目的研究水通道蛋白4 (AQP4)在体外培养大鼠星形胶质细胞中的表达规律。方法取新生1 d Wistar大鼠大脑皮层行星形胶质细胞原代培养,分别于原代培养3 d、5 d、7 d、9 d及传代培养9 d行胶质纤维酸性蛋白(GFAP)免疫荧光染色;实时荧光定量聚合酶链反应及AQP4免疫荧光染色检测AQP4 mRNA及蛋白的表达。结果原代及传代培养不同时间点95%以上细胞GFAP免疫荧光染色阳性,且GFAP表达量逐渐增加。原代培养3 d有AQP4 mRNA表达,3 d和5 d AQP4 mRNA表达水平无显著性差异(P>0.05),7 d AQP4 mRNA表达升高(P<0.05),9 d持续升高(P<0.05);传代培养9 d与原代培养9 d AQP4 mRNA表达无显著性差异(P>0.05)。AQP4蛋白表达与AQP4 mRNA表达一致。结论大鼠星形胶质细胞原代培养3 d即有AQP4表达,之后逐渐增加,9 d左右表达渐趋稳定。
ObjectiveTo explore the expression of aquaporin 4 (AQP4) in primary and secondary cultured rat astrocytes. MethodsRat cortical astrocytes from a newborn (one day) Wistar rat were cultured. Astrocytes were identified with immunofluorescence staining of glial fibrilillary acidic protein (GFAP). The expression of AQP4 was determined with real-time quantitative polymerase chain reaction and immunofluorescence staining three, five, seven and nine days of primary culture, and nine days of secondary culture. ResultsThe purity of GFAP-positive cells was more than 95%. The expression of AQP4 mRNA was found three days of primary culture, remained unchanged five days of primary culture (P>0.05), and increased seven and nine days of primary culture (P<0.05). The expression of AQP4 mRNA was not different between nine days of primary culture and nine days of secondary culture (P>0.05). AQP4 immunofluorescence staining showed the same trend of AQP4 mRNA. ConclusionAQP4 may express since three days of primary culture in rat astrocytes in vitro, and increase slowly until nine days of primary culture.
[1] Papadopoulos MC, Verkman AS. Aquaporin water channels in the nervous system [J]. Nat Rev Neurosci, 2013, 14(4): 265-277.
[2] Filippidis AS, Carozza RB, Rekate HL. Aquaporins in brain edema and neuropathological conditions [J]. Int J Mol Sci, 2016, 18(1): 55.
[3] Verkman AS, Anderson MO, Papadopoulos MC. Aquaporins: important but elusive drug targets [J]. Nat Rev Drug Discov, 2014, 13(4): 259-277.
[4] 徐仟,孙振荣,李桂林,等. 人颞叶内侧癫痫海马组织星形胶质细胞水通道蛋白4和内向整流性钾离子通道4.1的再分布[J]. 中国康复理论与实践, 2012, 18(3): 215-218.
[5] Nielsen S, Nagelhus EA, Amiry-Moghaddam M, et al. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain [J]. J Neurosci, 1997, 17(1): 171-180.
[6] Yamamoto N, Yoneda K, Asai K, et al. Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation [J]. Brain Res Mol Brain Res, 2001, 90(1): 26-38.
[7] McCarthy KD, Vellis JD. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue [J]. J Cell Biol, 1980, 85(3): 890-902.
[8] Yuan F, Wang T. Glutamate-induced swelling of cultured astrocytes is mediated by metabotropic glutamate receptor [J]. Sci China C Life Sci, 1996, 39(5): 517-522.
[9] Shi ZF, Zhao WJ, Xu LX, et al. Downregulation of aquaporin 4 expression through extracellular signal-regulated kinases1/2 activation in cultured astrocytes following scratch-injury [J]. Biomed Environ Sci, 2015, 28(3): 199-205.
[10] 王晓璇,孙振荣,吴敏,等. 人颞叶内侧癫痫海马组织星形胶质细胞Syntrophin的表达变化[J]. 中国康复理论与实践, 2017, 23(3): 292-297.
[11] Shi Z, Zhang W, Lu Y, et al. Aquaporin 4-mediated glutamate-induced astrocyte swelling is partially mediated through metabotropic glutamate receptor 5 activation [J]. Front Cell Neurosci, 2017, 11: 116.
[12] Stokum JA, Kurland DB, Gerzanich V, et al. Mechanisms of astrocyte-mediated cerebral edema [J]. Neurochem Res, 2015, 40(2): 317-328.
[13] Thrane AS, Rangroo Thrane V, Nedergaard M. Drowning stars: reassessing the role of astrocytes in brain edema [J]. Trends Neurosci, 2014, 37(11): 620-628.
[14] Nuriya M, Hirase H. Involvement of astrocytes in neurovascular communication [J]. Prog Brain Res, 2016, 225: 41-62.
[15] Becerra-Calixto A, Cardona-Gómez GP. The role of astrocytes in neuroprotection after brain stroke: potential in cell therapy [J]. Front Mol Neurosci, 2017, 10: 88.
[16] Potokar M, Jorgacevski J, Zorec R. Astrocyte aquaporin dynamics in health and disease [J]. Int J Mol Sci, 2016, 17(7): 1121.
[17] 师忠芳,袁芳. 脑内水通道蛋白4表达调节的研究进展[J]. 中国康复理论与实践, 2007, 13(8): 724-726.
[18] Assentoft M, Larsen BR, MacAulay N. Regulation and function of AQP4 in the central nervous system [J]. Neurochem Res, 2015, 40(12): 2615-2627.
[19] Chu H, Huang C, Ding H, et al. Aquaporin-4 and cerebrovascular diseases [J]. Int J Mol Sci, 2016, 17(8): 1249.
[20] Tang G, Yang GY. Aquaporin-4: a potential therapeutic target for cerebral edema [J]. Int J Mol Sci, 2016, 17(10): 1413.
[21] Yang Z, Wang KK. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker [J]. Trends Neurosci, 2015, 38(6): 364-374.
[22] Hol EM, Pekny M. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system [J]. Curr Opin Cell Biol, 2015, 32: 121-130.
[23] 吴敏,方庆,师忠芳,等. 亚甲基蓝对大鼠局灶性脑缺血再灌注后血脑屏障的保护作用[J]. 中国康复理论与实践, 2016, 22(2): 125-131.
[24] Wen H, Nagelhus EA, Amiry-Moghaddam M, et al. Ontogeny of water transport in rat brain: postnatal expression of the aquaporin-4 water channel [J]. Eur J Neurosci, 1999, 11(3): 935-945.
[25] Li X, Gao J, Ding J, et al. Aquaporin-4 expression contributes to decreases in brain water content during mouse postnatal development [J]. Brain Res Bull, 2013, 94: 49-55.
