脂噬对奶牛乳腺上皮细胞脂滴大小的调控研究

Regulation of Lipophagy on the Size of Lipid Droplets in Bovine Mammary Epithelial Cells

脂噬是一种新的自噬形式,可以选择性识别脂滴并将其降解,在维持细胞内脂质稳态等过程中发挥着重要的作用。本研究旨在探讨奶牛乳腺上皮细胞经饥饿诱导脂噬后对细胞脂滴大小的调控作用。试验用100μmol·L^(-1)的亚油酸处理细胞24 h,刺激细胞脂滴蓄积,然后分别饥饿诱导细胞0、6、12、24、48 h,油红O染色观察细胞内脂滴的尺寸和数量随饥饿时间的变化情况,随后饥饿诱导24 h通过免疫荧光观察脂滴和自噬蛋白LC3的共定位,蛋白免疫印迹检测细胞内自噬蛋白LC3和P62的表达,透射电镜观察脂滴自噬情况。结果发现:随着饥饿处理时间的延长,脂滴直径从1.57μm显著增加到2.12μm,每个细胞的脂滴数量显著减少,从39个·cell^(-1)减少到24个·cell^(-1),大脂滴比例显著增加,小脂滴比例显著减少(P<0.05),免疫荧光发现LC3与脂滴发生共定位,透射电镜观察发现自噬溶酶体包裹着脂滴,饥饿处理24 h后LC3Ⅱ/LC3Ⅰ蛋白的比值显著增加1倍,P62蛋白表达显著下降(P<0.05)。以上结果表明,脂噬对奶牛乳腺上皮细胞脂滴的大小发挥调控作用。

Lipophagy is a new form of autophagy,which can selectively recognize and degrade lipid droplets,and plays an important role in maintaining intracellular lipid homeostasis.The aim of this study was to investigate the effect of starvation induced lipophagy on the size of lipid droplets in bovine mammary epithelial cells.The cells were treated with 100μmol·L^(-1) linoleic acid for 24 h to establish a lipid accumulation cell model,and then the cells were starved for 0,6,12,24,and 48 h,respectively.Oil red O staining was used to observe the changes in the size and number of intracellular lipid droplets with starvation time.Immunofluorescence was used to observe the co-localization of lipid droplets and autophagy protein LC3.Western blot was used to detect the expression of autophagy-related proteins LC3 and P62.The results showed that the diameter of lipid droplets significantly increased from 1.57μm to 2.12μm,the number of lipid droplets per cell significantly decreased from 39 to 24·cell^(-1),the proportion of large lipid droplets significantly increased,and the proportion of small lipid droplets significantly decreased with the extension of starvation treatment time(P<0.05),immunofluorescence showed that LC3 co-localized with lipid droplets,and transmission electron microscopy showed that lipid droplets were encapsulated in autolysosomes.After 24 hours of starvation,the ratio of LC3Ⅱ/LC3Ⅰprotein increased by 1 fold,and the expression of P62 protein decreased significantly(P<0.05).These results suggest that lipophagy plays a regulatory role in the size of lipid droplets in the bovine mammary epithelium.