【作者】 林叶；

【导师】 李庆章；

【作者基本信息】 东北农业大学 ， 动物生物化学与分子生物学， 2006， 博士

【摘要】 瘦素是主要由体脂合成和分泌的细胞因子，也可以由乳腺脂肪垫和乳腺上皮细胞旁分泌或自分泌生成。瘦素对乳腺的发育和泌乳具有一定的调控作用，它可以影响青春期乳腺导管发育，还可以刺激小鼠和人乳腺上皮细胞增殖。目前对于乳腺中瘦素及其受体的研究仅限于其mRNA水平的表达以及瘦素对培养的乳腺上皮细胞增殖的影响，尚缺乏对乳腺中瘦素及其受体在蛋白质水平上的表达变化规律以及瘦素对乳腺发育、泌乳及退化的作用研究，乳腺中瘦素的信号转导途径也不清楚。 本实验以小鼠为实验材料，运用激光扫描共聚焦显微技术首次对乳腺发育、泌乳和退化各个时期乳腺自身产生的瘦素(leptin)及其长型受体(OB-Rb)的蛋白表达、定位进行系统研究。结果表明：乳腺中瘦素的表达在青春期最高，妊娠期开始下降，妊娠16d乳腺中瘦素的表达稍有回升，随后降低，并在整个泌乳期维持在低水平，幼仔离乳后，乳腺腺泡上皮细胞凋亡，乳腺脂肪垫重新生成，瘦素的表达上调，在乳腺退化13d恢复到青春期水平。在整个发育周期中，OB-Rb的表达变化趋势与瘦素相似，呈显著正相关，表明在乳腺发育、泌乳和退化各个时期，瘦素的表达可以正向诱导OB-Rb的表达。青春期和妊娠早期，瘦素主要在乳腺脂肪细胞、导管上皮细胞以及围绕导管的基膜中被检测到，OB-Rb在乳腺脂肪细胞和导管上皮细胞中表达，妊娠晚期和泌乳期，瘦素存在于腺泡上皮细胞靠近基底侧和细胞外基质中，OB-Rb在腺泡上皮细胞靠近基底侧的细胞膜上特异性表达，退化期，乳腺脂肪垫重新生成，瘦素重新在乳腺脂肪细胞和导管上皮细胞中被检测到，OB-Rb也重新在乳腺脂肪细胞和导管上皮细胞中表达。瘦素和OB-Rb在乳腺发育循环中表达部位的相似性表明OB-Rb是瘦素的专一性配体，瘦素通过与OB-Rb结合发挥其在乳腺发育、泌乳和退化过程中的调控作用。 为阐明乳腺不同发育阶段瘦素及其受体的生物学功能，本实验采用乳腺组织体外培养的方法研究瘦素对妊娠期、泌乳期和退化期乳腺结构变化以及泌乳功能的影响。体外培养妊娠中期的乳腺组织5d，培养结束后通过石蜡切片H．E．染色观察乳腺结构变化，结果显示：与对照组相比，瘦素处理使乳腺导管分支增多，表明妊娠期瘦素可以促进乳腺导管上皮细胞的增殖和分化；体外培养泌乳中期的乳腺组织5d，分别在培养24h、48h、72h、96h和120h采用毛细管电泳方法检测培养基和乳腺组织匀浆中β-酪蛋白含量的变化，结果表明：在培养96h内，瘦素可以明显诱导乳腺中β-酪蛋白基因的表达；体外培养退化5d的乳腺组织，5d后通过石蜡切片H．E．染色观察乳腺组织结构变化，结果显示：对照组乳腺仍然维持导管结构，而瘦素处理组乳腺导管消失，仅残留上皮细胞簇样结构，表明瘦素可以诱导退化期乳腺组织重建。 为揭示乳腺中瘦素的作用机理，采用Western blot方法分别对妊娠期、泌乳期和退化期乳腺中瘦素信号转导通路成分的磷酸化状态进行检测。结果表明：虽然在乳腺发育的各个时期，乳腺中均存在JAK-STAT3，JAK-STAT5和JAK-MAPK信号转导通路成分，但是瘦素并不能同时激活所有的信号分子。妊娠期，瘦素专一性地诱导JAK-MAPK信号转导通路中p42MAPK／p44MAPK磷酸化，说明妊娠期瘦素与OB-Rb结合，通过激活JAK-MAPK信号转更多还原

【Abstract】 Leptin was a cytokine which was synthesized and secreted by body fat. It was also produced by mammary fat pad and mammary epithelial cells through paracrine and autocrine. Leptin controlled the development and lactation of mammary gland. It influenced the development of duct in virgin and stimulated the proliferation of mammary epithelial cells. At present, the research about leptin and the long form of leptin receptor (OB-Rb) was only the expression of them in mRNA and the impact of leptin on the proliferation of mammary epithelial cells in culture. There were no results about the expression of leptin and OB-Rb in protein and the impacts of leptin on the development, lactation and involution of mammary gland. It was also not clear about the signal transduction pathway of leptin in mammary gland.In these experiments, for the first time, we investigated the expression and localization of leptin and OB-Rb in protein which were produced by mouse mammary gland itself in virgin, pregnancy, lactation and involution systematically by Confocal laser scanning microscope. These results showed that in virgin, the expression of leptin was highest and degraded at the beginning of pregnancy. On pregnancy 16d, the expression of leptin in mammary gland upgraded slightly then degraded and kept at the low level during the whole lactation. In involution, mammary epithelial cells were apoptosis and adipose tissue restored again, the expression of leptin increased and recovered to the original level about virgin on involution 13d. In the whole development cycle, the expression of OB-Rb was similar with leptin and showed significant positive correlation. This indicated that the expression of leptin induced the expression of OB-Rb positively in development, lactation and involution mammary gland. In virgin and early pregnancy, leptin was found in adipocytes, ductal epithelial cells and basal lamina around the duct and OB-Rb was found in adipocytes and ductal epithelial cells; in late pregnancy and lactation, leptin was detected in alveolus epithelial cells near to the basal lamina and extracellular matrix, OB-Rb was detected on the membrane of alveolus epithelial cells near to the basal lamina; in involution, mammary fat pad restored and leptin was existed in adipocytes and ductal epithelial cells again and OB-Rb was also existed in adipocytes and ductal epithelial cells.The localization of leptin and OB-Rb were similar suggested that in each development period of mammary gland, leptin affected the development and physiology function of mammary gland by binding OB-Rb.In order to clarify the biological function of leptin and OB-Rb in the development cycle of mammary gland, explants of mammary gland were cultured in a synthetic medium with various更多还原