The effect of exosomes in mesenchymal stem cell differentiation

doi:10.3969/j.issn.2095-4344.2016.41.020

Abstract

Abstract:

BACKGROUND: Currently, what we know about exosomes is that it can be produced by a variety of cells and transfer a variety of materials, producing subsequent function of regulation.
OBJECTIVE: To review the function of exosomes in mesenchymal stem cell differentiation, in order to provide reference for further in-depth study.
METHODS: With the key words of “exosome, mesenchymal stem cell, differentiation” in Chinese and English, respectively, a computer-based search was performed for articles published in CNKI, Medline and Embase databases from January 2001 to September 2016. After the initial screening, the reserved articles were further detailed, summarized and concluded.
RESULTS AND CONCLUSION: Exosomes are a kind of vesicles 40-100 nm in diameter, which can be secreted by a variety of cell types, containing functional products, such as functional protein, gene product, lipid and so on. As a bridge of adjacent cells transferring functional products, exosomes are becoming an issue of concern in the microenvironment for cell interaction. In the differentiation of mesenchymal stem cells, exosomes also play an indispensable role via pathway regulation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Exosomes, Mesenchymal Stem Cells, Cell Differentiation, Tissue Engineering

CLC Number:

R318

Hou Xiao-can, Jia Yan-jie, Peng Tao. The effect of exosomes in mesenchymal stem cell differentiation[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(41): 6209-6215.

