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星形胶质细胞外泌体对缺氧缺血神经元的保护作用
黄静兰, 屈艺, 唐军, 邹蓉, 李世平, 李雅斐, 张莉, 夏斌, 母得志
中国当代儿科杂志 ›› 2018, Vol. 20 ›› Issue (5) : 397-402.
PDF(1375 KB)
PDF(1375 KB)
星形胶质细胞外泌体对缺氧缺血神经元的保护作用
Protective effect of astrocyte exosomes on hypoxic-ischemic neurons
目的 探索星形胶质细胞外泌体对缺氧缺血神经元的影响。方法 体外培养大鼠星形胶质细胞，通过差速离心法获取细胞上清液中的外泌体。采用透射电镜、Nanosight和Western blot鉴定外泌体；采用BCA法检测外泌体蛋白浓度。体外培养大鼠神经元，分为对照组、外泌体组、氧糖剥夺（OGD）组和OGD+外泌体组（n=3）。OGD组和OGD+外泌体组给予无糖培养基和缺氧处理；外泌体组和OGD+外泌体组分别给予终浓度为22 μg/mL的外泌体处理，对照组和OGD组给予等体积PBS处理。采用ELISA法检测神经元乳酸脱氢酶（LDH）水平；TUNEL法检测神经元凋亡指数。结果 外泌体鉴定结果显示差速离心法提取的外泌体符合外泌体特点；与对照组和外泌体组相比，OGD组LDH值和凋亡指数显著增加（P < 0.05）；与OGD组相比，OGD+外泌体组LDH值和凋亡指数均显著降低（P < 0.05）。结论 星形胶质细胞来源的外泌体对神经元缺氧缺血损伤有保护作用。
Objective To study the effect of astrocyte exosomes on hypoxic-ischemic neurons. Methods Rat astrocytes were cultured in vitro, and differential centrifugation was used to obtain the exosomes from the cell supernatant. Transmission electron microscopy, Nanosight, and Western blot were used for the identification of exosomes. BCA method was used to measure the concentration of exosomes. Rat neurons were cultured in vitro and then divided into control group, exosome group, oxygen glucose deprivation (OGD) group, and OGD+exosome group (n=3 each). The OGD and OGD+exosome groups were cultured in glucose-free medium under the hypoxic condition. The exosome and OGD+exosome groups were treated with exosomes at a final concentration of 22 μg/mL. The control and OGD groups were given an equal volume of phosphate-buffered saline. ELISA was used to measure the level of lactate dehydrogenase (LDH) in neurons. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to measure the apoptotic index of neurons. Results The identification of exosomes showed that the exosomes extracted by differential centrifugation had the features of exosomes. Compared with the control and exosome groups, the OGD group had significant increases in LDH level and apoptotic index (P < 0.05). Compared with the OGD group, the OGD+exosome group had significant reductions in LDH level and apoptotic index (P < 0.05). Conclusions The exosomes from astrocytes have a protective effect on neurons with hypoxic-ischemic injury.
星形胶质细胞 / 外泌体 / 神经元 / 缺氧 / 缺血 / 大鼠
[1] Shankaran S, Laptook AR, Ehrenkranz RA, et al. Wholebody hypothermia for neonates with hypoxic-ischemic encephalopathy[J]. N Engl J Med, 2005, 353(15):1574-1584.
[2] Edwards AD, Brocklehurst P, Gunn AJ, et al. Neurological outcomes at 18 months of age after moderate hypothermia for perinatal hypoxic ischaemic encephalopathy:synthesis and meta-analysis of trial data[J]. BMJ, 2010, 340:c363.
[3] Pappas A, Shankaran S, McDonald SA, et al. Cognitive outcomes after neonatal encephalopathy[J]. Pediatrics, 2015, 135(3):e624-e634.
[4] 黄静兰, 康冰瑶, 屈艺, 等. 外泌体对缺血再灌注器官损伤的保护作用[J]. 中国修复重建外科杂志, 2017, 31(6):751-754.
[5] Verkhratsky A, Matteoli M, Parpura V, et al. Astrocytes as secretory cells of the central nervous system:idiosyncrasies of vesicular secretion[J]. EMBO J, 2016, 35(3):239-257.
[6] Théry C, Amigorena S, Raposo G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids[J]. Curr Protoc Cell Biol, 2006, 30(1):3.22.1-3.22.29.
[7] Qu Y, Mao M, Zhao F, et al. Proapoptotic role of human growth and transformation-dependent protein in the developing rat brain after hypoxia-ischemia[J]. Stroke, 2009, 40(8):2843-2848.
[8] Qu Y, Shi J, Tang Y, et al. MLKL inhibition attenuates hypoxiaischemia induced neuronal damage in developing brain[J]. Exp Neurol, 2016, 279:223-231.
[9] Zhu J, Qu Y, Lin Z, et al. Loss of PINK1 inhibits apoptosis by upregulating α-synuclein in inflammation-sensitized hypoxicischemic injury in the immature brains[J]. Brain Res, 2016, 1653:14-22.
[10] Ophelders DR, Wolfs TG, Jellema RK, et al. Mesenchymal stromal cell-derived extracellular vesicles protect the fetal brain after hypoxia-ischemia[J]. Stem Cells Transl Med, 2016, 5(6):754-763.
[11] Zhang Y, Chopp M, Liu XS, et al. Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons[J]. Mol Neurobiol, 2017, 54(4):2659-2673.
[12] Li Y, Yang YY, Ren JL, et al. Exosomes secreted by stem cells from human exfoliated deciduous teeth contribute to functional recovery after traumatic brain injury by shifting microglia M1/M2 polarization in rats[J]. Stem Cell Res Ther, 2017, 8(1):198.
[13] Huang S, Ge X, Yu J, et al. Increased miR-124-3p in microglial exosomes following traumatic brain injury inhibits neuronal inflammation and contributes to neurite outgrowth via their transfer into neurons[J]. FASEB J, 2018, 32(1):512-528.
[14] Zhang L, Zhang S, Yao J, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth[J]. Nature, 2015, 527(7576):100-104.
[15] Cai S, Shi GS, Cheng HY, et al. Exosomal miR-7 mediates bystander autophagy in lung after focal brain irradiation in mice[J]. Int J Biol Sci, 2017, 13(10):1287-1296.
[16] Nafar F, Williams JB, Mearow KM. Astrocytes release HspB1 in response to amyloid-β exposure in vitro[J]. J Alzheimers Dis, 2016, 49(1):251-263.
国家自然科学基金（81330016；81630038；81771634）；国家科技部重大专项（2017YFA0104200）；四川省科技计划项目（2016TD0002）；国家临床重点专科（儿科新生儿专业）建设项目（1311200003303）。
