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| 健康成人24 h尿液细胞外囊泡定量及大小分布 | |
| 引用本文： | 林韩翡,刘新宇,徐小蒙,Luca Musante,Harry Holthofer,邹和群. 健康成人24 h尿液细胞外囊泡定量及大小分布[J]. 南方医科大学学报, 2015, 35(11): 1530 |
| 作者姓名： | 林韩翡 刘新宇 徐小蒙 Luca Musante Harry Holthofer 邹和群 |
| 摘 要： | 目的研究观察健康成人24 h尿液细胞外囊泡（uEVs）数量、大小分布及个体间差异。方法利用液压透析滤过法分离9名 健康成年人24 h uEVs，通过western blot（WB）和透射电镜（TEM）技术验证uEVs的分离效果，应用BCA蛋白定量法及纳米微粒 跟踪分析技术分别对富集的uEVs样品进行蛋白定量测定和囊泡数量及大小分布的分析。结果TEM下可见大小不等、形状不 一的囊泡。WB技术检测到外泌体的标记物—人肿瘤易感基因（TSG101）。9名健康成人24 h尿液分离的uEVs样品蛋白定量 范围为132.50~760.70 ng/mL。纳米微粒跟踪分析技术检测结果显示24 h uEVs数量为（3.56~5.12）×1012个，CV=14.23%。24 h 分泌的uEVs中，直径在<40 nm范围内的囊泡占所检测到的总囊泡数的0.04~0.69%，数量为（1.80~26.49）×109 个；符合外泌体 直径范围（40~100 nm）的囊泡占总囊泡数的22.07%~42.08%，数量为（1.00~1.77）×1012个。符合微囊泡直径范围（100~1000 nm） 的囊泡约占总囊泡数的57.88%~77.85%，数量为（2.09~3.86）×1012个。结论新型液压透析滤过法能高效、便捷地实现大样本尿 液uEVs的分离。健康成人分泌的24 h uEVs个体间差异较小，是uEVs相关研究较理想的标本来源。 |
Quantification and size distribution of 24-hour urinary extracellular vesicles from healthy adults |
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| Abstract: | Objective To analyze the quantity and size distribution of 24-hour urinary extracellular vesicles (uEVs) from healthy adults. Methods The 24-hour uEVs from 9 healthy adults were isolated by hydrostatic filtration dialysis (HFD). The effectiveness of uEVs enrichment was evaluated using Western blotting and transmission electron microscopy (TEM). The quantity and size distribution of the uEVs was analyzed with BCA protein quantification, TEM, and nanoparticle tracking analysis (NTA). Results uEVs with different sizes and morphologies were observed under TEM. Western blotting confirmed the expression of TSG101 in all the uEV fractions from the 9 donors, ranging from 132.50 to 760.70 ng/mL. NTA results showed that the number of 24-hour uEVs amount ranged from 3.56 × 1012 particles to 5.12 × 1012 particles, with a CV of 14.23%. The proportion of the vesicles with a diameter <40 nm was 0.04%-0.69% with a number range of (1.80-26.49) × 109 particles; the proportion of vesicles with a diameter of 40-100 nm (which is consistent with the size of exosomes)was 22.07%-42.08% with a number range of (1.00-1.77)×1012 particles. The proportion of vesicles with a diameter of 100-1000 nm (consistent with the size of microvesicles) was 57.88%-77.85% with a number range of (2.09-3.86) × 1012 particles. Conclusion The established HFD method allows efficient and convenient isolation of uEVs from a large amount of urine samples. The 24-hour uEVs from healthy adults show narrow differences between individuals and thus can be an ideal source of samples for relevant studies. |
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