一株耐ＶＣＲ人舌鳞癌细胞系Ｔｃａ８１１３／Ｖ１００。对其药物敏感性、倍增时间、裸鼠成瘤性、超微结构及耐多药基因等生物学特性进行了研究。结果Ｔｃａ８１１３／Ｖ１００在含ＶＣＲ１００μｍｏｌ／Ｌ的培养基中稳定生长，其对ＶＣＲ的耐药性为Ｔｃａ８１１３细胞系的１９２倍，对其他几种抗癌药物也有程度不同的耐药性。Ｔｃａ８１１３／Ｖ１００倍增时间３７２小时，具有裸鼠成瘤性，病理诊断为鳞状细胞癌，超微结构观察其微绒毛短粗，ｍｄｒ１ｍＲＮＡ表达水平明显增高。结论Ｔｃａ８１１３／Ｖ１００细胞系已具备多药耐药性的特点，为口腔癌多药耐药性研究提供了细胞模型。     

金属蛋白酶及其组织抑制剂在涎腺腺样囊性癌细胞株表达的分子生物学研究

为了研究金属蛋白酶（ｍｅｔａｌｏｐｒｏｔｅｉｎａｓｅ，ＭＭＰ）及其组织抑制剂（ｔｉｓｓｕｅｉｎｈｉｂｉｔｏｒｏｆｍｅｔａｌｏｐｒｏｔｅｉｎａｓｅ，ＴＩＭＰ）对涎腺腺样囊性癌细胞（ａｄｅｎｏｉｄｃｙｓｔｉｃｃａｒｃｉｎｏｍａ，ＡＣＣ）转移的影响，本研究应用斑点印迹杂交的分子生物学方法检测ＡＣＣ２细胞系及高转移细胞株ＡＣＣＭ中ＭＭＰ２、ＭＭＰ９和ＴＩＭＰ２的表达。结果显示ＭＭＰ２，ＭＭＰ９在ＡＣＣＭ表达较高，在ＡＣＣ２表达较低，而ＴＩＭＰ２在ＡＣＣ２表达较高，在ＡＣＣＭ表达较低，证实ＭＭＰ２，ＭＭＰ９促进转移的发生，而ＴＩＭＰ２则抑制转移的发生。提示ＭＭＰ与ＴＩＭＰ平衡关系可能是ＡＣＣ转移的关键机制之一。     

Parotid secretory granules: crossroads of secretory pathways and protein storage

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins-including amylase, proline-rich proteins, and parotid secretory protein (PSP)-to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.     

Regulatory aspects of N-linked glycoproteins

Activation of beta-adrenoreceptors in rat parotid acinar cells leads to copious exocrine protein secretion. Additionally, beta-adrenergic stimulation dramatically increases specific secretory protein synthesis and enhances N-linked glycosylation of secretory glycoproteins. Recently, efforts have been directed toward understanding the mechanisms underlying these biosynthetic events. We have been particularly interested in the receptor-mediated regulation of glycosylation. In this report, we evaluate available mechanistic information from the rat parotid gland and present initial data examining the ability of various regulatory agents to modulate N-linked glycosylation in enzymatically-dispersed cell aggregates from surgical specimens of human parotid glands. We conclude that glycosylation of human parotid N-linked glycoproteins may be regulated by extracellular signaling similar to that operative in the rat parotid gland.     

Response of rat parotid acini to barium.

The effect of barium on isolated acini was tested. Barium in the 0.1-10 mM concentration range non-competitively inhibited the efflux of 86Rb+ stimulated by carbamylcholine or substance P. This inhibition was independent of the presence of calcium in the extracellular medium. In the same preparation, barium did not affect the efflux of 45Ca2+ but, at a 10 mM concentration, it increased amylase release by 70%. Removal of extracellular calcium decreased basal amylase release and the response to carbamylcholine. Adding back calcium or barium to the incubation medium increased basal and carbamylcholine-stimulated amylase secretion, but calcium was more effective than barium. These results suggest that barium has two opposite effects on calcium-regulated processes in rat parotid gland: (1) it is an inhibitor of calcium-activated potassium channels; (2) it is a partial agonist of calcium-activated amylase secretion.     

