【作者】 安倩；

【导师】 吴补领；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2002， 硕士

【摘要】 Calbindin-D28k是一种与Ca²⁺有高度亲和力的细胞内可溶性钙结合蛋白，因其与骨、牙釉质、牙本质等硬组织形成关系密切而受到关注。目前Calbindin-D28k的功能尚不清楚。传统观点认为Calbindin-D28k在细胞内充当钙转运载体或保护性钙缓冲剂，但近来大量事实表明，Calbindin-D28k可能还参与调节细胞内许多重要的生理活动，如：作为钙敏感调节蛋白参与Ca²⁺信号转导系统相关作用；作为激活蛋白调节多种蛋白和酶的活性以激活细胞内的一系列反应；促进细胞分化；抑制细胞凋亡，等等。牙齿是含Calbindin-D28k较丰富的组织之一。有研究表明，Calbindin-D28k在亚细胞定位上与肌动蛋白微丝关系密切，而Ca²⁺是影响微丝骨架变化的重要因素。本研究拟通过免疫组化技术观察Calbindin-D28k和肌动蛋白在大鼠磨牙及其牙胚、成年大鼠下切牙、人正常和龋坏恒牙和损伤后大鼠磨牙牙髓中的表达和动态分布特征：应用免疫荧光双标记结合激光共聚焦显微镜技术观察两种蛋白在培养人牙乳头细胞中的共定位情况，并通过免疫沉淀方法检测二者之间是否存在直接相互作用，以期揭示Calbindin-D28k与肌动蛋白在牙齿发育、矿化和损伤修复中的作用及功能相关性。 1、Calbindin-D28k与肌动蛋白在大鼠磨牙及其发育过程中的分布 将成年大鼠及胎鼠、幼鼠颌骨组织制石蜡切片，采用免疫组化方法观察成年大鼠磨牙牙髓及牙胚发育不同时期Calbindin-D28k和肌动蛋白的分布情况。结果：Calbindin-D28k在整个牙胚发育过程中，只有硬组织形成期的成釉细胞呈强阳性，染色部位为整个胞浆；成年大鼠磨牙牙髓未见阳性染色。肌动蛋白在蕾状期成釉器细胞、钟状期外釉上皮细胞阳性，分布于整个胞浆；钟状晚期中间层细胞与成釉细胞之间、成釉细胞与成牙本质细胞之间出现阳性染色；硬组织形成期成釉细胞Tome’s突和成牙本质细胞胞浆为阳性；成年大鼠磨牙成牙本质细胞阳性染色部位为 第四军医大学硕士学位论文 核以上胞浆及细胞突；牙乳头细胞及牙髓细胞未见阳性染色。提示： Calbindin028k与肌动蛋白都与釉质形成有关，但功能并不完全重叠；肌 动蛋白参与了牙本质的形成。 人Calbindin·D28k与肌动蛋白在大民切牙成牙本质细胞中的分布 将成年大鼠下颌切牙制石蜡切片，采用免疫组化方法观察 Calbindin－D28k和肌动蛋白在成牙本质细胞中的分布情况。结果： CalbindinD28k与肌动蛋白在大鼠切牙成牙本质细胞中的分布规律具有 一致性。唇侧冠方2／3区域的成牙本质细胞两种蛋白均呈阳性，且阳性部 位都集中在成牙本质细胞突；根尖区及舌侧成牙本质细胞二者均为阴性c 提示：Calbindin028k和肌动蛋白与牙本质的形成关系密切。 3、Calbindin＊28k与肌动蛋自在钻磨后大鼠唇牙牙函中的表达 将24只成年大鼠随机分为8组，第一磨牙钻磨穿髓后分别于Zh、sh、 id、Zd、sd、sd、lid、14d取材制石蜡切片，寸狈同名牙作为正常寸照， 采用免疫组化方法观察Calbindin－D28k和肌动蛋白在钻磨刺激后不同时 间牙髓中的表达和分布情况。结果：Calbindin－D28k在各组牙髓染色均为 阴性。肌动蛋白在正常组成牙本质细胞及细胞突阳性，血管内皮细胞及 部分管周细胞弱阳性；牙髓受到钻磨刺激后，在创伤或炎症累及区域成 牙本质细胞肌动蛋白转阴，同时细胞排列紊乱且与前期牙本质分离；在 炎症发展的前沿区，血管内皮细胞及其外周一些细胞呈强阳性。提示： 肌动蛋白在维持成牙本质细胞的排列方式及正常生理功能中有重要意 义：肌动蛋白在血管内皮细胞及管周细胞中的表达上调可能与牙髓损伤 的早期修复有关。 4、Calbindin－D28k与肌动蛋白在人健康及琢坏牙齿中的分布 将因治疗需要而拔除的第三磨牙依据临床体征和离体探察情况分为 正常、中龋和深龋三组，制石蜡切片，采用免疫组化方法观察 Calbindin－D28k和肌动蛋白在正常和龋坏刺激下牙髓中的表达特征。结 果：正常组CalbindinD28k在成牙本质细胞和牙髓细胞阴性，前期牙本 质远髓213区域染色阳性；肌动蛋白在成牙本质细胞及位于矿化牙本质中 的细胞突呈阳性，牙髓细胞弱阳性；中龋组Calbindin－D28k在龋坏部位 一3 一 第四军医大学硕士学位论文 所对应的前期牙本质全层呈阳性且较正常组明显增强，位于前期牙本质 中的成牙本质细胞突呈强阳性；肌动蛋白在龋坏部位所对应的成牙本质 细胞胞浆及位于前期牙本质中的细胞突起呈强阳性：深龋组龋坏部位所 对应的中心区域成牙本质细胞变?更多还原

