论文部分内容阅读
摘 要：脂多糖（Lipopolysaccharide LPS）能调控mTOR信号通路影响炎症反应及乳蛋白的合成。而L-精氨酸（L-Arg）可以经Arg-NO途径及mTOR途径发挥免疫作用并促进蛋白质的沉积。该试验采用LPS刺激中国荷斯坦奶牛乳腺上皮细胞建立免疫模型，研究应激条件下L-精氨酸（L-Arg）对奶牛乳腺上皮细胞炎症反应及β-酪蛋白基因表达的影响，旨在探讨L-Arg能否缓解乳腺上皮细胞的免疫应激作用。复苏2代泌乳奶牛乳腺上皮细胞，待长满90%经消化后调整密度接种于6孔板。细胞贴壁后饥饿培养16 h，分为四组（每组3个重复，每个孔为一个重复）分别用DMEM/F12培养基（对照组），终浓度为100 g/LL-Arg、10 g/LLPS、100 g/LL-Arg+10g/LLPS的DMEM/F12培养基培养细胞，分别于12 h、24 h提取细胞总RNA。以β-actin为内参采用Real-time PCR法检测Toll样受体4（TLR4），细胞核因子（NFκB），白介素-1（IL-1β），白介素-6（IL-6），诱导型一氧化氮合成酶（iNOS），哺乳动物雷帕霉素靶蛋白（mTOR），β-酪蛋白（CSN2）mRNA的相对表达。12 h时与对照组相比较，添加LPS组能显著上调TLR4、NFκB、IL-1β、IL-6、iNOS、mTOR的表达（P<0.05）；而与LPS组相比，添加L-Arg+LPS组中TLR4，mTOR的表达显著降低（P<0.05）。24 h时与对照组相比，添加L-Arg组中mTOR的上调差异显著（P<0.05），添加LPS组NFκB、IL-1β、IL-6、iNOS的表达上调仍然显著（P<0.05），而mTOR的表达差异不显著（P>0.05），CSN2的表达显著降低（P<0.05）；与L-Arg组相比添加L-Arg+LPS组中TLR4、NFκB、IL-1β、IL-6、iNOS、mTOR表达显著上调（P<0.05），而CSN2的表达差异不显著（P>0.05）；与LPS组相比添加L-Arg+LPS组中TLR4、NFκB及炎症因子IL-1β、IL-6的表达显著降低（P<0.05），β-酪蛋白基因CSN2的表达显著增加（P<0.05）。LPS刺激泌乳奶牛乳腺上皮细胞促进了炎症因子的表达，并降低β-酪蛋白的表达，而L-Arg能缓解乳腺上皮细胞的炎症反应及β-酪蛋白表达的抑制作用。
关键词：乳腺上皮细胞 精氨酸 脂多糖 免疫 酪蛋白
Effect of L-Arg on Inflammatory Response and Beta-casein Expression when LPS Chanllenged Bovine Mammary Epithelial Cell
Wu Tianyou Wang Hongrong
（Yangzhou University）
Abstract：According to the recent researches， Lipopolysaccharide （LPS） is involved in the the mammalian target of rapamycin （mTOR） pathway to affect the activity of inflammatory response and regulation of protein synthesis in the mammary gland. Arginine （L-Arg）regulated the immune response via Arg/NO pathway and promoted the deposits of protein thought mTOR pathway.The objective of this study was to investigate the effect of L-Arg on inflammatory response and gene express of beta-casein （β-CN） during LPS chanllenge of bovine mammary epithelial cell （bMEC）. Primary culture of bMEC was induced by LPS to establish inflammatory model. When cells were grown to 90% confluence in 6 well plates. After 16h starve culture， cells were divided into 4 treatments： （1） group of control， DMEM/F12 culture medium 2ml； （2） group of Arg， 100 g/L L-Arg 2ml； （3） group of LPS， 10g/L LPS 2ml； （4） group of Arg +LPS， 100 g/L L-Arg+10g/L LPS. The total RNA was extracted after 12 h and 24 h.and the mRNA express levels of Toll-Like Receptor 4 （TLR-4）， Nuclear factor kappa B （NF-κB）， Interlukin 1（IL-1β）， Interlukin 6 （IL-6）， Inducible nitric oxide synthase（ iNOS）， mTOR， CSN2 were evaluated by real-time quantitative PCR. The results indicated that mRNA expression of TLR4， NFκB， IL-1β， IL-6， iNOS， mTOR increased to their highest values （P<0.05） at 12 h after LPS challenge， and the same as 24 h except for mTOR was slight up-regulate. Expression of CSN2 was decreased （P<0.01） at 12 h， but no significant change at 24 h. Expression of mTOR in group of Arg was elevated at 24 h when compare with the control quarters. Decreased （P<0.01） gene expression of TLR4， NFκB， IL-1β， IL-6， and increased of CSN2 in Arg +LPS treated quarters when compare with LPS-treated quarters. The results of this study demonstrate that inflammatory reaction was induced， and casein were decreased when LPS chanllenged bovine mammary epithelial cell. L-Arg would relieve those symptom.
