本文選題：植物性功能食品 + 雌激素受體��； 參考：《南京醫(yī)科大學(xué)》2013年碩士論文

【摘要】：隨著社會的發(fā)展和人們保健意識的提高，一些傳統(tǒng)的食用食物和中草藥被廣泛的應(yīng)用于植物性功能食品中，例如涼茶，其主要原料就有甘草、布渣葉、金銀花、菊花、仙草、雞蛋花、夏枯草等。這些植物性功能食品的發(fā)展極大地擴(kuò)大了人群對各種植物化學(xué)物質(zhì)的暴露范圍。植物中含有豐富的化學(xué)物質(zhì)，包括植物性來源的雌激素和雄激素，這些是植物體內(nèi)天然的激素物質(zhì)，具有一定的內(nèi)分泌干涉效應(yīng)。部分植物性功能食品利用植物激素的內(nèi)分泌干預(yù)作用，獲得相應(yīng)的疾病預(yù)防作用，如大豆異黃酮預(yù)防腫瘤、骨質(zhì)疏松和心血管疾病。但是對于特定的人群，例如正在生長發(fā)育的兒童、更年期婦女、乳腺癌或者前列腺癌人群來說，攝入了這些植物類激素就有可能產(chǎn)生潛在的風(fēng)險。目前，甘草和當(dāng)歸就被報道具有雌激素效應(yīng)，能夠促進(jìn)乳腺癌細(xì)胞的增長，而對其他大部分植物性功能食品（飲料）的原料的內(nèi)分泌干擾效應(yīng)尚缺乏了解，尤其是雄激素效應(yīng)方面。所以，為了更安全的食用這些植物性功能食品，就有必要對它們是否具有內(nèi)分泌干擾效應(yīng)及其機(jī)制進(jìn)行研究。 目的：本研究采用雌激素依賴性乳腺癌細(xì)胞（MCF-7）和雄激素依賴性前列腺癌細(xì)胞(22RV1)為模型，研究植物性功能食品的原料是否具有內(nèi)分泌干擾效應(yīng)并探討其機(jī)制。 方法：采用MTS實(shí)驗(yàn)檢測植物性功能食品原料提取物對細(xì)胞活性的影響；采用RT-PCR檢測提取物對雌激素受體（ER）和雄激素受體(AR)靶基因的影響；通過Western blot檢測提取物對ERα、ERK/1/2蛋白表達(dá)的影響和ERα及ERK1/2蛋白的分別在漿核的表達(dá)情況；應(yīng)用免疫熒光方法檢測ERα及ERK1/2的漿核定位；采用免疫沉淀實(shí)驗(yàn)檢測ERα和ERK1/2蛋白是否相互結(jié)合。 結(jié)果：對八種植物提取物（當(dāng)歸、布渣葉、甘草、金銀花、菊花、仙草、雞蛋花、夏枯草）進(jìn)行篩檢，在雌激素依賴性乳腺癌細(xì)胞MCF-7中，MTS實(shí)驗(yàn)結(jié)果顯示，與對照相比布渣葉、當(dāng)歸提取物在一定濃度下能夠增加MCF-7細(xì)胞的活性，且這種活性能夠被ERα的拮抗劑ICI182，780（ICI）和MEK1/2抑制劑U0126所抑制，這些結(jié)果表明ERα和ERK1/2信號通路參與了提取物介導(dǎo)的細(xì)胞活性的增加；Western blot顯示當(dāng)歸和布渣葉在MCF-7細(xì)胞中能夠下調(diào)ERα蛋白的水平，且能夠介導(dǎo)ER的靶基因pS2表達(dá)的增加，加入ICI或者U0126之后，提取物介導(dǎo)的pS2水平明顯下降；當(dāng)歸和布渣葉能誘導(dǎo)延遲的ERK1/2激活，并呈現(xiàn)時間效應(yīng)，當(dāng)加了U0126后，這種誘導(dǎo)作用被阻斷，而ICI對提取物介導(dǎo)的ERK1/2的激活沒有影響，表明ERK1/2激活位于ERα介導(dǎo)的信號通路的上游；當(dāng)歸和布渣葉能夠促進(jìn)ERα的核轉(zhuǎn)位，誘導(dǎo)磷酸化的ERK1/2和ERα蛋白的結(jié)合。在雄激素依賴型前列腺癌細(xì)胞22RV1中，對布渣葉、甘草和當(dāng)歸三種植物提取物進(jìn)行篩檢，布渣葉提取物與對照相比，能夠促進(jìn)22RV1細(xì)胞的活性，并且能夠誘導(dǎo)雄激素受體靶基因PSA的表達(dá)，當(dāng)加入雄激素受體抑制劑后，細(xì)胞活性和雄激素受體靶基因水平均明顯下降，當(dāng)歸和甘草提取物與對照相比并沒有明顯改變；當(dāng)加入ERK1/2的抑制劑U0126后，同樣抑制了布渣葉提取物介導(dǎo)的細(xì)胞活性和PSA的表達(dá)。 結(jié)論：當(dāng)歸、布渣葉具有雌激素效應(yīng)，布渣葉同時還具有雄激素效應(yīng)；這些提取物介導(dǎo)的內(nèi)分泌效應(yīng)的發(fā)生是通過ERα或AR介導(dǎo)；且延遲的ERK1/2的激活是當(dāng)歸、甘草和布渣葉介導(dǎo)的雌激素效應(yīng)所需要的。
[Abstract]:With the development of society and the improvement of people's awareness of health care, some traditional food and Chinese herbal medicine are widely used in plant functional food, such as herbal tea. The main materials are Glycyrrhiza, dregs leaves, honeysuckle, chrysanthemum, fairy grass, egg flower, and Prunella vulgaris. The development of these plant functional food has greatly expanded the population to the population. The exposure range of various plant chemicals. Plants contain abundant chemicals, including phytoestrogens and androgens, which are natural hormone substances in plants and have certain endocrine interference effects. Some plant functional foods use the endocrine intervention of plant hormones to obtain corresponding diseases. Anti - effects, such as soy isoflavones, prevent cancer, osteoporosis, and cardiovascular disease. But for a particular population, such as growing children, menopause women, breast or prostate cancers, the intake of these plant hormones may lead to potential risks. Hormone effects can promote the growth of breast cancer cells, but the endocrine disrupting effects on the raw materials of most other plant functional foods (beverages) are still lacking, especially in the androgenic effect. Therefore, it is necessary to have endocrine disrupting effects and whether it is necessary to eat these plant functional foods more safely. The mechanism is studied.
