【作者】 李峰；

【导师】 柳巨雄；

【作者基本信息】 吉林大学 ， 基础兽医学， 2022， 硕士

【摘要】 随着我国日益强盛,乳产业持续高速发展,国民对牛奶的需求也逐步攀升。如何进一步提升奶牛的泌乳性能和牛奶的品质以满足国民消费需求成为众多研究者关注的热点问题。奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)是奶牛乳腺中唯一具有产生和分泌乳汁功能的细胞。奶牛的产乳性能主要依靠BMECs增殖和合成乳汁的能力。乳蛋白和乳脂是牛奶中的主要营养成分,也被认为是评价奶牛产奶性能和牛奶质量的重要指标。BMECs中乳蛋白和乳脂的合成受环境、激素和生长因子等诸多因素的影响。脂肪酸(Fatty acids,FAs)是多种脂类合成的底物,也从信号传导、转录及翻译等多个层面参与调节机体内多种生物合成过程。硬脂酸(Stearic acid,SA)是一种含有18个碳的饱和长链脂肪酸,其作为瘤胃惰性脂肪酸,在调控奶牛泌乳方面具有巨大的潜力,并被作为饲料添加剂应用于奶牛养殖业。目前关于SA调控奶牛泌乳的研究主要集中在临床应用。然而,相关研究的结果不尽一致,且SA调控泌乳的具体机制也不清晰。因此,阐明SA对奶牛乳脂乳蛋白合成的影响,并揭示其潜在调控机制对奶牛养殖业合理应用SA调控奶牛泌乳、保障牛奶产业持续健康发展、提高我国牛奶产业在国际市场中的核心竞争力具有重要意义。为了阐明SA对奶牛乳脂乳蛋白合成的影响及潜在机制,本研究首先运用组织块接种法分离泌乳期荷斯坦奶牛的原代BMECs;通过差时消化法对分离的细胞进行纯化;并对上皮细胞标志蛋白角蛋白18(cytokeratin 18,CK-18)进行免疫荧光染色,鉴定本研究所获得细胞确为BMECs。使用不同浓度的SA(0、12.5、25、50、100、200μM)处理BMECs,结果发现该浓度范围内的SA对BMECs的活力没有显著影响,却显著提高了BMECs中β-casein和甘油三酯(triglycerides,TGs)的表达水平,在浓度为100μM时效果最为显著。调控机制的研究发现,SA显著激活BMECs中PI3K、m TOR和SREBP-1等信号分子的活性。为了明确这些信号分子在SA调控乳脂乳蛋白合成中的作用,m TOR的抑制剂——雷帕霉素(rapamycin,RAPA,200 n M)和PI3K的抑制剂——LY294002(15μM)分别被用来抑制m TOR和PI3K信号分子的活性。结果发现m TOR被抑制后,4EBP1、S6K和SREBP-1的活性显著降低,SA对BMECs乳脂乳蛋白合成的促进作用被阻断。抑制PI3K的活性后,SA对m TOR-4EBP1/S6K和m TOR-SREBP-1信号轴的激活作用显著减弱,这表明PI3K在SA刺激BMECs激活m TOR、SREBP-1的信号传导中具有关键作用。之前的研究发现细胞周期蛋白依赖性激酶1(cyclin-dependent kinases 1,CDK1)通过影响翻译因子的活性参与调控蛋白质合成。在本研究中SA显著上调了BMECs中CDK1的蛋白水平。因此,本研究构建了CDK1-si RNA和CDK1-pc DNA,探索其在SA调控BMECs乳脂乳蛋白合成中的作用。结果显示CDK1缺失后阻断了SA对BMECs中上述信号通路的激活及促进乳脂乳蛋白合成的作用;而过表达CDK1后,即便没有SA的刺激,PI3K-m TOR-4EBP1/S6K和m TOR-SREBP-1信号轴也被显著激活。这些结果均提示CDK1先于上述信号分子响应SA的刺激,对BMECs的乳脂乳蛋白合成具有积极作用。此外,SA属于长链脂肪酸(long chain fatty acids,LCFAs),需要转运蛋白的协助进入细胞。因此,本研究检测了在SA的刺激下,BMECs中脂肪酸转运蛋白(Fatty acid transport proteins,FATPs)的表达变化。结果发现,当使用100μM的SA刺激BMECs时,FATPs中FATP3和FATP4的m RNA水平被显著上调,并发现FATP4的m RNA基础表达丰度最高。以往的众多研究表明FATP3主要负责调控长链脂酰辅酶A合成酶的功能,转运LCFAs的能力较弱,而FATP4转运LCFAs的能力远高于FATP3,是主要的LCFAs转运蛋白。因此,本研究重点关注了FATP4,通过构建FATP4-si RNA,检测其对SA调控BMECs中乳脂乳蛋白合成的影响。结果显示FATP4缺失后,SA上调BMECs中CDK1的蛋白水平和PI3K-m TOR-4EBP1/S6K和m TORSREBP-1信号轴活性的作用被阻断,对乳脂乳蛋白合成的促进作用消失。这表明FATP4是介导SA跨膜转运进入BMECs的主要转运子。综上所述,本研究发现SA促进BMECs中乳脂乳蛋白的表达,明确了SA对奶牛泌乳具有积极影响;揭示了SA被FATP4转运入BMECs,上调CDK1的蛋白水平,继而激活PI3K-m TOR-4EBP1/S6K和m TOR-SREBP-1信号轴,最终促进BMECs中β-casein和TGs的合成。本研究的发现为奶牛养殖业合理使用SA调控奶牛泌乳提供了理论支撑,对我国牛奶产业合理健康发展具有重要意义。更多还原