Key Words：Bovine mammary epithelial cell；Arginine；LPS；Immune；Casein
阅读全文链接（需实名注册）：http：//www.nstrs.cn/xiangxiBG.aspx？id=87934&flag=1
关键词：乳腺上皮细胞 精氨酸 脂多糖 免疫 酪蛋白
Effect of L-Arg on Inflammatory Response and Beta-casein Expression when LPS Chanllenged Bovine Mammary Epithelial Cell
Wu Tianyou Wang Hongrong
（Yangzhou University）
Abstract：According to the recent researches， Lipopolysaccharide （LPS） is involved in the the mammalian target of rapamycin （mTOR） pathway to affect the activity of inflammatory response and regulation of protein synthesis in the mammary gland. Arginine （L-Arg）regulated the immune response via Arg/NO pathway and promoted the deposits of protein thought mTOR pathway.The objective of this study was to investigate the effect of L-Arg on inflammatory response and gene express of beta-casein （β-CN） during LPS chanllenge of bovine mammary epithelial cell （bMEC）. Primary culture of bMEC was induced by LPS to establish inflammatory model. When cells were grown to 90% confluence in 6 well plates. After 16h starve culture， cells were divided into 4 treatments： （1） group of control， DMEM/F12 culture medium 2ml； （2） group of Arg， 100 g/L L-Arg 2ml； （3） group of LPS， 10g/L LPS 2ml； （4） group of Arg +LPS， 100 g/L L-Arg+10g/L LPS. The total RNA was extracted after 12 h and 24 h.and the mRNA express levels of Toll-Like Receptor 4 （TLR-4）， Nuclear factor kappa B （NF-κB）， Interlukin 1（IL-1β）， Interlukin 6 （IL-6）， Inducible nitric oxide synthase（ iNOS）， mTOR， CSN2 were evaluated by real-time quantitative PCR. The results indicated that mRNA expression of TLR4， NFκB， IL-1β， IL-6， iNOS， mTOR increased to their highest values （P<0.05） at 12 h after LPS challenge， and the same as 24 h except for mTOR was slight up-regulate. Expression of CSN2 was decreased （P<0.01） at 12 h， but no significant change at 24 h. Expression of mTOR in group of Arg was elevated at 24 h when compare with the control quarters. Decreased （P<0.01） gene expression of TLR4， NFκB， IL-1β， IL-6， and increased of CSN2 in Arg +LPS treated quarters when compare with LPS-treated quarters. The results of this study demonstrate that inflammatory reaction was induced， and casein were decreased when LPS chanllenged bovine mammary epithelial cell. L-Arg would relieve those symptom.
Key Words：Bovine mammary epithelial cell；Arginine；LPS；Immune；Casein
阅读全文链接（需实名注册）：http：//www.nstrs.cn/xiangxiBG.aspx？id=87934&flag=1