组织外泌体提取

2023-04-12 14:20:29 316

一般,组织外泌体提取的方法包括三个过程。首先,组织切成小块,使组织具有更大的表面积。其次,在细胞培养基中利用组织解离酶与组织切片孵育,目的是使组织外泌体扩散到培养基中。最后,外泌体通过密度梯度离心、分子尺寸排阻等方法从培养基中分离出来。需要注意的是组织切片和组织消化过程中避免细胞的破碎,因为细胞破碎后会有大量的囊泡产生。


外泌体膜蛋白组质谱鉴定与分析

对EVs表面的膜蛋白进行生物素标记,然后利用链霉亲和素磁珠进行EVs膜蛋白的富集,再进行EVs膜蛋白的质谱鉴定分析,从而实现低丰度EVs膜蛋白的筛选。针对筛选的EVs膜蛋白,可以利用免疫胶体金电镜进一步验证


外泌体膜蛋白芯片分析

利用定制化的膜蛋白抗体芯片直接对样本中的外泌体进行捕获和检测,无需进行外泌体的提取。


外泌体蛋白质组学分析(Label-Free)


蛋白质组学(Proteomics)是指利用高分辨的蛋白质分离技术和高效的蛋白质鉴定技术在蛋白质水平上整体性、动态和定量地研究生命现象及规律的科学。外泌体蛋白组学即分析外泌体中所有蛋白质的集合。

质谱分析技术有着高灵敏度,高精准度等特点,能够快速准确地分析外泌体蛋白,适用于外泌体蛋白组学的研究。非标定量法(Label-Free)是通过比较质谱分析次数或质谱峰强度,分析不同来源样品蛋白的数量变化,认为肽段在质谱中被捕获检测的频率与其在混合物中的丰度正相关,因此蛋白质被质谱检测的计数反映了蛋白质的丰度,通过适当的数学公式可以将质谱检测计数与蛋白质的量联系起来,从而对蛋白质进行定量。


外泌体非编码RNA组学

非编码RNA(Non-coding RNA)是指不编码蛋白质的RNA,包括miRNA、lncRNA、circRNA、piRNA等。非编码RNA发挥功能的方式很多,可以与蛋白、DNA和RNA相互作用,参与多种细胞活动,主要包括基因的激活和沉默,RNA的剪接、修饰和编辑,蛋白质的翻译等。

Tissue typesCollection and pre-processingEV characterizationMethod of analysisKey findingsCitationAuthor影响因子备注
组织处理Pre-EV isolationEV-isolationMethodsMarkersPMIDYear


新鲜人/动物组织
2500g, 30min;
10000g, 35min;
0.2 μm过滤;
110000g, 2h;
NTA, WB, TEM, IEM, fluorescence staining, FCMCD9, CD63, CD8,
ESCRT proteins (Alix, TSG101)
/EVs分离鉴定方法;278015112016


人转移性黑色素瘤组织新鲜组织切碎到1-2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min;
70 μm过滤;
300g, 10min;
2000g, 20min;
16500g, 20min;
118000g, 2.5h;TEM, NTA, WB CD9, CD63, CD81, calnexin, flotillin-1NanoLC-MS/MS analysis分离EVs亚型方法;
ADAM10富集于LD EVs;
mitofilin富集于大EVs;
321280732020Crescitelli et al.

人黑色素瘤组织新鲜组织切碎到2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min,期间24rpm缓慢振荡;
300g, 10min;
2000g, 20min;
16500g, 20min;
118000g, 2.5h;
碘克沙醇密度梯度离心, 186000g, 16h;
TEM, WB, ExoView analysisCD63, CD81, CD9, flotillin-1, CalnexinRNA profile analysis, ExoView, proteomic analysis人黑色素瘤组织外泌体分离鉴定方案;334956262021Crescitelli et al.

新鲜人肠组织组织切碎到<0.5cm;
含collagenase I(300U/ml)的HBSS中孵育30min,期间缓慢振荡;
用含有蛋白酶抑制剂的PBS终止消化;
70 μm过滤;
1000g, 10min;
2000g, 20min;
5000g, 30min;
15000g, 1h;
0.2 μm PVDF过滤;
120000g, 130min;
碘克沙醇密度梯度离心, 288000g, 5h;
100000g, 70min;
NTA, TEM, WBCD9, CD63, CD81, Alix, Tsg101RT-qPCR分离肠组织外泌体并应用于肠缺血-再灌注(I/R)损伤研究;320905872020


人肾癌及癌旁组织组织切碎;
加入4ml DMEM,4°C孵育1h;
2000g, 20min;
16000g, 20min;
100000g, 90min;
100000g, 90min;
WB, TEM, NTACD9, CD63, CD81Quantitative LC/MS analysisTe‐EVs;
蛋白组分析;
AZU1
289756132018


