Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.
Quantitation of Exosomes and Their MicroRNA Cargos in Frozen Human Milk.
复制标题
DOI：
10.1097/pg9.0000000000000172
复制
发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
我们评估了分析冷冻人乳中的外来体和microRNA货物的可行性，作为乳汁外来体流行病学研究的先决条件。我们在产后医院护理期间的3个时间点收集了5对母亲-早产儿的乳汁，储存在-80 °C下。我们通过超离心纯化外泌体，使用免疫印迹探测标记蛋白，用纳米颗粒追踪器评估大小和计数，并用定量PCR定量3种microRNA。可检测到阳性外泌体标志物蛋白; β-酪蛋白是唯一可检测的污染物。从早期到晚期样品，外泌体计数和大小呈下降趋势（计数，2.3 × 109 ± 3.8 × 109至5.6 × 108 ± 9.7 × 108外泌体/mL;大小，117 ± 25至92 ± 16 nm）。仅在早期样本中可检测到两种microRNA; miR-30 d-5 p和miR-125 a-5 p的循环阈值等于28.7 ± 0.7; miR-423- 5 p不可检测。我们的结论是，外来体的分析和microRNA的定量在先前储存在-80 °C的人乳中是可行的。
We assessed feasibility of analyzing exosomes and microRNA cargos in frozen human milk as a prerequisite for epidemiological studies of milk exosomes. We collected milk from 5 mother-preterm infant dyads at 3 time points during postnatal hospital care for storage at −80 °C. We purified exosomes by ultracentrifugation, probed marker proteins using immunoblots, assessed size and counts with a nanoparticle tracker, and quantified 3 microRNAs with quantitative PCR. Positive exosome marker proteins were detectable; β-casein was the only detectable contaminant. Exosome count and size trended to decrease from early to late samples (count, 2.3 × 109 ± 3.8 × 109 to 5.6 × 108 ± 9.7 × 108 exosomes/mL; size, 117 ± 25 to 92 ± 16 nm). Two microRNAs were detectable in early samples only; cycle threshold values equaled 28.7 ± 0.7 for miR-30d-5p and miR-125a-5p; miR-423-5p was not detectable. We conclude that the analysis of exosomes and quantification of microRNAs is feasible in human milk previously stored at −80 °C.