Kanic acid effects on the differentiation and proliferation of neural stem cells

doi:10.3969/j.issn.2095-4344.2014.14.017

Abstract

Abstract:

BACKGROUND: The role of neural stem cells to repair brain tissue is very limited. About 80% of endogenous neural stem cells will die within the first 6 weeks, and only 0.2% of the cells continue to proliferate, differentiate, and participate in the brain repair.
OBJECTIVE: To study the effects of kanic acids at different concentrations on the differentiation and proliferation of neural stem cells．
METHODS: The neural stem cells from newborn Wistar rats were isolated and cultured in vitro, and then divided into two groups: blank control group and experimental group. We treated the second group by adding different concentration gradients of kainic acid. Then the cells were identified by immumofluorescence and immunohistochemistry methods, cell differentiation was detected by MTT colorimetric assay and the percentages of neurons and astrocytes were calculated.
RESULTS AND CONCLUSION: The adherent neurospheres in the experimental group proliferated more rapid and showed longer migration distance than those in the blank control group. After 5 days of differentiation, the number of astrocytes in the experimental group was higher, but the number of neuron-like cells was lower compared with the blank control group. The cultured cells had self-renew and multipotent differentiation potential. Kanic acid could induce the neural stem cells to differentiate toward astrocytes efficiently.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, neural stem cells, neurons, cell proliferation, cell differentiation

CLC Number:

R394.2

Sun Gui-fang, Yuan Zhi-hao, Peng Tao, Wang Jing-tao, Fu Zhen-qiang, Zhang Bo-ai . Kanic acid effects on the differentiation and proliferation of neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(14): 2238-2243.

Figures/Tables 2

2.1 神经干细胞鉴定巢蛋白抗原表达：巢蛋白免疫荧光检测显示：神经干细胞球经分散后形成单细胞，再增殖，传导至第3代形成的神经干细胞球呈巢蛋白抗原阳性(图1A)。多潜能分化特性：将传代3代以上的神经干细胞在撤去碱性成纤维生长因子的培养基中贴壁分化培养10 d后，使神经干细胞贴壁分化，形成神经元、星形胶质细胞、少突胶质细胞3种细胞，分别做抗神经元特异性烯醇化酶、胶质纤维酸性蛋白、2，3-环核苷酸磷酸二酯酶免疫荧光染色，用免疫荧光检测，可检测到神经干细胞分化后的3种神经细胞：神经元特异性烯醇化酶、胶质纤维酸性蛋白、2，3-核苷酸磷酸二酯酶阳性细胞(图1B，C，D)。 2.2 海人酸对神经干细胞分化的影响显微镜下观察结果：通过倒置显微镜下观察，发现空白对照组中悬浮神经球均可贴壁，折光性好。而海人酸组神经球贴壁率较低，培养孔中可见大量贴壁后又悬浮起来的神经球，神经球变黑，折光性变差，继续培养，这些未贴壁的神经球逐渐死亡，可见有絮状物和较多细胞碎片漂浮在培养基中，贴壁的神经球继续分化，但分化数天后，实验发现海人酸组贴壁的神经球分化速度较空白对照组快，在同一时间点进行观察，神经细胞的迁移距离较未处理组远。分化5 d后观察，海人酸组所分化的细胞中，星状细胞，胞体为扁平形，突起细长，突起伸展在细胞之间且交织成网状，较空白对照组多，而神经元样细胞相对较少(图2)。免疫组织化学法检测神经干细胞分化结果：实验结果显示：空白对照组神经元和胶质细胞平均比例为(23.07±1.51)%和(76.93±1.51)%；海人酸组神经元和胶质阳性细胞平均比例为(13.99±2.07)%和(86.01±2.07)%，相比差异有显著性意义(F=377.4，P < 0.01，表1)。 2.3 MTT法检测神经干细胞在加入海人酸后细胞的分化速度的改变与空白对照组相比，在第2天时海人酸组 (1 mmol/L海人酸组)细胞A值明显下降，与空白对照组相比在2，3 d时A值差异有显著性意义(P < 0.05)，但海人酸组细胞分化较快，从第4天起A值与空白对照组比较，差异无显著性意义(P > 0.05，表2)。"