人转移性黑色素瘤组织组织切碎到1-2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min;
70 μm过滤;
300g, 10min;
2000g, 20min;
16500g, 20min;
110000g, 2.5h;TEM, WB, ELISA, particle measurementCD9, CD81MS analysis, RNA detection黑色素瘤组织外泌体富含线粒体膜蛋白;314972642019


尸检脑组织组织冰上切碎,冰上解冻;
在含有collagenase III (75 U/mL)的Hibernate-E培养基中37°C孵育20min,期间缓慢振荡;
加入蛋白酶抑制剂(PI/PS)终止消化;
37°C水浴振荡20min;
300g, 5min;
2000g, 10min;
10000g, 5min;
Triple sucrose cushion;
180000g, 3h;
TEM, WB, IBCalnexinSmall RNA sequencingAD脑组织EVs miRNA分析;329226922020


毫米大小癌及癌旁组织组织切碎;
在含有双抗的RPMI1640培养基中37°C孵育24h;
500g, 10min;
3000g, 20min;
12000g, 20min;
100000g, 70min;
1 ml sucrose density cushion,
100000g, 70min;
WB, TEM, NTACD9, CD81, TSG101MS蛋白组pan-EVP markers (ACTB, MSN, RAP1B);
肿瘤特异性EVP蛋白;
327954142020
66
小鼠黑色素瘤组织组织切碎到<3mm, PBS洗一遍;
500g, 4min;
组织解离为单细胞悬液(含有25 μg/ml DNase I, 5 Wünsch units of Liberase的PBS,37°C,20min,缓慢振荡);
70μm过滤;
PBS(含5 mM EDTA,25 μg/ml DNase I)洗一遍;

500g, 10min;
500g, 10min;
2000g, 15min;
2000g, 15min;
16.5K EVs:
16500g, 24min (×2);
100K EVs: 100000g,70min (×2);
TEM, WBCalnexin, cytochrome-C, GM130LC-MS/MS, transcriptomics analysisCells from two types of melanoma phenocopy migratory behaviour through EV exchange.299076952018


结直肠癌及癌旁组织新鲜组织冰上切碎;
在含有collagenase I (250 units/ml) RPMI1640 培养基中37°C孵育30min;
置于冰上,加入含多种蛋白酶抑制剂的PBS;
吹散细胞;
60mm筛网;
40mm筛网;
400g, 10min;
2000g, 20min;
15000g, 40min;
0.22 μm PES过滤;
120000g, 4h (×2);
碘克沙醇密度梯度离心;
IBCD63, CD81, Syntenin-1, CalreticulinMS, RNA sequencing, DNA analysis, direct immunoaffinity CaptureCD9, CD63, CD81, Annexin V在外泌体中缺失;
Annexin A1和A2为非外泌体EVs新markers;
309516702019
66
小鼠结直肠癌组织PBS洗一遍;
组织在含2 mM EDTA的PBS中切碎;
70μm细胞筛网;
500g, 5min;
3000g, 10min;
10000g, 30min;
110000g, 70min;
134000g, 70min;
TEM, NTA, WB, Flow cytometryCD9, CD81, ALIXLC-MS/MS, miRNA profiling, Lipidome analysisTAM-EVs诱导炎症及抗肿瘤反应;311671482019


AD小鼠脑组织在Hibernate-E培养基中37°C孵育20min;
研磨(loose-fit Dounce homogenizer);
500g, 5min;
2000g, 10min;
10000g, 30min;
0.45 μm 过滤;
100000g, 2h;
1.5 ml of 0.25 M sucrose buffer for gradient purification; floatation density gradient.
WBCD63, CD81, Alix, TSG101, HSC70, Rab8aMS, enrichment analysisTi-EVs分离方法;298947262018


人脑组织在Hibernate-E培养基(木瓜蛋白酶,20 units/ml)中37°C孵育15min;40 μm 筛网;
300g, 10min;
2000g, 10min;
10000g, 10min;
0.22 μm 过滤;
100000g, 70min;
0.475 M of sucrose solution
NTA, TEM/Label-free Nano-LC-MS/MS analysisTau、Aβ1-42上调;323015812020


人脑组织PBS中切碎,涡旋;300g, 10min;
1200g, 10min(×2);
0.22 μm 过滤;
10000g, 30min(×2);
22000g, 22h;WB, TEM, Immunogold Labeling,CD63, GAPDH, flotillin-2MiRNA expression analysis, qPCR analysisSZ样品miR-497上调;
BD样品miR-29c上调;
233827972013


恒河猴脑组织在Hibernate-A培养基(木瓜蛋白酶,20 units/ml)中37°C振荡孵育15min;40 μm 筛网;
5 μm 筛网;
0.22 μm 筛网;
300g, 10min;
2000g, 10min;
10000g, 30min;
100000g,60min (×2);
2 ml of 0.95 M sucrose solution and inserted inside a sucrose step gradient column; 200000g,16h;
TEM, WBCD9, CD63, CD81, HSP70, flotillin, TSG101Small RNA sequencing, qRT-PCRNeurotoxicity triggered by EV-miR-21 was not influenced by apoptosis inhibition but restricted by necrostatin-1.261541332015