【Abstract】 Calbindin-D28k is an intracellular soluble vitamin D-dependent calcium binding protein, which has a high affinity for Ca2"1". Previous studies have demonstrated the presence of Calbindin-D28k and its mRNA in mineralized tissues, such as bone and tooth, and suggested the involvement of Calbindin-D28k in the mineralization. Although there have been several hypotheses for the role of Calbindin-D28k, the exact function of this protein is still to be established. Traditionally, Calbindin-D28k has been regarded as calcium transporter or intracellular calcium buffer. But recently, the regulatory effects of Calbindin-D28k have been proposed. Some studies showed that Calbindin-D28k subcellularly co-localized with actin filaments in ameloblasts and odontoblasts. In the present study, the distribution of Calbindin-D28k and actin in tooth germs and adult pulps obtained either from rats or human beings was observed and compared by immunochemical method. To further investigate the relationship between the two proteins, double immunofluorescence method was applied to observe the co-localization of Calbindin-D28k and actin in cultured dental papilla cells, and then, the interaction between the two proteins was clarified by immunoprecipitation technique.1 Distribution of Calbindin-D28k and actin in the development of rat molarsIn this study, fetal rats (17-19 days of gestation) and new born pups (from 1 to 14 days old) were utilized to investigate the localization of Calbindin-D28k and actin in the developing molars. Immunohistochemical- 6 -results showed that Calbindin-D28k was detected only in the whole cytoplasm of the differentiated ameloblasts. However, actin was present in the enamel organ as early as at the bud stage. During the period of hard tissue formation, actin was distributed in the Tome’s process of ameloblast and throughout the cytoplasm of odontoblast. In adult rat, odontoblasts were still positively stained for actin but only in the cytoplasm abovethe nucleus. These findings tended to suggest that both Calbindin-D28k and actin could be involved in enamel formation but they might function differently. In addition, actin also participateed in dentin formation.2 Distribution of Calbindin-D28k and actin in rat incisor odontoblastsImmunohistochemical method was adopted for examining and comparing the distribution of Calbindin-D28k and actin in the odontoblasts of rat’s continuously erupting lower incisor. The results of the present study demonstrated that Calbindin-D28k and actin shared a similar expression pattern, that is, both proteins were detected in the odontoblasts, particularly in the processes of labial side within the 2/3 crownal portion whereas neither in those of lingual side and apical region. This could indicate that the two proteins could be in close relation to dentin formation.3 Distribution of Calbindin-D28k and actin in burred rat dental pulpsIn order to investigate the expression of Calbindin-D28k and actin during pulp restoration, we established the model of rat tooth injury and examined Calbindin-D28k and actin in healthy pulp and the pulps from 2 hours to 14 days after burred. No immunostaining was found for Calbindin-D28k in all the pulps examined. As for actin, positive staining was localized in odontoblasts and vessel endothelia in the healthy pulp. 2h after injury, the odontoblasts near the exposure point turned negative while the vessel endothelia and the nearby cells were strongly labelled. Such strongly immunoreactive vessels always occurred in the area where pulpitis first attacked in the 8h~5d groups. The present study tended to suggest that actincould play an important role in sustaining the odontoblasts in their specificlining style and physical function, and that its up-regulation in vessels mighthave something to do with the early restoration of pulp after injury.4 Distribution of Calbindin-D28k and actin in healthy and carious humanteeth34 wisdom teeth extracted for therapeutic reasons were collected and divided into three groups: n更多还原