Wnt信号通路激活剂BIO在山羊乳腺上皮细胞形成腺泡样结构中的调控作用

发布时间：2018-06-15 02:19

本文选题：6-溴靛玉红-3-肟 + Wnt信号通路　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：乳腺是一种复杂的分泌器官,它的主要功能是泌乳。其中乳腺导管和小叶腺泡是乳腺执行泌乳过程所必须的,乳腺导管主要参与乳汁的运输而乳腺腺泡则具有泌乳功能。所以对乳腺上皮细胞构成乳腺导管结构和乳腺腺泡结构的机制的研究是非常重要的。在体试验中Wnt信号通路在乳腺腺泡发育和乳腺导管发育方面的作用机制已经被广泛研究,并且取得了很大的进展。由于在体试验中乳腺发育中调控因素较多,它们会对研究Wnt信号通路在乳腺上皮细胞形成腺泡样结构中的调控作用有影响。因此,在体外条件下研究Wnt信号通路对乳腺上皮细胞分化与功能的影响是非常有意义的。为了研究体外条件下Wnt信号通路对乳腺上皮细胞分化与功能的影响,本研究使用关中奶山羊的乳腺上皮细胞作为试验材料,在二维和三维的培养条件下研究Wnt信号通路激活剂BIO对山羊乳腺上皮细胞生物学特性以及发育能力的影响。研究结果如下:1.通过组织块法和差速消化法分离、纯化得到具有酪蛋白和乙酰辅酶A羧化酶mRNA表达的山羊乳腺上皮细胞。在二维培养条件下,Wnt信号通路激活剂BIO可促进山羊乳腺上皮细胞形成能够表达酪蛋白和乙酰辅酶A羧化酶mRNA的腺泡样结构;在三维培养条件下,BIO可以促进山羊乳腺上皮细胞形成能表达酪蛋白和乙酰辅酶A羧化酶的mRNA的乳腺腺泡样结构和导管样结构;与常规三维培养相比,在添加BIO的培养液中培养的腺泡结构体积更大,并且成腔速度更快。2.通过单细胞克隆培养法分离得到了能表达酪蛋白和乙酰辅酶A羧化酶mRNA的两种增殖特性的山羊乳腺上皮细胞,即扁平状增殖的乳腺上皮细胞和蜂窝状增殖的乳腺上皮细胞。在二维培养条件下对两种增殖特性的山羊乳腺上皮细胞的生物学特性、形态结构和特异性基因表达功能进行了研究,其中扁平状增殖的乳腺上皮细胞高表达N-钙粘素,并且具有雌激素受体α、雌激素受体β和孕酮受体mRNA的表达能力;蜂窝状增殖的乳腺上皮细胞仅能表达雌激素受体β。在三维培养条件下,蜂窝状增殖的乳腺上皮细胞较扁平状增殖的乳腺上皮细胞所能形成的腺泡样结构数量更多,体积更大。3.在二维培养条件下,Wnt信号通路激活剂BIO可以促进蜂窝状增殖的山羊乳腺上皮细胞形成腺泡样结构,但不能促进扁平状增殖的山羊乳腺上皮细胞形成腺泡样结构。在三维培养条件下,BIO可促进蜂窝状增殖的山羊乳腺上皮细胞形成有功能的腺泡样结构和导管样结构;而扁平状增殖的山羊乳腺上皮细胞所形成的腺泡样结构数量较少,体积较小。
[Abstract]:Mammary gland is a kind of complex secretory organ, its main function is lactation. The mammary duct and lobular acinus are necessary for the mammary gland to carry out the lactation process. The mammary duct is mainly involved in the transport of milk and the mammary acinus has the function of lactation. Therefore, it is very important to study the mechanism of mammary gland epithelial cells forming mammary duct structure and mammary acinar structure. The mechanism of Wnt signaling pathway in breast acinar development and mammary duct development has been extensively studied in vivo and great progress has been made. Since there are many regulatory factors in breast development in vivo, they may play an important role in the study of the regulation of Wnt signaling pathway in the formation of acinar structure in mammary epithelial cells. Therefore, it is significant to study the effects of Wnt signaling pathway on the differentiation and function of breast epithelial cells in vitro. In order to study the effect of Wnt signaling pathway on the differentiation and function of mammary epithelial cells in vitro, we used the mammary epithelial cells of Guanzhong dairy goats as experimental materials. The effects of Wnt signal pathway activator bio on the biological characteristics and developmental ability of goat mammary epithelial cells were studied under two and three dimensional culture conditions. The results are as follows: 1. Goat mammary epithelial cells with casein and acetyl coenzyme A carboxylase mRNA expression were purified by tissue block method and differential digestion method. In two dimensional culture, BIO, an activator of Wnt signaling pathway, promoted goat mammary epithelial cells to form acinar like structures capable of expressing casein and acetyl coenzyme A carboxylase mRNA. Under the condition of three-dimensional culture, BIO can promote goat mammary epithelial cells to form mammary acinar like structure and catheter-like structure which can express casein and acetyl coenzyme A carboxylase mRNA. The acinar structure cultured in the medium supplemented with bio was larger in volume and faster in cavity formation. Goat mammary epithelial cells which express casein and acetyl coA carboxylase mRNA were isolated by single cell clone culture method, namely flat proliferating mammary gland epithelial cells and honeycomb mammary gland epithelial cells which can express casein and acetyl coenzyme A carboxylase mRNA. The biological characteristics, morphological structure and specific gene expression function of goat mammary epithelial cells with two proliferative characteristics were studied in two-dimensional culture. Moreover, estrogen receptor 伪, estrogen receptor 尾 and progesterone receptor mRNA were expressed in breast epithelial cells, and only estrogen receptor 尾 could be expressed in honeycombed mammary epithelial cells. Under the condition of three-dimensional culture, the number and volume of acinar like structure of mammary epithelial cells with honeycomb proliferation was more than that of flattened mammary epithelial cells, and the volume of acinar structure was larger than that of flattened mammary epithelial cells. Under the condition of two-dimensional culture, BIO, a signal pathway activator, could promote the formation of acinar structure in goat mammary epithelial cells with honeycomb proliferation, but could not promote the formation of acinar structure in flat proliferating goat mammary epithelial cells. Under the condition of three-dimensional culture, BIO could promote the formation of functional acinar structure and catheter-like structure in goat mammary epithelial cells with honeycomb proliferation, while the number of acinar like structures formed by flat proliferating goat mammary epithelial cells was less. Small in size.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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