小鼠脑组织
40 μm 筛网;
0.2 μm 筛网;
300g, 10min;
2000g, 10min;
10000g, 30min;
100000g,70min (×2);
2 ml of 0.95 M sucrose solution centrifuged at 200000g for 16 h.
Immunoelectron microscopyTSG101Gene expression analysis, image analysis小神经胶质细胞通过外泌体扩散tau;264369042015
28
人脑组织冰冻组织冰上切碎;
在Hibernate-E培养基(collagenase III ,75 U/ml )中37°C振荡孵育20min;
蛋白酶抑制剂终止消化(PhosSTOP和Complete Protease Inhibitor);
300g, 15min;
2000g, 15min;
0.2 μm 筛网;
10000g, 30min;
Sucrose density gradient ultracentrifugation (SDGU);
110000g,70min;
ultrafiltration through a 10 kDa MWCO protein concentrator.
TEM, NTA, WB, NanoFCM flow analysisCD9, CD63, CD81, Rab27, TSG101, Syntenin, Calnexin, GM130Small RNA sequencing, MS3个物种脑组织Ti-EVs分离比较,强调了不同物种的分离参数;329441742020


人脂肪组织
800g, 10min;
2000g, 10min;
12000g, 30min;
0.2 μm 筛网;
100000g, 2h;Fluorescence NTA, WB, EMCD9, CD63, TSG101, Grp94 (a negative control)MS analysis, Ingenuity pathway analysisAdipose Ti-EVs mediate changes in placental functions in GDM and are involved in some pregnancy complications.305176762019


脂肪组织切碎至1-2mm;
加入到含双抗的α-MEM培养基中,37°C 100rpm/min振荡孵育2天;
2000g, 10min;
5000g, 30min;
5000g, 30min;
Exosome Isolation TM reagent 4°C孵育过夜;
10000g, 1h;
WB, TEM, Zetasizer Nano ZSCD9, CD63, ALIX, TSG101MS analysis, Bioinformatic analysis, Real-time PCRNPM3, STEAP3, DAD1与脂肪形成325976612020


Human obese white adipose tissue explant
1800g, 5min;
0.22 μm 筛网;
10000g, 20min;
100000g,90min (×2);TEM, NTA, IBCD9, CD63, CD81, negative control GRP94MS DDA qualitative analysisObese AT release functional EVs carrying AT and obesity-specific biomarkers depending on the original AT.334654892022


小鼠脂肪组织切碎至>4mm;
加入到含抗生素和10%FBS(已去除外泌体)的DMEM培养基中,37°C 培养;
200g, 10min;
500g, 10min(×2);
2000g, 15min;
10000g, 30min;
70000g, 60min;
5 ml of 2.6 M sucrose at 270000g 16 h;
Gradient fractions were centrifugated at 70000g 1h.
//Fluorescence activated cell sorter (FACS) analysisAdipose Ti-ELVs mediate crosstalk between AT and macrophages; ObELV induced TNF-α and IL-6 activation and insulin resistance require the TLR4/TRIF pathway.196751372009


小鼠脂肪组织灌洗以去除组织中血液;
切碎;
37°C解离组织1h(100 mM HEPES, 1.5% BSA, 5 mM glucose, 1 mM calcium and 1mg/ml collagenase D, 2.4U/ml dispase II);
2 mM EGTA中止消化;
100 μm 筛网;
600g, 5min;
1200g, 15min;
10000g, 15min;
0.22 μm 筛网;
100000g, 90min (×2);NTA, TEM, WBCD63, Alix, TSG101Proteomics analysis, LC‐MS lipidomics analysis.Adipose Ti‐EVs are involved in the response to changes in systemic nutrient conditions.30293865201866

小鼠脂肪组织剪碎;
1000g,5min,清洗组织;
培养基中37 °C孵育30min;
换液,培养基中37 °C孵育2h以释放外泌体;
1000g for 5 min at room temperature and re‐dissolved in medium; incubated for 30 min at 37°C, 5% CO2.Medium exchange, tissues were incubated for 2 h at 37°C, 5% CO2. The supernatant was used for EVs isolation according to the manufacturer's instruction.WB, TEMCD63, Hsp70, CytoC, α‐TubulinMiRNA profiling外泌体miR‐92a与人BAT活性负相关;271178182016


小鼠肝组织通过灌注胶原酶来解离肝脏组织;肝灌注;
70 μm 筛网;
50g, 10min;
300g, 10min;
2000g, 20min;
10000g, 70min;
100000g, 70min (×2);NTA, tunable resistive pulse sensing// An optimum and replicable procedure for the isolation of hepatic Ti‐EVs.314983232019

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