全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

3T3-fGFP细胞摄取神经干细胞胞外泌囊泡的途径

, PP. 1193-1199

Keywords: 小鼠神经干细胞,胞外囊泡,T-fGFP细胞,网格蛋白介导通路

Full-Text   Cite this paper   Add to My Lib

Abstract:

目的以超速离心方案提取胞外囊泡的鉴定为基础,初步探求小鼠神经干细胞C17.2胞外囊泡与小鼠胚胎成纤维细胞3T3-fGFP细胞之间的通讯交流通路。方法分别用超速离心法获得胞外囊泡(EV)和TotalExosomeIsolationKit试剂盒获得外泌体(EXO);用透射电镜和原子力显微镜分别对两种样品进行形态学比较;用SDS-PAGE和Westernblot分别对两种样品进行生物化学比较;用染料标记超速离心法获得胞外囊泡;将标记的胞外囊泡与3T3-fGFP细胞共孵育,对3T3-fGFP细胞摄取胞外囊泡进行研究;通过药理实验筛选3T3-fGFP细胞摄取胞外囊泡的主要通路。结果C17.2细胞生长良好,形态规则未出现明显分化现象。在透射电镜下两种样品均观察到圆形双层膜结构和“杯状”外形的囊泡,直径分布多集中在300nm以内,并且发现胞外囊泡的平均直径大于外泌体。原子力显微镜的结果显示,两种样品的高度分布多集中在2~20nm以内,直径分布多集中在300nm以内,并且外泌体的平均高度和平均直径均大于胞外囊泡。SDS-PAGE结果显示两种样品所含蛋白种类、表达丰度接近。在相同上样量下HSP70的表达强度两者接近,但CD63、CD9和CD813种蛋白在胞外囊泡中表达更强。摄取实验表明C17.2细胞胞外囊泡可以被3T3-fGFP细胞内化,被包裹在囊泡内,转运到细胞核周围的区域,并且摄取数量随着时间延长而增加(P<0.05)。通过药理实验确定网格蛋白(Clathrin)介导的内吞通路为3T3-fGFP细胞摄取C17.2细胞胞外囊泡的主要通路。结论小鼠神经干细胞外泌体可能主要经网格蛋白介导的通路参与了神经干细胞与3T3-fGFP细胞的通讯。

References

[1]  De-Feo D, Merlini A, Laterza C, et al. Neural stem cell transplantation in central nervous system disorders: from cell replacement to neuroprotection[J]. Curr Opin Neurol, 2012, 25(3): 322-333. [2]Fruhbeis C, Frohlich D, Kuo W P, et al. Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication[J]. PLoS Biol, 2013, 11(7): e1001604. [3]Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends[J]. J Cell Biol, 2013, 200(4): 373-383. [4]Cossetti C, Iraci N, Mercer T R, et al. Extracellular vesicles from neural stem cells transfer IFN-gamma via Ifngr1 to activate Stat1 signaling in target cells[J]. Mol Cell, 2014, 56(2): 193-204. [5]Palanisamy V, Sharma S, Deshpande A, et al. Nanostructural and transcriptomic analyses of human saliva derived exosomes[J]. PLoS One, 2010, 5(1): e8577. [6]Burns G, Brooks K, Wildung M, et al. Extracellular vesicles in luminal fluid of the ovine uterus[J]. PLoS One, 2014, 9(3): e90913. [7]Valadi H, Ekstrom K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells[J]. Nat Cell Biol, 2007, 9(6): 654-659. [8]Grant R, Ansa-Addo E, Stratton D, et al. A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma[J]. J Immunol Methods, 2011, 371(1/2): 143-151. [9]Tauro B J, Greening D W, Mathias R A, et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes[J]. Methods, 2012, 56(2): 293-304. [10]Aalberts M, van-Dissel-Emiliani F M, van-Adrichem N P, et al. Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans[J]. Biol Reprod, 2012, 86(3): 82. [11]van-der-Pol E, Hoekstra A G, Sturk A, et al. Optical and non-optical methods for detection and characterization of microparticles and exosomes[J]. J Thromb Haemost, 2010, 8(12): 2596-2607. [12]Schneider A, Simons M. Exosomes: vesicular carriers for intercellular communication in neurodegenerative disorders[J]. Cell Tissue Res, 2013, 352(1): 33-47. [13]Ghosh A, Davey M, Chute I C, et al. Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins[J]. PLoS One, 2014, 9(10): e110443. [14]Tian T, Wang Y, Wang H, et al. Visualizing of the cellular uptake and intracellular trafficking of exosomes by live-cell microscopy[J]. J Cell Biochem, 2010, 111(2): 488-496. [15]Mathivanan S, Ji H, Simpson R J. Exosomes: extracellular organelles important in intercellular communication[J]. J Proteomics, 2010, 73(10): 1907-1920. [16]Canton I, Battaglia G. Endocytosis at the nanoscale[J]. Chem Soc Rev, 2012, 41(7): 2718-2739.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133