本文關鍵詞：Ghrelin對奶牛乳腺上皮細胞β-酪蛋白合成的影響及其機理的研究　出處：《吉林大學》2016年博士論文　論文類型：學位論文

  更多相關文章： 奶牛乳腺上皮細胞 �；膅hrelin CSN2 非�；痝hrelin

【摘要】：乳蛋白是構成牛奶營養(yǎng)品質(zhì)的主要物質(zhì)基礎,乳蛋白的含量既關系牛奶的質(zhì)量與消費者的健康,同時又是奶業(yè)核心競爭力的重要標志。�；�(acylated ghrelin,AG)和非酰化(unacylated ghrelin,UAG)是ghrelin主要的兩種生物活性形式。有研究表明除了調(diào)節(jié)GH分泌、攝食、炎癥、能量代謝等重要生命活動之外,AG還能調(diào)節(jié)體內(nèi)或體外培養(yǎng)的不同動物乳腺上皮細胞(mammary epithelial cells,MECs)和乳腺組織塊中β-酪蛋白(β-casein,CSN2)的表達,然而其具體機制目前還不清楚。在奶牛血清中,UAG的濃度比AG高很多倍。與AG不同,UAG對MECs和乳腺組織中CSN2表達的影響目前還沒有相關研究。此外,革蘭氏陰性菌引起的乳房炎破壞乳腺組織中正常乳汁的生成。然而,在乳房炎中乳蛋白的合成是如何被抑制的目前也不清楚。因此,在本論文中,我們利用分離培養(yǎng)的奶牛乳腺上皮細胞(bovine mammary epithelial cells,BMECs)、乳腺組織塊以及LPS誘導的乳房炎細胞模型,系統(tǒng)研究了AG或UAG對CSN2合成的影響及機制。首先通過組織塊培養(yǎng)法分離培養(yǎng)了BMECs,免疫熒光和流式細胞術結果表明分離的BMECs純度高達99%。在分離的BMECs中穩(wěn)定表達各種乳蛋白、AG及其受體,同時細胞生長曲線呈典型的“S”型,這些結果證明分離的BMECs可用于后續(xù)試驗。用不同濃度的AG或UAG(0-100 ng/m L)處理BMECs不同時間點后發(fā)現(xiàn),與NT組相比,AG和UAG顯著促進BMECs中CSN2基因和蛋白的表達,而GHSR1a(ghrelin特異性受體)和Gαs蛋白的特異性抑制劑顯著抑制AG或UAG的作用。AG或UAG通過GHSR1a和Gαs蛋白激活BMECs中ERK1/2、AKT和JNK信號通路,而抑制ERK1/2、AKT和JNK的磷酸化則抑制AG或UAG誘導的CSN2合成。此外,AG或UAG還通過上述通路促進BMECs的增殖。最后,AG或UAG也通過上述通路促進乳腺組織中CSN2的表達。這些結果說明AG和UAG激活GHSR1a,活化的GHSR1a與Gαs蛋白耦合,通過ERK1/2、AKT和JNK通路促進細胞增殖,進而增加CSN2的合成。最后我們檢測了AG或UAG對LPS誘導的乳房炎細胞模型中CSN2合成影響。結果發(fā)現(xiàn)與NT組相比,LPS能顯著增加炎性因子的表達,然而抑制CSN2的表達。進一步研究發(fā)現(xiàn)AG或UAG能緩解LPS誘導的炎癥,AG或UAG的抗炎作用是通過GHSR1a和Gαs蛋白介導的ERK1/2和AKT信號通路發(fā)揮的。在LPS誘導的乳房炎細胞模型中,AG或UAG促進LPS誘導的BMECs中CSN2表達,抑制ERK1/2和AKT的磷酸化則抑制AG或UAG促CSN2表達的作用;LPS顯著誘導BMECs中GHSR1a表達,而GHSR1a和Gαs蛋白的特異性抑制劑封閉ERK1/2和AKT的磷酸化,抑制CSN2的表達。AG和UAG還通過p53/Bax/Bcl-2通路抑制LPS誘導的細胞凋亡,增加CSN2的表達。這些結果說明AG或UAG能通過GHSR1a依賴的方式抑制LPS誘導的乳房炎細胞模型中炎性因子的產(chǎn)生,同時AG和UAG還通過p53/Bax/Bcl-2通路抑制LPS誘導的細胞凋亡,最終增加CSN2的表達。綜上所述,這些結果證明AG和UAG通過GHSR1a和Gαs蛋白介導的ERK1/2、AKT或JNK通路誘導體外培養(yǎng)的BMECs中CSN2的表達;并且這一結果在體外培養(yǎng)的乳腺組織塊得到了進一步的驗證。AG或UAG能通過GHSR1a依賴的ERK1/2和AKT通路抑制LPS誘導的乳房炎細胞模型中炎性因子的產(chǎn)生,增加CSN2的表達;同時AG和UAG還通過p53/Bax/Bcl-2通路抑制LPS誘導的細胞凋亡,從而促進CSN2的表達。這些結果為深入研究乳蛋白合成調(diào)控機制及乳房炎的防治提供了科學依據(jù),為通過人工干預手段來提高牛奶營養(yǎng)品質(zhì)奠定了理論基礎。
[Abstract]:Milk protein is an important material basis for the nutritional quality of milk, milk protein content and quality of consumers is not only related to the health of milk, but also is an important symbol of the core competitiveness of the dairy industry. Acylation (acylated ghrelin, AG) and non acylated (unacylated ghrelin, UAG ghrelin) is the main form of two biological activity. Studies have shown that in addition to regulating the secretion of GH, feeding, inflammation, other major life activities of energy metabolism, AG can also regulate the cultured in vitro or in vivo in different animal mammary epithelial cells (mammary epithelial cells, MECs) and breast tissue in beta casein (beta -casein, CSN2) expression, but its mechanism is not sure. In serum, the concentration of UAG many times higher than the AG. Unlike AG, UAG and MECs effect on the expression of CSN2 in breast tissue has not been studied yet. In addition, gram negative bacteria cause mastitis broken The formation of normal milk bad breast tissue. However, in the synthesis of mastitis milk protein is to be suppressed is still unclear. Therefore, in this thesis, we use the cultured bovine mammary epithelial cells (bovine mammary epithelial cells, BMECs), mastitis mammary tissue and cell model induced by LPS. Study on the system and mechanism of AG or UAG effect on CSN2 synthesis. Firstly through tissue culture