Differentiating myoepithelial and acinar cells in rat neonatal parotid gland and histogenetic concepts for salivary gland tumors

Histogenetic concepts for salivary gland tumors are predicated on the presence of reserve or undifferentiated cells in normal glands, presumably the source for cell renewal and induction of tumors. Developing rat parotid gland, which remains fetal-like at birth, provides the opportunity to study differentiation and observe whether cytologically undifferentiated cells do or do not have functional indicators of specific differentiation pathways. Immunohistochemistry and immune-electron microscopy, when applied to parotid gland at birth, at 12 days of age and in the adult gland, indicate that commitment to myoepithelial cell differentiation occurs prior to development of structural changes characteristic of these cells. Conversely, secretory granules are evident in differentiating acinar cells prior to synthesis of amylase. The results suggest that an appearance of undifferenliation does not confer reserve cell status either in the normal salivary gland or their tumors.     

小型猪与小鼠腮腺、泪腺、垂体超微结构的观察与比较

目的 研究小型猪及小鼠腮腺的超微结构特点,并将腮腺与泪腺、垂体的超微结构对比观察,旨为涎腺基因治疗转基因表达的生物学特性提供腺体形态学的依据.方法选用成年小型猪、昆明种小鼠各5只,分别取腮腺、泪腺、垂体进行超微结构观察.结果小型猪较小鼠的腮腺腺泡细胞内分泌颗粒密度大,质地均匀,细胞器少见,血管内皮细胞内有较多分泌颗粒存在.小型猪与小鼠泪腺超微结构均与各自腮腺相似,小型猪与小鼠垂体结构特点为细胞排列散在,细胞间充满血窦及毛细血管.结论小型猪腮腺腺泡细胞分泌颗粒多,其毛细血管内皮细胞中也可见较多分泌颗粒,提示这些分泌颗粒可能进入血液发挥内分泌功能.     

过氧化物酶体增殖物激活受体γ（PPAR-γ）在腮腺癌中的表达及意义

目的观察过氧化物酶体增殖物激活受体γ（PPAR-γ）在腮腺癌中的表达状况,探讨其临床意义。方法利用免疫组织化学技术检测89例腮腺癌（腺泡细胞癌40例,恶性混合瘤26例,腺样囊性癌23例）组织中PPAR-γ的表达状况,以肿瘤周围正常腮腺组织作为对照进行研究。结果正常粘膜、腮腺癌标本中PPAR-γ的阳性表达率分别为86.52%（77/89）、20.22%（18/89）,与正常腮腺组织比较,腮腺癌组PPAR-γ表达率下降,差异有显著性（P（0.05）;PPAR-γ低表达与组织学类型无相关性（P〉0.05）;与肿瘤淋巴结转移和临床病理分期密切相关（P〈0.05）。结论PPAR-γ在腮腺癌中的表达低于正常腮腺组织,腮腺癌组织中PPARγ的异常表达提示其与肿瘤的发生、发展密切相关。     

Sialin(SLC17A5)在腮腺、颌下腺及颌下腺细胞系HSG表达的初步分析

目的研究唾液酸转运蛋白Sialin在正常涎腺组织和颌下腺细胞系HSG的表达.方法应用基因芯片检测正常人腮腺、颌下腺组织Sialin编码基因SLC17A5的表达;提取腮腺、颌下腺组织及颌下腺细胞系(HSG)总RNA,通过RT-PCR在基因水平上验证SLC17A5基因的表达;提取HSG细胞总蛋白,用免疫印迹法检测Sialin在蛋白水平表达.结果基因芯片检测到Sialin编码基因SLC17A5基因在正常人腮腺、颌下腺均有表达,表达比值分别为226、206.9,不存在表达差异;RT-PCR验证芯片结果并在其他涎腺样本及颌下腺细胞系HSG中能扩增出其全部翻译区1485bp的片段,应用商业化的抗体能够在HSG细胞检测到Sialin相应的约54KDa蛋白.结论正常腮腺、颌下腺组织和颌下腺细胞系HSG均表达SLC17A5基因,但是唾液酸转运蛋白Sialin在组织、细胞内定位和功能需要进一步研究.     

