Ghrelin对奶牛乳腺上皮细胞β-酪蛋白合成的影响及其机理的研究

发布时间：2017-12-31 10:24

  本文关键词：Ghrelin对奶牛乳腺上皮细胞β-酪蛋白合成的影响及其机理的研究　出处：《吉林大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 奶牛乳腺上皮细胞 酰化的ghrelin CSN2 非酰化ghrelin

【摘要】：乳蛋白是构成牛奶营养品质的主要物质基础,乳蛋白的含量既关系牛奶的质量与消费者的健康,同时又是奶业核心竞争力的重要标志。酰化(acylated ghrelin,AG)和非酰化(unacylated ghrelin,UAG)是ghrelin主要的两种生物活性形式。有研究表明除了调节GH分泌、摄食、炎症、能量代谢等重要生命活动之外,AG还能调节体内或体外培养的不同动物乳腺上皮细胞(mammary epithelial cells,MECs)和乳腺组织块中β-酪蛋白(β-casein,CSN2)的表达,然而其具体机制目前还不清楚。在奶牛血清中,UAG的浓度比AG高很多倍。与AG不同,UAG对MECs和乳腺组织中CSN2表达的影响目前还没有相关研究。此外,革兰氏阴性菌引起的乳房炎破坏乳腺组织中正常乳汁的生成。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,在本论文中,我们利用分离培养的奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)、乳腺组织块以及LPS诱导的乳房炎细胞模型,系统研究了AG或UAG对CSN2合成的影响及机制。首先通过组织块培养法分离培养了BMECs,免疫荧光和流式细胞术结果表明分离的BMECs纯度高达99%。在分离的BMECs中稳定表达各种乳蛋白、AG及其受体,同时细胞生长曲线呈典型的“S”型,这些结果证明分离的BMECs可用于后续试验。用不同浓度的AG或UAG(0-100 ng/m L)处理BMECs不同时间点后发现,与NT组相比,AG和UAG显著促进BMECs中CSN2基因和蛋白的表达,而GHSR1a(ghrelin特异性受体)和Gαs蛋白的特异性抑制剂显著抑制AG或UAG的作用。AG或UAG通过GHSR1a和Gαs蛋白激活BMECs中ERK1/2、AKT和JNK信号通路,而抑制ERK1/2、AKT和JNK的磷酸化则抑制AG或UAG诱导的CSN2合成。此外,AG或UAG还通过上述通路促进BMECs的增殖。最后,AG或UAG也通过上述通路促进乳腺组织中CSN2的表达。这些结果说明AG和UAG激活GHSR1a,活化的GHSR1a与Gαs蛋白耦合,通过ERK1/2、AKT和JNK通路促进细胞增殖,进而增加CSN2的合成。最后我们检测了AG或UAG对LPS诱导的乳房炎细胞模型中CSN2合成影响。结果发现与NT组相比,LPS能显著增加炎性因子的表达,然而抑制CSN2的表达。进一步研究发现AG或UAG能缓解LPS诱导的炎症,AG或UAG的抗炎作用是通过GHSR1a和Gαs蛋白介导的ERK1/2和AKT信号通路发挥的。在LPS诱导的乳房炎细胞模型中,AG或UAG促进LPS诱导的BMECs中CSN2表达,抑制ERK1/2和AKT的磷酸化则抑制AG或UAG促CSN2表达的作用;LPS显著诱导BMECs中GHSR1a表达,而GHSR1a和Gαs蛋白的特异性抑制剂封闭ERK1/2和AKT的磷酸化,抑制CSN2的表达。AG和UAG还通过p53/Bax/Bcl-2通路抑制LPS诱导的细胞凋亡,增加CSN2的表达。这些结果说明AG或UAG能通过GHSR1a依赖的方式抑制LPS诱导的乳房炎细胞模型中炎性因子的产生,同时AG和UAG还通过p53/Bax/Bcl-2通路抑制LPS诱导的细胞凋亡,最终增加CSN2的表达。综上所述,这些结果证明AG和UAG通过GHSR1a和Gαs蛋白介导的ERK1/2、AKT或JNK通路诱导体外培养的BMECs中CSN2的表达;并且这一结果在体外培养的乳腺组织块得到了进一步的验证。AG或UAG能通过GHSR1a依赖的ERK1/2和AKT通路抑制LPS诱导的乳房炎细胞模型中炎性因子的产生,增加CSN2的表达;同时AG和UAG还通过p53/Bax/Bcl-2通路抑制LPS诱导的细胞凋亡,从而促进CSN2的表达。这些结果为深入研究乳蛋白合成调控机制及乳房炎的防治提供了科学依据,为通过人工干预手段来提高牛奶营养品质奠定了理论基础。
[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.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823
，

本文编号：1359360