【摘要】：目的:頜下腺具有內分泌腺的功能,能分泌多種生物活性物質,因為甲狀腺和頜下腺以及胃腸胰腺同是由內胚層分化形成的,它們在結構和功能方面可能密切相關。最近研究報道頜下腺能分泌降鈣素(calcitonin,CT),由此推測頜下腺分泌的降鈣素參與了機體鈣磷代謝調節(jié)。本實驗通過對大鼠乳鼠頜下腺細胞用自然培養(yǎng)的方法進行體外培養(yǎng),并應用免疫細胞化學方法,對培養(yǎng)細胞進行免疫化學染色和原位雜交法證實降鈣素的存在,用酶聯(lián)免疫吸附實驗測定降鈣素的含量,驗證大鼠頜下腺腺細胞分泌降鈣素,并進一步證實頜下腺的內分泌功能。 方法:(1)分別取大鼠乳鼠的頜下腺組織,制做石蠟組織標本切片,進行免疫組織化學染色,從組織學方面定位頜下腺細胞分泌降鈣素。(2)建立大鼠原代頜下腺細胞體外培養(yǎng)體系并傳代,用免疫細胞化學方法,分別對培養(yǎng)的細胞進行細胞角蛋白(cytokeratin)、CT染色,觀察細胞的特征和降鈣素在細胞內的分布。(3)用地高辛標記的降鈣素DNA探針原位雜交法,驗證頜下腺細胞分泌降鈣素并進行基因定位。(4)酶聯(lián)免疫吸附實驗(ELISA)測定降鈣素的含量。 結果:(1)對大鼠頜下腺組織標本切片免疫化學染色觀察發(fā)現(xiàn):大鼠頜下腺的顆粒性曲管(granular convoluted tubule,GCT)、閏管、紋狀管和小葉間導管上皮細胞分別呈CT免疫反應陽性,反應產物分布在細胞漿中。(2)在大鼠乳鼠頜下腺細胞的培養(yǎng)中,培養(yǎng)生長的細胞呈扁平的三角形,方形,圓形,索形等,似鋪路石狀粘貼在瓶壁上,并且被上皮細胞的特異標志cytokeratin著染。(3)在對細胞化學染色和地高辛標記的降鈣素DNA探針原位雜交實驗中觀察到:分泌降鈣素的DNA分布在細胞漿中,細胞核無表達。(4)在以上實驗的基礎上用酶聯(lián)免疫吸附實驗(ELISA)測定了細胞分泌降鈣素的水平。 結論:(1)在組織標本切片中明確了大鼠頜下腺分泌降鈣素的細胞及其形態(tài)結構和組成。(2)建立了大鼠原代頜下腺細胞體外培養(yǎng)體系并傳代,免疫細胞化學染色和原位雜交證實頜下腺細胞分泌降鈣素。(3)ELISA實驗測定了頜下腺細胞分泌降鈣素的含量。
[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.
【學位授予單位】：延安大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R33

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