胚胎小鼠肠道神经元的分离和原代培养
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摘要
目的:建立一种简便的胚胎小鼠肠道神经元分离和培养方法。方法:分离12～13 d胎鼠肠道组织,制备单细胞悬液,用神经元专用培养基进行培养,观察神经元的生长情况。培养第3、5、7、9、14 d使用免疫细胞化学染色法,以β微管蛋白(Tu J1)反映神经元突起形态并用Sholl分析进行量化,以神经元核心抗原(Neu N)为标志物反映神经元的纯度。结果:培养24 h神经元贴壁,随培养天数延长神经元突起长度增加,数目增多,形态逐渐复杂,与邻近细胞突起相互连接,形成网络。与培养第3 d相比,第7、9、14 d神经元纯度明显增高(P <0.05)。结论:本方法分离培养的神经元纯度高,在神经元专用培养基中存活状态良好,未见胶质细胞明显增生。
Objective: To establish the method of isolation and culture of embryonic mice intestinal neurons. Methods:Guts of E12. 5 were dissociated and made single cell suspension,which were cultured in neuron specific medium observing the growth of neurons. The neurons were stained with immunocytochemistry on the day 3,5,7,9 and 14 of culturing in vitro. The morphological changes of neurons were displayed by anti-β-III tubulin( Tu J1) and analyzed quantitatively by Sholl analysis. Neuron specific protein( Neu N) was used as a marker to reflect the purity of neurons.Results: The neurons adhered to the wall at 24 hours after culturing in vitro. With the increase of the length and number of neurites,the number of primary neurites increased,and the morphology became more and more complicated. The neurites connected with the adjacent cells to form a network after several days. Compared with the 3 rd day of culture,the purity of neurons on the 7 th,9 th and 14 th day were significantly higher( P < 0. 05). Conclusion: Neurons isolated and cultured using this mothed had a high-purity. They were in good conditions without glial cells in neuron specific medium.
Objective: To establish the method of isolation and culture of embryonic mice intestinal neurons. Methods:Guts of E12. 5 were dissociated and made single cell suspension,which were cultured in neuron specific medium observing the growth of neurons. The neurons were stained with immunocytochemistry on the day 3,5,7,9 and 14 of culturing in vitro. The morphological changes of neurons were displayed by anti-β-III tubulin( Tu J1) and analyzed quantitatively by Sholl analysis. Neuron specific protein( Neu N) was used as a marker to reflect the purity of neurons.Results: The neurons adhered to the wall at 24 hours after culturing in vitro. With the increase of the length and number of neurites,the number of primary neurites increased,and the morphology became more and more complicated. The neurites connected with the adjacent cells to form a network after several days. Compared with the 3 rd day of culture,the purity of neurons on the 7 th,9 th and 14 th day were significantly higher( P < 0. 05). Conclusion: Neurons isolated and cultured using this mothed had a high-purity. They were in good conditions without glial cells in neuron specific medium.
引文
[1]Grubi2i'c V,Verkhratsky A,Zorec R,et al.Enteric glia regulate gut motility in health and disease[J].Brain Res Bull,2018,136:109-117.DOI:10.1016/j.brainresbull.2017.03.011.
[2]Schneider S,Wright CM,Heuckeroth RO.Unexpected roles for the second brain:enteric nervous system as master regulator of bowel function[J].Annu Rev Physiol,2019,81.DOI:10.1146/annurev-physiol-021317-121515.
[3]Muller PA,Koscso B,Rajani GM,et al.Crosstalk between muscularis macrophages and enteric neurons regulates gastrointestinal motility[J].cell,2014,158(2):300-313.DOI:10.1016/j.cell.2014.04.050.
[4]Bassotti G,Villanacci V,Maurer CA,et al.The role of glial cells and apoptosis of enteric neurons in the neuropathology of intractable slow transit constipation[J].Gut,2006,55(1):41-46.DOI:10.1136/gut.2005.073197.
