Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs
Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs
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DOI：
10.1016/j.bbrc.2010.04.135
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发表时间：
2010-05-28
影响因子：
3.1
通讯作者：
Aoki, Naohito
中科院分区：
文献类型：
--
作者：
Hata, Taketoshi;Murakami, Kosuke;Aoki, Naohito
通过一系列的离心法和超速离心法，我们可以从牛乳中分离出直径约为100 nm的微囊。我们还发现，从6毫升初乳和成熟乳中分离出的微囊中分别含有约1700和1000 ng的总RNA，其中小RNA是主要成分，尽管牛奶中的核糖核酸酶活性很高。微囊中存在主要乳蛋白和翻译延伸因子-1α(EF-1α)的多聚腺苷化基因转录本，部分转录本的完整性通过实时定量聚合酶链式反应和体外翻译分析得到证实。此外，乳源微泡中还含有大量的乳腺和免疫相关的microRNAs。酸化牛奶以模拟胃肠道对RNA的产量和质量影响不大。当与乳源微囊孵育时，在培养细胞中检测到与乳汁相关的基因转录，这表明细胞摄取了包括RNA在内的微囊内容物。我们的发现表明，牛母乳中含有能够转移到活细胞中的RNA，并参与了小牛胃肠道和免疫系统的发育。(C)2010 Elsevier Inc.保留所有权利。
By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1 alpha (EF-1 alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems. (C) 2010 Elsevier Inc. All rights reserved.