A型肉毒素对腮腺分泌功能影响的研究

目的 探讨大鼠腮腺注射A型肉毒素(botulinum toxin type A,BTX-A)后组织学和免疫变化的机制.方法 18只雌性Wistar大鼠分为A、B组.A组动物(n=6)右侧腮腺内注射0.1 ml的生理盐水;B组动物(n=12)右侧腮腺注射BTX-A(2.5 U/0.1 ml).两组动物分别在注射后第7、12、35天处死并行组织学及免疫组化观察.结果 A组、B组注射后第7天部分腺泡细胞轻度萎缩,血管活性肠肽免疫反应(vasoactive intestinal polypeptide immunoreaction,VIP-IR)阳性纤维密度显著减低(n=4,P＜0.05);12 d萎缩的腺泡细胞和腺管明显增多,VIP-IR阳性纤维密度减低更明显(n=4,P＜0.001);35 d腺细胞和VIP-IR纤维密度与生理盐水组基本相同.结论 局部注射BTX-A可以导致大鼠腮腺腺泡细胞和腺管短时性萎缩,VIP-IR表达减少,提示BTX-A通过抑制调控腮腺分泌的血管活性肠肽(vasoactive intestinal polypeptide,VIP)释放导致腺组织非损伤性萎缩,而产生腺体分泌减少.     

Diabetes reduces statherin in human parotid: immunogold study and comparison with submandibular gland

Oral Diseases (2012) 18 , 360–364 Background and Objective: Alteration of salivary gland secretion is one of the consequences of diabetes. In a recent study on the submandibular gland of diabetic subjects, we found changed expression of statherin, a salivary protein of fundamental importance in preserving tooth integrity, whose reduction was related with the high incidence of oral diseases in patients with diabetes. The goal of this report is to extend the study to human parotid gland and to compare the effects of diabetes on statherin expression with those previously described in submandibular gland. Materials and Methods:  Fragments of parotid glands obtained from diabetic and non‐diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The staining density was expressed as number of gold particles per μm² and statistically evaluated. Results and Conclusions:  In all samples, statherin reactivity was specifically localized in secretory granules of acinar cells. The statistical analysis showed that labelling density was significantly lower in diabetic than in non‐diabetic parotid glands and that diabetes affects protein expression at identical extent in parotid and submandibular glands. The results strengthen the hypothesis that a reduced statherin secretion may be responsible for the higher incidence of oral disorders in diabetic subjects.     

Effect of NaF on cAMP accumulation, cAMP-dependent protein kinase activity in, and amylase secretion from, rat parotid gland cells

Stimulation of amylase secretion from parotid glands by beta-adrenergic agonists is mediated by the activation of adenylate cyclase and the resultant increase in cellular cAMP. Since NaF is known to increase adenylate cyclase activity and cAMP accumulation in intact cells, we investigated whether it would stimulate amylase secretion from isolated rat parotid gland cells. The results provide evidence that the addition of NaF (0.01-10 mmol/L) increased cAMP concentration (1.5-2.8-fold) in, and amylase secretion (16-93%) from, isolated parotid gland acinar cells. NaF was found to increase cAMP-dependent protein kinase activity ratios (51-84%) in a concentration- and time-dependent manner. The data suggest that the stimulation of amylase secretion from parotid gland cells by NaF may be mediated by an increase in the cellular cAMP concentration, which exerts its effect, at least in part, by increasing the activity of cAMP-dependent protein kinase.