References

[1] Nochi R, Kato T, Kaneko J,et al. Involvement of metabotropic glutamate receptor 5 signaling in activity-related proliferation of adult hippocampalneural stem cells. Eur J Neurosci. 2012; 36(3):2273-2283.
[2] Bunk EC, König HG, Bonner HP, et al.NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation. Brain Res. 2010;1359:22-32.
[3] Ciceroni C, Mosillo P, Mastrantoni E, et al. mGLU3 metabotropic glutamate receptors modulate the differentiation of SVZ-derived neural stem cells towards the astrocytic lineage. Glia. 2010;58(7):813-822.
[4] Kantrowitz JT, Javitt DC. N-methyl-d-aspartate (NMDA) receptor dysfunction or dysregulation: the final common pathway on the road to schizophrenia? Brain Res Bull. 2010; 83(3-4):108-121.
[5] Yamashita T, Ninomiya M, Hernandez Acosta P, et al. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. J Neurosci. 2006;26(24):6627-6636.
[6] Tania Aguado, Eva Romero, Krisztina Monory, et al. The CB1 cannabinoid receptor mediates excitotoxicity-induced neural progenitor proliferation and neurogenesis. Biol Chem. 2007; 282:23892-23898.
[7] Jin K, Minami M, Lan JQ, et al. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Aead Sci.2001;98: 4710-4715.
[8] Jin K, Peel AL, Mao XO, et al. Increased hippocampal neurogenesis in Alzheimer's disease. Proc Natl Acad Sci U S A. 2004;101(1):343-347.
[9] Azami Tameh A, Clarner T, et al. Regional regulation of glutamate signaling during cuprizone-induced demyelination in the brain. Ann Anat. 2013;195(5):415-423.
[10] Gu N, Jackson J, Goutagny R, et al.NMDA-dependent phase synchronization between septal and temporal CA3 hippocampal networks. J Neurosci. 2013;33(19):8276-8287.
[11] Kim HJ, Kim TH, Choi SJ, et al. Fluoxetine suppresses synaptically induced[Ca2+]i spikes and excitotoxicity in cultured rat hippocampal neurons. Brain Res. 2013;1490: 23-34.
[12] Gibson CJ, Meyer RC, Hamm RJ. Traumatic brain injury and the effects of diazepam, diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus. J Biomed Sci. 2010;17(1): 38.
[13] Shacka JJ, Lu J, Xie ZL, et al. Kanic acid induces early and transient autophagic stress in mouse hippocampus. Neurosci Lett. 2007; 414(1):57-60.
[14] Koga K, Sim SE, Chen T,et al. Kainate receptor-mediated synaptic transmissions in the adult rodent insular cortex. J Neurophysiol. 2012;108(7):1988-1998.
[15] Meade AJ, Meloni BP, Mastaglia FL, et al. AP-1 inhibitory peptides attenuate in vitro cortical neuronal cell death induced by kainic acid. Brain Res. 2010;1360:8-16.
[16] Sun Y, Jin K, Childs JT, et al. Vascular endothelial growth factor-B ( VEGFB ) stimulates neurogenesis: evidence from knockout mice and growth factor administration. Dev Biol. 2006;289(2):329-335.
[17] Li Hua, Chen ZJ, Zhou SP. Apoptos,s in glioma-bearing rats after neural stem cell transplantation. Neural Regeneration Research. 2013,8(19):1793-1802.
[18] 张军,张琰君,贾云嶂,等. 碱性成纤维细胞生长因子对放射性诱导小鼠C17.2神经干细胞凋亡的影晌[J].神经解剖学杂志,2014, 30(1):98-102.
[19] 邓鑫,宋来君,郭新宾.血管内皮生长因子165对神经干细胞生长及分化的影响[J].中华脑科疾病与康复杂志(电子版),2013,3(3): 23-26.
