氧糖剥夺-再灌注星型胶质细胞内皮素-1过表达对神经干/祖细胞增殖的影响
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摘要
目的探讨氧糖剥夺-再灌注(OGD-R)星型胶质细胞内皮素(ET)-1过表达对神经干/祖细胞(NSPCs)增殖的影响。方法构建阴性对照星型胶质细胞(C6-Mock)和ET-1过表达的星形胶质细胞(C6-ET-1)OGD-R模型,并构建星型胶质细胞与NSPCs Transwell共培养体系。对星型胶质细胞及原代NSPCs进行形态观察及鉴定;将细胞分为以下4组进行共培养:C6-Mock+NSPCs组,OGD-R+C6-Mock+NSPCs组,C6-ET-1+NSPCs组,OGD-R+C6-ET-1+NSPCs组,共培养0、24、48、72 h,分析各组NSPCs神经球直径。结果 C6-Mock及C6-ET-1均呈现Ⅰ型星型胶质细胞的纤维状形态,神经胶质酸性纤维蛋白(GFAP)表达于这两种细胞的胞质中。原代NSPCs的巢蛋白(nestin)染色阳性。共培养后的48 h及72 h,OGD-R+C6-Mock+NSPCs组神经球的直径明显大于C6-Mock+NSPCs组的直径,OGD-R+C6-ET-1+NSPCs组神经球的直径明显大于C6-ET-1+NSPCs组的直径,同时OGD-R+C6-ET-1+NSPCs组神经球的直径明显大于OGD-R+C6-Mock+NSPCs组的直径,差异均有统计学意义(均为P<0.05)。结论 OGD-R星型胶质细胞促进NSPCs的增殖,且ET-1过表达进一步促进了NSPCs的增殖。
Objective To investigate the effect of oxygen glucose deprivation-reperfusion(OGD-R) in astrocytes overexpressing endothelin(ET)-1 on the proliferation of neural stem/progenitor cells(NSPCs). Methods OGD-R models of negative control astrocytes(C6-Mock) and astrocytes over-expressing ET-1(C6-ET-1) were constructed. Transwell coculture system of astrocytes and NSPCs was established. Morphologic observation and identification of the astrocytes and primary NSPCs were performed. The cells were divided into four groups: C6-Mock+NSPCs, OGD-R+C6-Mock+NSPCs, C6-ET-1+NSPCs and OGD-R+C6-ET-1+NSPCs groups and co-cultured for 0, 24, 48 and 72 h respectively. The diameter of neurosphere was measured in each group. Results In the C6-Mock and C6-ET-1 cells, type Ⅰ astrocytes in fibrous morphology were observed. Glial fibrillary acidic protein(GFAP) was expressed in the cytoplasm of these two types of cells. Primary NSPCs were positive for nestin staining. After co-culture for 48 and 72 h, the neurosphere diameter in the OGD-R+C6-Mock+NSPCs group was significantly greater than that in the C6-Mock+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was considerably greater than that in the C6-ET-1+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was significantly greater compared with that in the OGD-R+C6-Mock+NSPCs group(all P<0.05). Conclusions OGD-R astrocytes can promote the proliferation of NSPCs. ET-1 over-expression further accelerates the proliferation of NSPCs.
Objective To investigate the effect of oxygen glucose deprivation-reperfusion(OGD-R) in astrocytes overexpressing endothelin(ET)-1 on the proliferation of neural stem/progenitor cells(NSPCs). Methods OGD-R models of negative control astrocytes(C6-Mock) and astrocytes over-expressing ET-1(C6-ET-1) were constructed. Transwell coculture system of astrocytes and NSPCs was established. Morphologic observation and identification of the astrocytes and primary NSPCs were performed. The cells were divided into four groups: C6-Mock+NSPCs, OGD-R+C6-Mock+NSPCs, C6-ET-1+NSPCs and OGD-R+C6-ET-1+NSPCs groups and co-cultured for 0, 24, 48 and 72 h respectively. The diameter of neurosphere was measured in each group. Results In the C6-Mock and C6-ET-1 cells, type Ⅰ astrocytes in fibrous morphology were observed. Glial fibrillary acidic protein(GFAP) was expressed in the cytoplasm of these two types of cells. Primary NSPCs were positive for nestin staining. After co-culture for 48 and 72 h, the neurosphere diameter in the OGD-R+C6-Mock+NSPCs group was significantly greater than that in the C6-Mock+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was considerably greater than that in the C6-ET-1+NSPCs group. The neurosphere diameter in the OGD-R+C6-ET-1+NSPCs group was significantly greater compared with that in the OGD-R+C6-Mock+NSPCs group(all P<0.05). Conclusions OGD-R astrocytes can promote the proliferation of NSPCs. ET-1 over-expression further accelerates the proliferation of NSPCs.
引文
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[21]黄红云,毛更生,陈琳,等.神经修复临床细胞治疗现状与展望[J/CD].中华细胞与干细胞杂志(电子版),2017,7(3):162-167.DOI:10.3877/cma.j.issn.2095-1221.2017.03.008.HUANG HY,MAO GS,CHEN L,et al.Current status and prospect of cell therapy in neurorestoration[J/CD].Chin J Cell Stem Cell(Electr Edit),2017,7(3):162-167.DOI:10.3877/cma.j.issn.2095-1221.2017.03.008.
[22]BARONE FC,GLOBUS MY,PRICE WJ,et al.Endothelin levels increase in rat focal and global ischemia[J].J Cereb Blood Flow Metab,1994,14(2):337-342.
[2]BUTTI E,CUSIMANO M,BACIGALUPPI M,et al.Neurogenic and non-neurogenic functions of endogenous neural stem cells[J].Front Neurosci,2014,8:92.DOI:10.3389/fnins.2014.00092.
