本文選題：6-溴靛玉紅-3-肟 + Wnt信號通路��； 參考：《西北農(nóng)林科技大學》2015年碩士論文

【摘要】：乳腺是一種復雜的分泌器官,它的主要功能是泌乳。其中乳腺導管和小葉腺泡是乳腺執(zhí)行泌乳過程所必須的,乳腺導管主要參與乳汁的運輸而乳腺腺泡則具有泌乳功能。所以對乳腺上皮細胞構成乳腺導管結構和乳腺腺泡結構的機制的研究是非常重要的。在體試驗中Wnt信號通路在乳腺腺泡發(fā)育和乳腺導管發(fā)育方面的作用機制已經(jīng)被廣泛研究,并且取得了很大的進展。由于在體試驗中乳腺發(fā)育中調控因素較多,它們會對研究Wnt信號通路在乳腺上皮細胞形成腺泡樣結構中的調控作用有影響。因此,在體外條件下研究Wnt信號通路對乳腺上皮細胞分化與功能的影響是非常有意義的。為了研究體外條件下Wnt信號通路對乳腺上皮細胞分化與功能的影響,本研究使用關中奶山羊的乳腺上皮細胞作為試驗材料,在二維和三維的培養(yǎng)條件下研究Wnt信號通路激活劑BIO對山羊乳腺上皮細胞生物學特性以及發(fā)育能力的影響。研究結果如下:1.通過組織塊法和差速消化法分離、純化得到具有酪蛋白和乙酰輔酶A羧化酶mRNA表達的山羊乳腺上皮細胞。在二維培養(yǎng)條件下,Wnt信號通路激活劑BIO可促進山羊乳腺上皮細胞形成能夠表達酪蛋白和乙酰輔酶A羧化酶mRNA的腺泡樣結構;在三維培養(yǎng)條件下,BIO可以促進山羊乳腺上皮細胞形成能表達酪蛋白和乙酰輔酶A羧化酶的mRNA的乳腺腺泡樣結構和導管樣結構;與常規(guī)三維培養(yǎng)相比,在添加BIO的培養(yǎng)液中培養(yǎng)的腺泡結構體積更大,并且成腔速度更快。2.通過單細胞克隆培養(yǎng)法分離得到了能表達酪蛋白和乙酰輔酶A羧化酶mRNA的兩種增殖特性的山羊乳腺上皮細胞,即扁平狀增殖的乳腺上皮細胞和蜂窩狀增殖的乳腺上皮細胞。在二維培養(yǎng)條件下對兩種增殖特性的山羊乳腺上皮細胞的生物學特性、形態(tài)結構和特異性基因表達功能進行了研究,其中扁平狀增殖的乳腺上皮細胞高表達N-鈣粘素,并且具有雌激素受體α、雌激素受體β和孕酮受體mRNA的表達能力;蜂窩狀增殖的乳腺上皮細胞僅能表達雌激素受體β。在三維培養(yǎng)條件下,蜂窩狀增殖的乳腺上皮細胞較扁平狀增殖的乳腺上皮細胞所能形成的腺泡樣結構數(shù)量更多,體積更大。3.在二維培養(yǎng)條件下,Wnt信號通路激活劑BIO可以促進蜂窩狀增殖的山羊乳腺上皮細胞形成腺泡樣結構,但不能促進扁平狀增殖的山羊乳腺上皮細胞形成腺泡樣結構。在三維培養(yǎng)條件下,BIO可促進蜂窩狀增殖的山羊乳腺上皮細胞形成有功能的腺泡樣結構和導管樣結構;而扁平狀增殖的山羊乳腺上皮細胞所形成的腺泡樣結構數(shù)量較少,體積較小。
[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 偽, 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.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S827

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