[20] 张君度,龙大宏,陈艳,等.胶质细胞源性神经营养因子诱导神经干细胞分化过程中bHLH转录因子的表达变化[J].解剖学研究, 2013, 35(6):427-430.
[21] Sharma S, Rakoczy S, Dahlheimer K, et al.The hippocampus of Ames dwarf mice exhibits enhanced antioxidative defenses following kainic acid-induced oxidative stress. Exp Gerontol. 2010;45(12):936-949.
[22] Spaethling J, Le L, Meaney DF. NMDA receptor mediated phosphorylation of GluR1 subunits contributes to the appearance of calcium-permeableAMPA receptors after mechanical stretch injury. Neurobiol Dis. 2012;46(3):646-654.
[23] Lee KH, Cho JH, Choi IS, et al. Pregnenolone sulfate enhances spontaneous glutamate release by inducing presynaptic Ca2+-induced Ca2+ release. Neuroscience. 2010;171(1):106-116.
[24] Santiago AR, Carvalho CM, Carvalho AP, et al. Differential contribution of L-, N-, and P/Q-type calcium channels to [Ca2+]i changes evoked by kainate in hippocampal neurons. Neurochem Res, 2008;33(8):1501-1508.
[25] Pilli J, Kumar SS. Triheteromeric N-methyl-D-aspartate receptors differentiate synaptic inputs onto pyramidal neurons in somatosensory cortex: involvement of the GluN3A subunit. Neuroscience. 2012;222:75-88.
[26] Tsang SW, Vinters HV, Cummings JL, et al. Alterations in NMDA receptor subunit densities and ligand binding to glycine recognition sites are associated with chronic anxiety in Alzheimer's disease. Neurobiol Aging. 2008;29(10): 1524-1532.
[27] Rangel A, Burgaya F, Gavín R, et al. Enhanced susceptibility of Prnp-deficient mice to kainate-induced seizures, neuronal apoptosis, and death: Role of AMPA/kainate receptors. J Neurosci Res. 2007 ;85(12):2741-2755.
[28] Li Y, Chopp M. Temporal profile of nestin expression after focal cerebralischemia in adult rat. Brain Res. 1999;838 (1-2): 1-10.
[29] Panaqiotakos G, ALshamy G, Chan B, et al. Long-term impact of radiation on the stem cell and oligodendrocyte precursors in the brain. PLoS ONE. 2007;2(7): e588.
[30] Ruocco LA, Gironi Carnevale UA, Sica A, et al. Differential prepuberal handling modifies behaviour and excitatory amino acids in the forebrain of the Naples High-Excitability rats. Behav Brain Res. 2009;198(1):29-36.
[31] Daadi MM, Davis AS, Arac A,et al. Human neural stem cell grafts modify microglial response and enhance axonal sprouting in neonatal hypoxic-ischemic brain injury. Stroke. 2010;41(3):516-523.
[32] Csoknya M, Dénes V, Wilhelm M. Glial cells in the central nervous system of earthworm, Eisenia fetida.Acta Biol Hung. 2012;63 Suppl 1:114-128.
[33] Sivakumar V, Ling EA, Lu J, et al. Role of glutamate and its receptors and insulin-like growth factors in hypoxia induced periventricular white matter injury.Glia. 2010;58(5):507-523.
[34] Park DH, Eve DJ, Sanberg PR, et al. Increased neuronal proliferation in the dentate gyrus of aged rats following neural stem cell implantation. Stem Cells Dev.2010;19(2):175-180.
[35] Daadi MM, Davis AS, Arac A, et al. Human neural stem cell grafts modify microglial response and enhance axonal sprouting in neonatal hypoxic-ischemic brain injury. Stroke. 2010 ;41(3):516-523.