【Abstract】 With the growing prosperity of my country and the continuous rapid development of the dairy industry,the national demand for milk has gradually increased.How to further improve the lactation performance of dairy cows and the quality of milk to meet the needs of national consumption has become a hot issue concerned by many researchers.Bovine mammary epithelial cells(BMECs)are the only cells in the mammary gland that have the function of producing and secreting milk.The milk production performance of dairy cows mainly depends on the ability of BMECs to proliferate and synthesize milk.Milk fat and milk protein are the main nutrients in milk and are also considered as important indicators for evaluating milk production performance and milk quality of dairy cows.The synthesis of milk fat and milk protein in BMECs is affected by many factors such as environment,hormone,growth factor and so on.Fatty acids(FAs)are substrates for the synthesis of various lipids,and also participate in the regulation of various biosynthetic processes in the body from multiple levels such as signal transduction,transcription,and translation.Stearic acid(SA)is a saturated long-chain fatty acid containing 18 carbons.As a rumen inert fatty acid,SA has great potential in regulating the lactation of dairy cows,and is often used as a feed additive in the dairy industry.At present,the research on the regulation of dairy cow lactation by SA is mainly focused on clinical application.However,the results of related studies are not consistent,and the specific mechanism of SA regulating lactation is not clear.Therefore,to clarify the effect of SA on synthesis of milk fat and mlik protein and reveal its potential regulation mechanism is of great significance for the dairy industry to reasonably apply SA to regulate cow lactation,ensure the sustained and healthy development of the milk industry,and improve the core competitiveness of China’s milk industry in the international market.In order to clarify the effect and its potential mechanism of SA on synthesis of milk fat and milk protein,the primary BMECs of Holstein cows during lactation was isolated by tissue block inoculation method,the isolated cells were purified by differential digestion method,and cytokeratin18(CK-18)of the epithelial cell marker protein was stained with immunofluorescence to identify that the cells obtained in this study were indeed BMECs.Then,BMECs was treated with different concentrations of SA(0,12.5,25,50,100,200 μM).The results showed that SA within this concentration range had no significant effect on the viability of BMECs,but significantly increased the expression levels of β-casein and triglycerides(TGs)in BMECs,with the most significant effect at a concentration of 100 μM,which confirmed the positive effect of SA on lactation of dairy cows.The subsequent mechanism study found that SA significantly activated the activity of signal molecules such as PI3 K,m TOR and SREBP-1 in BMECs.In order to clarify the role of these signal molecules in the regulation of synthesis of milk fat and milk protein by SA,m TOR inhibitors-rapamycin(RAPA,200 n M)and PI3 K inhibitorsLY294002(15 μM)were used to inhibit the activity of m TOR and PI3 K signal molecules,respectively.The results showed that after m TOR was inhibited,the activities of 4EBP1,S6 K and SREBP-1 were significantly reduced,and the promoting effect of SA on synthesis of milk fat milk protein in BMECs was significantly blocked.After inhibiting the activity of PI3 K,the activation of m TOR-4EBP1/S6 K and m TORSREBP-1 signaling axes by SA was significantly attenuated,indicating that PI3 K plays a key role in SA-stimulated BMECs to activate m TOR and SREBP-1 signaling.Previous studies have found that cyclin-dependent kinase 1(CDK1)participates in the regulation of protein synthesis by affecting the activity of translation factors.In this study,SA significantly up-regulated the protein level of CDK1 in BMECs.Therefore,in this study,CDK1-si RNA and CDK1-pc DNA were constructed to explore the role in the regulation of synthesis of milk fat milk protein in BMECs by SA.The results showed that CDK1 deletion blocked the activation of SA on the above signal pathways and the synthesis of milk protein and milk fat;after overexpression of CDK1,even without SA stimulation,PI3K-m TOR-4EBP1/S6 K and m TOR-SREBP-1 signal axes were significantly activated.These results suggest that CDK1 responds to the stimulation of SA before the above signal molecules and plays a positive role in the milk synthesis of BMECs.In addition,SA belongs to long-chain fatty acids(LCFAs)and needs the assistance of transporters to enter cells.Therefore,this study examined the expression of fatty acid transport proteins(FATPs)in BMECs stimulated by SA.It was found that the m RNA levels of FATP3 and FATP4 in FATPs were significantly upregulated when BMECs were stimulated with 100 μM SA,and the basal m RNA expression abundance of FATP4 was found to be the highest.Many previous studies have shown that FATP3 is mainly responsible for regulating the function of long-chain fatty acyl-Co A synthase,and has a weak ability to transport LCFAs,while FATP4 has a much higher ability to transport LCFAs than FATP3,and is the main LCFAs transporter.Therefore,this study focused on FATP4,through the construction of FATP4-si RNA to detect the effect of SA on the regulation of synthesis of milk fat and milk protein BMECs.The results showed that in the absence of FATP4,the effect of SA on up-regulating CDK1 protein level and PI3K-m TOR-4EBP1/S6 K and m TORSREBP-1 signaling axis activities in BMECs was blocked,and the promoting effect on synthesis of milk fat and milk protein disappeared.This suggests that FATP4 is the main transporter mediating the transmembrane transport of SA into BMECs.To sum up,this study found that SA promotes the expression of milk fat and milk protein in BMECs,which confirms that SA has a positive effect on the lactation of dairy cows,and revealed that SA is transported into BMECs through FATP4,and activates PI3K-m TOR-4EBP1/S6 K and m TOR-SREBP-1 signal axis by up-regulated the protein level of CDK1,promotes the synthesis of β-casein and TGs in BMECs.The findings of this study provide a theoretical support for the rational use of SA in dairy industry to regulate cow lactation,and are of great significance to the rational and healthy development of dairy industry in my country.更多还原