發(fā)布時(shí)間：2018-10-23 13:52

【摘要】：目的:頜下腺具有內(nèi)分泌腺的功能,能分泌多種生物活性物質(zhì),因?yàn)榧谞钕俸皖M下腺以及胃腸胰腺同是由內(nèi)胚層分化形成的,它們?cè)诮Y(jié)構(gòu)和功能方面可能密切相關(guān)。最近研究報(bào)道頜下腺能分泌降鈣素(calcitonin,CT),由此推測(cè)頜下腺分泌的降鈣素參與了機(jī)體鈣磷代謝調(diào)節(jié)。本實(shí)驗(yàn)通過(guò)對(duì)大鼠乳鼠頜下腺細(xì)胞用自然培養(yǎng)的方法進(jìn)行體外培養(yǎng),并應(yīng)用免疫細(xì)胞化學(xué)方法,對(duì)培養(yǎng)細(xì)胞進(jìn)行免疫化學(xué)染色和原位雜交法證實(shí)降鈣素的存在,用酶聯(lián)免疫吸附實(shí)驗(yàn)測(cè)定降鈣素的含量,驗(yàn)證大鼠頜下腺腺細(xì)胞分泌降鈣素,并進(jìn)一步證實(shí)頜下腺的內(nèi)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(biāo)本切片,進(jìn)行免疫組織化學(xué)染色,從組織學(xué)方面定位頜下腺細(xì)胞分泌降鈣素。(2)建立大鼠原代頜下腺細(xì)胞體外培養(yǎng)體系并傳代,用免疫細(xì)胞化學(xué)方法,分別對(duì)培養(yǎng)的細(xì)胞進(jìn)行細(xì)胞角蛋白(cytokeratin)、CT染色,觀察細(xì)胞的特征和降鈣素在細(xì)胞內(nèi)的分布。(3)用地高辛標(biāo)記的降鈣素DNA探針原位雜交法,驗(yàn)證頜下腺細(xì)胞分泌降鈣素并進(jìn)行基因定位。(4)酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)測(cè)定降鈣素的含量。 結(jié)果:(1)對(duì)大鼠頜下腺組織標(biāo)本切片免疫化學(xué)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(dǎo)管上皮細(xì)胞分別呈CT免疫反應(yīng)陽(yáng)性,反應(yīng)產(chǎn)物分布在細(xì)胞漿中。(2)在大鼠乳鼠頜下腺細(xì)胞的培養(yǎng)中,培養(yǎng)生長(zhǎng)的細(xì)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(xì)胞的特異標(biāo)志cytokeratin著染。(3)在對(duì)細(xì)胞化學(xué)染色和地高辛標(biāo)記的降鈣素DNA探針原位雜交實(shí)驗(yàn)中觀察到:分泌降鈣素的DNA分布在細(xì)胞漿中,細(xì)胞核無(wú)表達(dá)。(4)在以上實(shí)驗(yàn)的基礎(chǔ)上用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)測(cè)定了細(xì)胞分泌降鈣素的水平。 結(jié)論:(1)在組織標(biāo)本切片中明確了大鼠頜下腺分泌降鈣素的細(xì)胞及其形態(tài)結(jié)構(gòu)和組成。(2)建立了大鼠原代頜下腺細(xì)胞體外培養(yǎng)體系并傳代,免疫細(xì)胞化學(xué)染色和原位雜交證實(shí)頜下腺細(xì)胞分泌降鈣素。(3)ELISA實(shí)驗(yàn)測(cè)定了頜下腺細(xì)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(xué)位授予單位】：延安大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R33

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