中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2015年
8期
557-561
,共5页
李卓%康炜%李蕊%郝晓柯%马越云
李卓%康煒%李蕊%郝曉柯%馬越雲
리탁%강위%리예%학효가%마월운
Exosome%血清%miRNA
Exosome%血清%miRNA
Exosome%혈청%miRNA
Exosome%Serum%miRNA
目的：分离、鉴定人血清中的Exosome，探讨将Exosomal miRNA 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Western Blot 对其进行形态学和分子表型鉴定，使用Agilent 2100生物分析仪进行Exosomal RNA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40～100 nm，最大分布峰值为58 nm，Western Blot检测可见热休克蛋白HSP70和四跨膜蛋白CD63表达；Agilent 2100生物分析仪结果显示，该Exosome中的RNA组分主要为约25 nt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 Exosomal miR-141水平，结果显示3组样本中Exosomal miR-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Exosomal miR-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。（中华检验医学杂志，2015，38：557-561）
目的：分離、鑒定人血清中的Exosome，探討將Exosomal miRNA 作為疾病分子標誌物的可能性。方法迴顧性研究。選擇2013年1月西京醫院門診體檢的健康男性血清10名，併收集2013年1月至2014年12月確診的前列腺癌患者、良性前列腺增生患者和健康對照者血清各20例。採用ExoQuick從血清中分離Exosome，分彆用透射電鏡、NanoSight納米顆粒分析儀和Western Blot 對其進行形態學和分子錶型鑒定，使用Agilent 2100生物分析儀進行Exosomal RNA質量分析。通過實時熒光定量聚閤酶鏈反應（ qRT-PCR）檢測 miRNAs，併採用非參數分析法對血清不同組分中的miRNA錶達量進行比較。結果採用ExoQuick可以從人血清中分離到Exosome ，其直徑主要集中在40～100 nm，最大分佈峰值為58 nm，Western Blot檢測可見熱休剋蛋白HSP70和四跨膜蛋白CD63錶達；Agilent 2100生物分析儀結果顯示，該Exosome中的RNA組分主要為約25 nt的小RNA；qRT-PCR檢測證實4種常見miRNA在人血清Exosome中均有錶達，且Exosome中miRNA的錶達量高于全血清樣本（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）；對前列腺癌（PCa）、良性前列腺增生（BPH）和正常對照人群各20名檢測血清中循環miR-141和 Exosomal miR-141水平，結果顯示3組樣本中Exosomal miR-141的錶達水平均顯著高于血清中的遊離miR-141（對照組：U＝66，P＝0.0003；BPH組：U＝83，P＝0.0016；PCa組：U＝54，P＜0.0001）；併且PCa組的Exosomal miR-141錶達水平顯著高于BPH組和正常對照人群（分彆為3.85倍，U＝74，P＝0.0007和4.06倍，U＝70，P＝0.0005）。結論人血清中可以有效分離得到Exosome。與全血清樣本相比，分離Exosome可以顯著提高循環miRNA類的疾病標誌物的檢齣。（中華檢驗醫學雜誌，2015，38：557-561）
목적：분리、감정인혈청중적Exosome，탐토장Exosomal miRNA 작위질병분자표지물적가능성。방법회고성연구。선택2013년1월서경의원문진체검적건강남성혈청10명，병수집2013년1월지2014년12월학진적전렬선암환자、량성전렬선증생환자화건강대조자혈청각20례。채용ExoQuick종혈청중분리Exosome，분별용투사전경、NanoSight납미과립분석의화Western Blot 대기진행형태학화분자표형감정，사용Agilent 2100생물분석의진행Exosomal RNA질량분석。통과실시형광정량취합매련반응（ qRT-PCR）검측 miRNAs，병채용비삼수분석법대혈청불동조분중적miRNA표체량진행비교。결과채용ExoQuick가이종인혈청중분리도Exosome ，기직경주요집중재40～100 nm，최대분포봉치위58 nm，Western Blot검측가견열휴극단백HSP70화사과막단백CD63표체；Agilent 2100생물분석의결과현시，해Exosome중적RNA조분주요위약25 nt적소RNA；qRT-PCR검측증실4충상견miRNA재인혈청Exosome중균유표체，차Exosome중miRNA적표체량고우전혈청양본（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）화거제Exosome적혈청상청（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）；대전렬선암（PCa）、량성전렬선증생（BPH）화정상대조인군각20명검측혈청중순배miR-141화 Exosomal miR-141수평，결과현시3조양본중Exosomal miR-141적표체수평균현저고우혈청중적유리miR-141（대조조：U＝66，P＝0.0003；BPH조：U＝83，P＝0.0016；PCa조：U＝54，P＜0.0001）；병차PCa조적Exosomal miR-141표체수평현저고우BPH조화정상대조인군（분별위3.85배，U＝74，P＝0.0007화4.06배，U＝70，P＝0.0005）。결론인혈청중가이유효분리득도Exosome。여전혈청양본상비，분리Exosome가이현저제고순배miRNA류적질병표지물적검출。（중화검험의학잡지，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 CD 63.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 (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, 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-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).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 helpful to improve the detection of circulating miRNA.
