大鼠颌下腺和离体培养细胞中降钙素的定位研究

发布时间：2018-10-23 13:52

【摘要】：目的:颌下腺具有内分泌腺的功能,能分泌多种生物活性物质,因为甲状腺和颌下腺以及胃肠胰腺同是由内胚层分化形成的,它们在结构和功能方面可能密切相关。最近研究报道颌下腺能分泌降钙素(calcitonin,CT),由此推测颌下腺分泌的降钙素参与了机体钙磷代谢调节。本实验通过对大鼠乳鼠颌下腺细胞用自然培养的方法进行体外培养,并应用免疫细胞化学方法,对培养细胞进行免疫化学染色和原位杂交法证实降钙素的存在,用酶联免疫吸附实验测定降钙素的含量,验证大鼠颌下腺腺细胞分泌降钙素,并进一步证实颌下腺的内分泌功能。方法:(1)分别取大鼠乳鼠的颌下腺组织,制做石蜡组织标本切片,进行免疫组织化学染色,从组织学方面定位颌下腺细胞分泌降钙素。(2)建立大鼠原代颌下腺细胞体外培养体系并传代,用免疫细胞化学方法,分别对培养的细胞进行细胞角蛋白(cytokeratin)、CT染色,观察细胞的特征和降钙素在细胞内的分布。(3)用地高辛标记的降钙素DNA探针原位杂交法,验证颌下腺细胞分泌降钙素并进行基因定位。(4)酶联免疫吸附实验(ELISA)测定降钙素的含量。结果:(1)对大鼠颌下腺组织标本切片免疫化学染色观察发现:大鼠颌下腺的颗粒性曲管(granular convoluted tubule,GCT)、闰管、纹状管和小叶间导管上皮细胞分别呈CT免疫反应阳性,反应产物分布在细胞浆中。(2)在大鼠乳鼠颌下腺细胞的培养中,培养生长的细胞呈扁平的三角形,方形,圆形,索形等,似铺路石状粘贴在瓶壁上,并且被上皮细胞的特异标志cytokeratin着染。(3)在对细胞化学染色和地高辛标记的降钙素DNA探针原位杂交实验中观察到:分泌降钙素的DNA分布在细胞浆中,细胞核无表达。(4)在以上实验的基础上用酶联免疫吸附实验(ELISA)测定了细胞分泌降钙素的水平。结论:(1)在组织标本切片中明确了大鼠颌下腺分泌降钙素的细胞及其形态结构和组成。(2)建立了大鼠原代颌下腺细胞体外培养体系并传代,免疫细胞化学染色和原位杂交证实颌下腺细胞分泌降钙素。(3)ELISA实验测定了颌下腺细胞分泌降钙素的含量。
[Abstract]:Objective: the submandibular gland has the function of endocrine gland and can secrete a variety of bioactive substances, because thyroid gland, submandibular gland and gastrointestinal pancreas are both formed by endoderm differentiation, which may be closely related in structure and function. Recently, it is reported that calcitonin (calcitonin,CT) can be secreted in submandibular gland, which suggests that calcitonin secreted by submandibular gland is involved in the regulation of calcium and phosphorus metabolism. In this experiment, the submandibular gland cells of neonatal rats were cultured in vitro by natural culture, and the existence of calcitonin was confirmed by immunocytochemical staining and in situ hybridization. The content of calcitonin was determined by enzyme-linked immunosorbent assay (Elisa) to verify the secretion of calcitonin by rat submandibular gland cells and to further confirm the endocrine function of submandibular gland. Methods: (1) the submandibular gland tissues of the neonatal rats were taken respectively and the paraffin tissue specimens were made and stained by immunohistochemistry. Histologically localization of calcitonin secretion in submandibular gland cells. (2) Establishment and passage of primary rat submandibular gland cells in vitro. Cytokeratin (cytokeratin), CT staining was performed on cultured cells by immunocytochemistry. The characteristics of cells and the distribution of calcitonin in cells were observed. (3) Digoxin labeled calcitonin DNA probe in situ hybridization was used to verify the secretion of calcitonin and gene localization in submandibular gland cells. (4) Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of calcitonin. Results: (1) Immunochemical staining of rat submandibular gland tissue showed that the granular convoluted tubules (granular convoluted tubule,GCT), intercalated duct, striated duct and interlobular ductal epithelial cells were positive for CT immunoreactivity. The reaction products were distributed in the cytoplasm. (2) in the culture of rat submandibular gland cells, the cultured cells were flat triangle, square, round, cord-shaped, etc. And stained by cytokeratin, a specific marker of epithelial cells. (3) in cytochemical staining and in situ hybridization with digoxigenin-labeled calcitonin DNA probes, it was observed that the DNA secreting calcitonin was distributed in the cytoplasm. (4) the level of calcitonin secretion was determined by enzyme linked immunosorbent assay (ELISA) on the basis of the above experiments. Conclusion: (1) the cells secreting calcitonin in rat submandibular gland were identified in tissue sections. (2) the primary submandibular gland cells were cultured in vitro. Immunocytochemical staining and in situ hybridization confirmed the secretion of calcitonin by submandibular gland cells. (3) the content of calcitonin secreted by submandibular gland cells was determined by ELISA assay.
【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R33

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