Figures/Tables 1

2.1 间充质干细胞来源的外泌体及作用 2010年，研究人员首次在鼠心肌缺血再灌注损伤模型中对间充质干细胞来源的外泌体展开了研究[11]。结果发现，间充质干细胞可较其他大多数细胞种类(如成肌细胞、人单核细胞白血病瘤细胞系、胚肾细胞等)分泌更多的外泌体[12]，而其在形态、提取及贮存上未发现与其他种类细胞明显的差异。与其他细胞来源的外泌体中蛋白成分一样，在间充质干细胞培养基中提取而来的外泌体中蛋白成分同样不稳定，通过400余种独立蛋白的波谱分析发现，仅154种蛋白可以被检测到[13]，这些蛋白与多种生物过程密切相关。另一方面，在间充质干细胞来源的外泌体中，miRNAs也是其重要组成成分，主要以其前体形式包裹在囊泡中。 2.1.1 miR-133b上调的间充质干细胞来源的外泌体[14] 大脑中动脉闭塞动物模型建立及脑组织的提取：通过血管腔内阻塞的方法建立大鼠右侧大脑中动脉阻塞模型，缺血模型建立24 h后，随机挑选6只大鼠接受尾静脉注射自体间充质干细胞移植(约3×109 L-1，持续注射5 min)，分别在缺血模型建立24，72，168 h后冰上取出闭塞动脉侧脑组织，添加含B27和谷氨酰胺的培养基培养，提取其培养上清，离心后取上清储存备用。间充质干细胞接受脑组织共培养处理及外泌体提取：将提取自同源大鼠股骨、胫骨干的骨髓间充质干细胞与上述分离出的脑组织(正常/大脑中动脉闭塞脑组织)以1︰10比例混合培养24 h，更换为新鲜普通培养基(包含B27及谷氨酰胺)再培养48 h，收集其培养基并提取其外泌体储存备用。外泌体中物质的检测：应用免疫印迹法检测外泌体内miR-133b表达情况。通过BCA检测相应蛋白浓度，应用miRNeasy Mini试剂盒提取外泌体中总RNA，通过实时定量PCR检测外泌体中miR-133b的表达水平，发现经上述过程提取的外泌体中miR-133b表达上调。 miR-133b作用的探究：为了探索miR-133b所起到的作用，通过质粒向间充质干细胞以及幼稚神经元细胞中转染miR-133b-inhibitor，探究miR-133b所起到的作用。神经元及胶质细胞神经分化检测：应用提取出的外泌体加入培养基中培养取自乳鼠大脑皮层的幼稚神经元及星形胶质细胞，通过细胞免疫荧光法分别检测转染miR133b inhibitor以及接受外泌体培养的幼稚神经元细胞内神经突形成相关蛋白β-Ⅲ-微管蛋白的表达，发现荧光只表达在成功转染miR-133b-inhibitor的神经元细胞上，而miR-133又具有下调RhoA表达的作用，后者表达抑制可产生促神经突形成的作用，进一步通过免疫印迹方法检测RhoA蛋白的表达，从而验证了该通路的存在。综上所述，该研究将经大脑中动脉闭塞后的大鼠皮质神经细胞作为同源骨髓间充质干细胞的处理因素，以达到miR-133b表达上调目的，进而发现miR-133b上调后的间充质干细胞，可上调突触间隙中外泌体的水平，后者可通过对RhoA表达抑制而在一定程度促进神经突形成及神经细胞增长的作用。 2.1.2 miR-124上调的间充质干细胞来源的外泌体[15] 间充质干细胞及神经前体细胞的转染以及标记：通过绿色荧光标记的携带miR-124慢病毒以合适的MOI值转染间充质干细胞及神经前体细胞，应用绿色荧光染料CMFDA标记神经前体细胞备用。间充质干细胞来源的外泌体的提取：转染miR-124 3 d后，应用外泌体提取试剂提取培养上清中的外泌体，并应用BCA试剂盒检测收集到的外泌体中的蛋白浓度，然后以2 mg/L的质量浓度将外泌体加入神经前体细胞的培养基中。间充质干细胞来源外泌体中RNA的检测：应用实时定量PCR方法检测外泌体中miR-124水平，并发现转染miR-124的间充质干细胞分泌的外泌体中miR-124上调。神经前体细胞中相关蛋白表达变化：应用双荧光素酶报告基因检测法、免疫印迹法检测接受外泌体培养以及转染miR-124的神经前体细胞中Sox-9、β3微管蛋白(β3-tubulin)、巢蛋白(nestin)以及谷氨酸盐转运蛋白EAAT1和EAAT2的表达，并发现均产生了有实际意义的上调或下调等相关变化。综合各步骤相关实验结果，该研究发现转染miR-124的间充质干细胞可通过包含miR-124外泌体的分泌，使神经前体细胞内β3微管蛋白上调、巢蛋白下调以及谷氨酸盐转运蛋白EAAT1和EAAT2的表达上调，并认为miR-124可作为间充质干细胞来源外泌体中对神经分化产生影响的一种具有意义的miRNA成分。 2.2 间充质干细胞分化过程相关的外泌体及作用[16] 2.2.1 外泌体在间充质干细胞神经分化的作用通过研究后发现，PC12细胞(神经元细胞系的一种)可通过外泌体的分泌对间充质干细胞产生诱导效应而分化为神经元样细胞，而对于其分泌的外泌体，miR-125b、miR-221、miR-222等均明显升高，且在间充质干细胞中神经样标志物(如NSE、MAP2)表达增多，即产生了诱导其分化成为神经元样细胞的作用。 PC12细胞来源外泌体的提取：首先PC12细胞以5.7×102/cm2密度接种，应用促生长培养基(由DMEM、体积分数为10%马血清、体积分数为5%胎牛血清， 100 U/mL青霉素链霉素混合液构成)培养2 d，然后更换为促分化培养基(由DMEM培养基、体积分数为2%马血清、100 μg/L神经生长因子100 U/mL青霉素链霉素组成)，每3 d换液1次，按说明步骤提取外泌体备用，将B16细胞(黑色素瘤细胞系)来源的外泌体作为对照，应用BCA试剂盒测定提取外泌体中蛋白的浓度。外泌体中RNA的检测：应用miRNeasy micro试剂盒提取外泌体中的RNA，根据相关microRNA分析试剂盒分析外泌体中miRNA的表达情况。骨髓来源间充质干细胞的提取与培养：分离培养新鲜骨髓间充质干细胞，以3.0×103/cm2密度接种，培养于DMEM培养基进行扩增(含体积分数为10%胎牛血清、1%MEM、100 U/mL青霉素链霉素混合液以及2 μg/L碱性成纤维生长因子)，然后用不含碱性成纤维生长因子的培养基继续培养，每天向培养基中加入外泌体(40 mg/L)，每3 d换液1次。间充质干细胞神经分化相关蛋白表达的测定：通过免疫细胞化学方法定性检测上述培养后的间充质干细胞中MAP-2(微管相关蛋白2)、NF160(神经丝160)、NSE(神经元特异性烯醇化酶)的表达情况，应用免疫印迹、qPCR方法定量检测MAP-2、NSE蛋白的表达水平。 2.2.2 外泌体在间充质干细胞成骨样分化的作用脂多糖处理后的人源性原始单核细胞来源的外泌体[17]：收集脂多糖活化的人源性原始单核细胞中的外泌体，将其作为间充质干细胞的培养成分，继续培养后发现，间充质干细胞中的RUNX2、BMP-2表达明显上调，而后者已经证实为成骨过程中关键的调节蛋白及标志物，即证明了该种外泌体存在一定促间充质干细胞成骨样分化的作用。进一步探索中发现，应用外泌体培养的细胞中此2种标志物的表达较直接共培养时偏低，而较应用普通培养基培养仍明显上调，故认为可能是外泌体提取过程中丢失一部分外泌体成分所致。间充质干细胞成骨样分化过程中的外泌体[18]：通过研究间充质干细胞成骨样分化过程中外泌体内各种成分的变化，发现miRNA成分let-7a、miR-199b、miR-218、miR-148a、miR-135b、miR-203、miR-219、miR-299-5p有不同程度明显升高，miR-221、miR-155、miR-885-5p、miR-181a、miR-320c则不同程度明显下降；mRNA成分RPS2、DGKA、ACIN1、DKK2、Xsox17、DDX6、Lsm2随着成骨样分化的进展出现了不同程度的表达，不过这些目前也仅停留在表达程度改变方面，对于其各自改变相应的生物学意义尚无更进一步的认识。 2.3 外泌体在间充质干细胞成纤维细胞分化的作用 2.3.1 胃癌细胞来源的外泌体 Gu等[19]在诱导脐带间充质干细胞向肿瘤相关成纤维细胞分化方面展开了研究，通过与上述大致相似的实验思路及方法提取胃癌细胞来源的外泌体，并应用其处理脐带来源的间充质干细胞，通过免疫荧光、免疫印迹、PCR等一系列实验方法检测处理后的间充质干细胞中成纤维细胞标志物FAP、α-SMA、N-钙黏着蛋白、波形蛋白等的表达，并均显示表达水平上调，从而达到分化成为成纤维细胞的效果，进一步研究及推断证明，外泌体中的转化生长因子β及其介导的Smad-2/3、P38通路激活参与了干细胞的分化过程。 2.3.2 乳腺癌细胞来源的外泌体 Cho等[13]应用乳腺癌的2株细胞MCF-7、MDA-MB-231，收集其各自分泌的外泌体，并应用其培养脂肪来源的间充质干细胞后，其α-平滑肌动蛋白表达升高，而后者是一种肌成纤维样细胞的标志蛋白。进一步对其中通路进行探究及反复验证表明，TGF-β-TGFR-SMAD2通路参与了该2种乳腺癌细胞来源的外泌体促干细胞分化为肌成纤维样细胞的过程，即脂肪来源的间充质干细胞向肌成纤维样细胞分化。"

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