摘要
目的分离、鉴定人血清中的Exosome，探讨将ExosomalmiRNA作为疾病分子标志物的可能性。方法回顾性研究。选择2013年1月西京医院门诊体检的健康男性血清10名，并收集2013年1月至2014年12月确诊的前列腺癌患者、良性前列腺增生患者和健康对照者血清各20例。采用ExoQuick从血清中分离Exosome，分别用透射电镜、NanoSight纳米颗粒分析仪和WesternBlot对其进行形态学和分子表型鉴定，使用Agilent2100生物分析仪进行ExosomalRNA质量分析。通过实时荧光定量聚合酶链反应（qRT—PCR）检测miRNAs，并采用非参数分析法对血清不同组分中的miRNA表达量进行比较。结果采用ExoQuick可以从人血清中分离到Exosome，其直径主要集中在d0—100nm，最大分布峰值为58nm，WesternBlot检测可见热休克蛋白HSP70和四跨膜蛋白CD63表达：Agilent2100生物分析仪结果显示，该Exosome中的RNA组分主要为约25nt的小RNA；qRT-PCR检测证实4种常见miRNA在人血清Exosome中均有表达，且Exosome中miRNA的表达量高于全血清样本（miR-21，U=16，P=0．0072：miR-16，U=3，P〈0．0001；miR-20a，U=2，P〈0．0001；let-7a，U=13，P=0．0032）和去除Exosome的血清上清（miR-21，U=15，P=0．0065；miR-16，U=2，P〈0．0001；miR-20a，U=1，P〈0．0001；let-7a，U=10，P=0．0028）；对前列腺癌（PCa）、良性前列腺增生（BPH）和正常对照人群各20名检测血清中循环miR-141和ExosomalmiR-141水平，结果显示3组样本中ExosomalmiR-141的表达水平均显著高于血清中的游离miR-141（对照组：U=66，P=0．0003；BPH组：U=83，P=0．0016；PCa组：U=54，P〈0．0001）；并且PCa组的ExosomalmiR-141表达水平显著高于BPH组和正常对照人群（分别为3．85倍，U=74，P=0．0007和4．06倍，U=70，P=0．0005）。结论人血清中可以有效分离得到Exosome。与全血清样本相比，分离Exosome可以显著提高循环miRNA类的疾病标志物的检出。r中华j硷验医学杂岙，2015，38：557-561）
Objective To isolate and identify exosomes from human serum, explore the feasibility of detecting exosomal miRNA in human serum. Methods Retrospective study. Serum samples from 10 healthy individuals in January 2013 were randomly selected. Besides, from January 2013 to December 2014, serum samples from prostate cancer（PCa） patients （n = 20 ）, benign prostatic hyperplasia（ BPH ） patients （n = 20） and healthy controls （ n = 20） were selected. Exosomes were isolated from these serum samples using ExoQuick, and then identified by using transmission electron microscopy, NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype. The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser. Then quantificational real-time polymerase chain reaction （ qRT- PCR） was carried out to detect miRNAs in different components of human serum, and nonparametric tests were used for difference analysis. Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40 - 100 nm, and with maximum peak distribution of 58 nm. Moreover, they expressed HSP70 and four transmembrane protein CD63. Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA. qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome, and the expression of miRNAs in exosome pellets were higher than the whole serum （ miRo21, U = 16, P = 0. 007 2 ; miR-16, U = 3, P 〈 0. 000 1 ; miR-2Oa, U = 2, P 〈 0. 000 1 ; let-7a, U = 13, P = 0. 003 2） and exosome-depleted supernatant （ miR-21, U = 15, P = 0. 006 5 ; miR-16, U=2,P〈0.000 1; miR-2Oa, U=I,P〈0.000 1; let-7a, U=lO,P=0.0028）. miR-141, the molecular marker of prostate cancer, were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients, 20 BPH patients and 20 healthy control people. The results showed that, in three groups, exosomal miR-141 expression were all significantly higher than serum circulating miR-141 （ Control group, U = 66, P = 0. 000 3 ; BPH group, U = 83, P = 0. 001 6 ; PCa group, U = 54-,P 〈 0. 000 1 ）. In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls （ 3.85 fold, U = 74, P = 0. 000 7 and 4.06 fold, U = 70,P = 0. 000 5 ）. Conclusion Exosome can be efficiently isolated from human serum. Compared with the whole serum, isolation of serum exosome may helnful to imorove the detection of circulating miRNA.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2015年第8期557-561,共5页
Chinese Journal of Laboratory Medicine
基金
陕西省教育厅专项科研计划项目（12JK0768）
