外泌体之家 | 细胞外膜泡领域核心平台—exosomes & microvesicles—小膜泡大作用

标题: 【文献编译】Cardiology:iPS来源的exosomes抑制缺血心肌细胞凋亡 [打印本页]
作者: 丁倩倩    时间: 2015-5-25 22:08
标题: 【文献编译】Cardiology:iPS来源的exosomes抑制缺血心肌细胞凋亡
Exosomes/microvesicles from induced pluripotent stem cells deliver cardioprotective miRNAs and prevent cardiomyocyte apoptosis in the ischemic myocardium
Background/objectives: Induced pluripotent stem cells (iPS) exhibit enhanced survival and proliferation in ischemic tissues. However, the therapeutic   application of iPS cells is limited by their tumorigenic potential. We hypothesized that iPS cells can transmit cytoprotective signals to cardiomyocytes via exosomes/microvesicles.
Methods: Exosomes/microvesicles secreted from mouse cardiac fibroblast (CF)-derived iPS cells (iPS-exo) were purified from conditioned medium and confirmed by electron micrograph, size distribution and zeta potential by particle tracking analyzer and protein expression of the exosome markers CD63 and Tsg101.
Results: We observed that exosomes are at low zeta potential, and easily aggregate. Temperature affects zeta potential (−14 to−15 mV at 23 °C vs−24 mV at 37 °C). The uptake of iPS-exo protects H9C2 cells against H2O2- induced oxidative stress by inhibiting caspase 3/7 activation (P b 0.05, n=6). Importantly, iPS-exo treatment can protect against myocardial ischemia/reperfusion (MIR) injury via intramyocardial injection into mouse ischemic myocardium before reperfusion. Furthermore, iPS-exo deliver cardioprotective miRNAs, including nanogregulated miR-21 and HIF-1α-regulated miR-210, to H9C2 cardiomyocytes in vitro.
Conclusions: Exosomes/microvesicles secreted by iPS cells are very effective at transmitting cytoprotective signals to cardiomyocytes in the setting of MIR. iPS-exo thus represents novel biological nanoparticles that offer the benefits of iPS cell therapy without the risk of tumorigenicity and can potentially serve as an “off-the-shelf” therapy to rescue ischemic cardiomyocytes in conditions such as MIR.
背景：iPS细胞，即Induced pluripotent stem cells（诱导性多功能干细胞）由日本的Yamanaka于2006年利用病毒载体将四个转录因子Oct4、Sox2、c-Myc、Klf4转入分化的小鼠体细胞中，使其重编程而得到的类似胚胎干细胞的一种细胞类型。该种干细胞因克服了其他类型干细胞来源困难以及伦理性的问题而受到广泛关注，但其难以预计的致瘤性则限制了IPS应用于临床治疗。这篇文献认为iPS可以通过exosomes/microvesicles传递细胞保护信号。
方法：首先分离纯化由小鼠心肌成纤维细胞诱导而来的iPS所分泌的exosomes/microvesicles，然后经电镜、nanosight、western进行鉴定。但是这篇文章中分离exosome时既没有用超离也没有用试剂盒，貌似是自制的一种溶液但看其步骤感觉原理应该是和SBI的试剂盒是一样的（哪位大神可以研究下，说不定以后大家都不用买试剂盒了）10 ml culture medium (CM) with 10% exosome-depleted FBS was added to CF or iPS cell cultures in 10 cm dishes. After 48 h, medium was centrifuged at 1000 r.p.m. for 10 min and the supernatant passed through 0.22 μm filters to remove cell debris. Exosomes/microvesicles were precipitated with 1/3 volume of polyethylene glycol (PEG) buffer (33.4% PEG 4000, 50 mM HEPES (pH 7.4), 1 M NaCl) overnight at 4 °C followed by centrifugation at 3000 rpm for 30 min. Exosomes/microvesicles were then resuspended in PBS。
结果：文章用了两种细胞分泌的exosome，分别是心肌成纤维细胞和心肌成纤维细胞诱导的IPS细胞（CF-Exo、iPS-Exo)
1、体外观察EXO摄取：PKH26标记的EXO与H9C2(心肌母细胞株）共培养，分别于0h、2h、10h、14h荧光显微镜下观察
2、EXO体外抗凋亡作用：分别用PBS、CF-Exo、iPS-Exo预处理H9C2，然后给予H2O2刺激，检测caspase-3/7 activation
3、EXO体内抗凋亡作用：建立心肌缺血再灌注模型，分别注射PBS、CF-Exo、iPS-Exo，TUNEL检测
4、iPS-Exo抑制缺血心肌促凋亡蛋白active caspase 3的表达
5、iPS-Exo介导心肌保护性miRNAs由iPS细胞转至H9C2细胞：首先比较了CF-Exo、iPS-Exo中miR-21 和 miR −210的含量，发现iPS-Exo中两种RNA的含量明显高于CF-Exo；然后让H9C2细胞分别与CF-Exo、iPS-Exo共培养14h后检测细胞中两种RNA的含量，发现iPS-Exo处理的H9C2细胞中RNA的含量高于对照组3-5倍。
讨论：（是我的讨论）1、文章中多次提到exosome的zeta potential，并指出其电势易受温度的影响，不知道这对exosome的提取、鉴定以及功能有什么影响？我们在提取、保存以及做功能实验时是否应考虑到其电势的变化对exosome稳定性的影响？2、文章中的体外实验是先用PBS、CF-Exo、iPS-Exo预处理H9C2，然后给予H2O2刺激，这跟先给予损伤刺激再用EXO处理相比，其机制是一样的吗？（还是我想多了）







处女座的处女作
第一次做文献编译，心情无比激动，久久不忍上传，希望大家多多指教











作者: 丁倩倩    时间: 2015-5-25 22:18
[attach]140[/attach]
作者: 惜名    时间: 2015-5-26 08:38
international journal of cardiology的IF已经蹿到6以上了，但貌似许多人对此颇有微辞。。。
作者: niceman1987    时间: 2015-5-26 19:01
翻译的挺好的，学习了



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