[3]YONEYAMA M,SHIBA T,HASEBE S,et al.Adult neurogenesis is regulated by endogenous factors produced during neurodegeneration[J].J Pharmacol Sci,2011,115(4):425-432.
[4]SIMS JR,LEE SW,TOPALKARA K,et al.Sonic hedgehog regulates ischemia/hypoxia-induced neural progenitor proliferation[J].Stroke,2009,40(11):3618-3626.DOI:10.1161/STROKEAHA.109.561951.
[5]NAKAGOMI T,TAGUCHI A,FUJIMORI Y,et al.Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice[J].Eur JNeurosci,2009,29(9):1842-1852.DOI:10.1111/j.1460-9568.2009.06732.x.
[6]GO HS,SHIN CY,LEE SH,et al.Increased proliferation and gliogenesis of cultured rat neural progenitor cells by lipopolysaccharide-stimulated astrocytes[J].Neuroimmunomodulation,2009,16(6):365-376.DOI:10.1159/000228911.
[7]LEE C,HU J,RALLS S,et al.The molecular profiles of neural stem cell niche in the adult subventricular zone[J].PLoS One,2012,7(11):e50501.DOI:10.1371/journal.pone.0050501.
[8]CAO X,LI LP,QIN XH,et al.Astrocytic adenosine5'-triphosphate release regulates the proliferation of neural stem cells in the adult hippocampus[J].Stem Cells,2013,31(8):1633-1643.DOI:10.1002/stem.1408.
[9]GUO Y,WEI Q,HUANG Y,et al.The effects of astrocytes on differentiation of neural stem cells are influenced by knock-down of the glutamate transporter,GLT-1[J].Neurochem Int,2013,63(5):498-506.DOI:10.1016/j.neuint.2013.08.003.
[10]TSANG MC,LO AC,CHEUNG PT,et al.Perinatal hypoxia-/ischemia-induced endothelin-1 mRNA in astrocyte-like and endothelial cells[J].Neuroreport,2001,12(10):2265-2270.
[11]YE H,WOLF RA,KURZ T,et al.Phosphatidic acid increases in response to noradrenaline and endothelin-1in adult rabbit ventricular myocytes[J].Cardiovasc Res,1994,28(12):1828-1834.
[12]SAH SK,KIM BH,PARK GT,et al.Novel isonahocol E(3)exhibits anti-inflammatory and anti-angiogenic effects in endothelin-1-stimulated human keratinocytes[J].Eur J Pharmacol,2013,720(1/2/3):205-211.
[13]HO MC,LO AC,KURIHARA H,et al.Endothelin-1protects astrocytes from hypoxic/ischemic injury[J].FASEB J,2001,15(3):618-626.
[14]RODRIGUEZ AL,BRUGGEMAN KF,WANG Y,et al.Using minimalist self-assembling peptides as hierarchical scaffolds to stabilise growth factors and promote stem cell integration in the injured brain[J].J Tissue Eng Regen Med,2018,12(3):e1571-e1579.DOI:10.1002/term.2582.
[15]NGUYEN HX,HOOSHMAND MJ,SAIWAI H,et al.Systemic neutrophil depletion modulates the migration and fate of transplanted human neural stem cells to rescue functional repair[J].J Neurosci,2017,37(38):9269-9287.DOI:10.1523/JNEUROSCI.2785-16.2017.
[16]PILTTI KM,FUNES GM,AVAKIAN SN,et al.Increasing human neural stem cell transplantation dose alters oligodendroglial and neuronal differentiation after spinal cord injury[J].Stem Cell Reports,2017,8(6):1534-1548.DOI:10.1016/j.stemcr.2017.04.009.
[17]李明军,张琦玮,王维峰,等.神经干细胞的应用[J].中国组织工程研究与临床康复,2008,12(29):5709-5714.LI MJ,ZHANG QW,WANG WF,et al.Application of neural stem cells[J].Chin J Clin Rehabil Tissue Eng Res,2008,12(29):5709-5714.
[18]LO AC,CHEN AY,HUNG VK,et al.Endothelin-1overexpression leads to further water accumulation and brain edema after middle cerebral artery occlusion via aquaporin 4 expression in astrocytic end-feet[J].J Cereb Blood Flow Metab,2005,25(8):998-1011.
[19]LI M,SUN L,LI Y,et al.Oxygen glucose deprivation/reperfusion astrocytes promotes primary neural stem/progenitor cell proliferation by releasing high-mobility group box 1[J].Neurochem Res,2014,39(8):1440-1450.DOI:10.1007/s11064-014-1333-z.
[20]ZHAO X,KUJA-PANULA J,ROUHIAINEN A,et al.High mobility group box-1(HMGB1;amphoterin)is required for zebrafish brain development[J].J Biol Chem,2011,286(26):23200-23213.DOI:10.1074/jbc.M111.223834.
[21]黄红云,毛更生,陈琳,等.神经修复临床细胞治疗现状与展望[J/CD].中华细胞与干细胞杂志(电子版),2017,7(3):162-167.DOI:10.3877/cma.j.issn.2095-1221.2017.03.008.HUANG HY,MAO GS,CHEN L,et al.Current status and prospect of cell therapy in neurorestoration[J/CD].Chin J Cell Stem Cell(Electr Edit),2017,7(3):162-167.DOI:10.3877/cma.j.issn.2095-1221.2017.03.008.
[22]BARONE FC,GLOBUS MY,PRICE WJ,et al.Endothelin levels increase in rat focal and global ischemia[J].J Cereb Blood Flow Metab,1994,14(2):337-342.