method isolated BMECs, immunofluorescence and flow cytometry showed that the isolated BMECs purity of 99%. in isolated BMECs stable expression of milk protein, AG and its receptor, and the cell growth curve showed a typical "S" type, these results demonstrate that BMECs can be used to separate the follow-up test. With different concentrations of AG or UAG (0-100 ng/m L) BMECs treatment at different time points after that, compared with NT, AG and UAG significantly promoted BMECs The expression of CSN2 gene and protein, and GHSR1a (ghrelin receptor) and G specific inhibitor of alpha s protein significantly inhibited AG or UAG.AG or UAG by GHSR1a and G alpha s protein activated BMECs ERK1/2, AKT and JNK signaling pathway, inhibition of ERK1/2, AKT and JNK phosphorylation is inhibition of CSN2 synthesis induced by AG or UAG. In addition, AG or UAG also promote BMECs through the pathway of proliferation. Finally, AG or UAG also promotes the expression of CSN2 in breast tissue through the pathway. These results indicate that AG and UAG activate GHSR1a, GHSR1a and G s protein coupling, activated by ERK1/2, promote cell the proliferation of AKT and JNK pathway, thereby increasing the synthesis of CSN2. Finally, we tested the effect of AG or UAG on the synthesis of CSN2 cell mastitis model induced by LPS. The results showed that compared with the NT group, LPS significantly increased the expression of inflammatory factors, however, further research on the inhibition of CSN2 expression. AG or UAG can alleviate LPS induced inflammation and anti-inflammatory effects of AG or UAG is ERK1/2 by GHSR1a and G s protein mediated and AKT signaling pathway. In cell mastitis model induced by LPS, AG or UAG promoted CSN2 expression induced by LPS in BMECs, inhibited ERK1/2 and AKT phosphorylation AG inhibited the expression of CSN2 or UAG to promote the role of the expression of GHSR1a in BMECs; LPS was induced, and specific inhibitor GHSR1a and G alpha s protein ERK1/2 and AKT phosphorylation, the expression of.AG and UAG inhibit CSN2 cell apoptosis through inhibition of p53/Bax/Bcl-2 pathway induced by LPS and increase the expression of CSN2. These results indicate that inflammatory factors in mastitis cell model of AG or UAG by GHSR1a dependent inhibition of LPS induced the generation, while AG and UAG through apoptosis inhibition of p53/Bax/Bcl-2 pathway induced by LPS, and ultimately increase the expression of CSN2. In summary, these. The result showed AG and UAG by GHSR1a and G s protein mediated ERK1/2 expression of CSN2, AKT or JNK signaling pathway induced by BMECs cultured in vitro; and the results of inflammatory factors in mastitis cell model of ERK1/2 and AKT pathway further validation of.AG or UAG by GHSR1a dependent inhibition induced by LPS in in cultured mammary tissue, increase the expression of CSN2 and AG; and UAG apoptosis through p53/Bax/Bcl-2 pathway inhibition induced by LPS, thus promoting the expression of CSN2. These results provide a scientific basis for the prevention and treatment of in-depth study of the mechanism of milk protein synthesis and regulation of mastitis, through artificial intervention means to improve the nutrition of milk quality has laid a theoretical foundation.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S823
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本文編號：1359360