Objective: in this study, estrogen dependent breast cancer cells (MCF-7) and androgen dependent prostate cancer cells (22RV1) were used as a model to study the endocrine disrupting effects of plant functional food materials and to explore its mechanism.
Methods: the effects of extracts from plant functional food materials on cell activity were detected by MTS, and the effects of extracts on the target genes of estrogen receptor (ER) and androgen receptor (AR) were detected by RT-PCR, and the effects of extracts on the expression of ER A and ERK/1/2 protein were detected by Western blot, and the ER alpha and ERK1/2 proteins were in the pulp nucleus respectively. The expression of ER and ERK1/2 was detected by immunofluorescence. The localization of ER and ERK1/2 protein was detected by immunoprecipitation assay.
Results: eight plant extracts (Angelica, dregs leaf, licorice, honeysuckle, chrysanthemum, chrysanthemum, chrysanthemum, egg flower, Xia Kucao) were screened, and in estrogen dependent breast cancer cell MCF-7, the results of MTS experiment showed that the extract of Angelica sinensis could increase the activity of MCF-7 cells at a fixed concentration, and the activity could be ER Inhibition of alpha antagonist ICI182780 (ICI) and MEK1/2 inhibitor U0126, these results suggest that ER alpha and ERK1/2 signaling pathway participates in the increase in activity of extracts mediated by extracts; Western blot shows that Angelica sinensis and cloth residue leaves can reduce the level of ER alpha in MCF-7 cells, and can mediate the increase of ER target gene pS2 expression. Or after U0126, the pS2 level of the extract was obviously decreased; Angelica and dregs leaves could induce delayed ERK1/2 activation and have a time effect. When U0126 was added, the induction was blocked, and ICI had no effect on the activation of ERK1/2, which indicated that ERK1/ 2 activated the upstream of the signal pathway mediated by ER a; Angelica sinensis. And cloth residue leaves can promote the nuclear transposition of ER A and induce the binding of phosphorylated ERK1/2 and ER alpha protein. In the androgen dependent prostate cancer cell 22RV1, the extracts of three kinds of plant extracts from the leaf, the licorice and Angelica sinensis were screened. The residue leaf extract of the cloth can promote the activity of 22RV1 cells and can induce the androgen receptor. When the target gene PSA was expressed, the level of cell activity and androgen receptor target gene decreased obviously when the androgen receptor inhibitor was added, and the extract of Angelica and Glycyrrhiza Glycyrrhiza did not significantly change compared with the control. When the inhibitor U0126 was added to ERK1/2, the cell activity and the expression of PSA were also inhibited by the leaf extract of the dregs.
Conclusion: Angelica, the leaf of dregs has estrogen effect, and the leaf of the cloth also has androgen effect; the endocrine effect mediated by these extracts is mediated by ER alpha or AR; and the delayed activation of ERK1/2 is necessary for the estrogen effect of angelica, licorice and cloth residue leaves.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R173

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本文編號：1798181