[5]Desmet A,Cirillo C,Tack J,et al.Live calcium and mitochondrial imaging in the enteric nervous system of Parkinson patients and controls[J].e Life,2017,6.pii:e26850.DOI:10.7554/e Life.26850.
[6]Bessac A,Cani PD,Meunier E,et al.Inflammation and gut-brain axis during type 2 Diabetes:focus on the crosstalk between intestinal immune cells and enteric nervous system[J].Front Neurosci,2018,12:725.DOI:10.3389/fnins.2018.00725.
[7]Guerrero-Alba R,Valdez-Morales E,Jimenez-Vargas N,et al.Stress activates pronociceptive endogenous opioid signalling in DRGneurons during chronic colitis[J].Gut,2017,66(12):2121-2131.DOI:10.1136/gutjnl-2016-311456.
[8]Gracie DJ,Guthrie EA,Hamlin PJ,et al.Bi-directionality of braingut interactions in patients with inflammatory bowel disease[J].Gastroenterology,2018,154(6):1635-1646.DOI:10.1053/j.gastro.2018.01.027.
[9]Filpa V,Moro E,Protasoni M,et al.Role of glutamatergic neurotransmission in the enteric nervous system and brain-gut axis in health and disease[J].Neuropharmacology,2016,111:14-33.DOI:10.1016/j.neuropharm.2016.08.024.
[10]De Vadder F,Grasset E,Manners Holm L,et al.Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks[J].Proc Natl Acad Sci USA,2018,115(25):6458-6463.DOI:10.1073/pnas.1720017115.
[11]杨小薇,刘安军,任安经,等.小鼠子宫内胚胎电转技术方法的改良[J].解剖学报,2018:391-394.DOI:10.16098/j.issn.0529-1356.2018.03.020.
[12]赵又谊,张力,杨吉平,等.Wnt/β-catenin信号参与慢性痛引起的小鼠海马神经元发生减少[J].神经解剖学杂志,2017,33(6):652-658.DOI:10.16557/j.cnki.1000-7547.2017.06.002.
[13]Day JS,O’Neill E,Cawley C,et al.Noradrenaline acting on astrocyticβ2-adrenoceptors induces neurite outgrowth in primary cortical neurons[J].Neuropharmacology,2014,77:234-248.DOI:10.1016/j.neuropharm.2013.09.027.
[14]Sarnat HB.Immunocytochemical markers of neuronal maturation in human diagnostic neuropathology[J].Cell Tissue Res,2015,359(1):279-294.DOI:10.1007/s00441-014-1988-4.
[15]Thiere M,Kliche S,Müller B,et al.Integrin activation through the hematopoietic adapter molecule ADAP regulates dendritic development of hippocampal neurons[J].Front Mol Neurosci,2016,9:91.DOI:10.3389/fnmol.2016.00091.
[16]Bardy C,van den Hurk M,Eames T,et al.Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro[J].Proc Natl Acad Sci USA,2015,112(25):E2725-E2734.DOI:10.1073/pnas.1509741112.
[17]Hu W,He Z,Yang L,et al.The Ca2+channel inhibitor 2-APBreversesβ-amyloid-induced LTP deficit in hippocampus by blocking BAX and caspase-3 hyperactivation[J].Br J Pharmacol.2015,172(9):2273-2285.DOI:10.1111/bph.13048.
[18]刘检,王宇红,徐雅岚,等.改良的胎鼠海马神经元原代培养及模拟糖尿病并发抑郁环境对其的损伤作用[J].神经解剖学杂志,2016,32(4):459-465.DOI:10.16557/j.cnki.1000-7547.2016.04.007.
[19]Nolan AM,Collins LM,Wyatt SL,et al.The neurite growth inhibitory effects of soluble TNFαon developing sympathetic neurons are dependent on developmental age[J].Differentiation,2014,88(4-5):124-130.DOI:10.1016/j.diff.2014.12.006.
[20]Ristanovi'c D,Milo2evi'c N T,2tuli'c V.Application of modified Sholl analysis to neuronal dendritic arborization of the cat spinal cord[J].J Neurosci Methods,2006,158(2):212-218.DOI:10.1016/j.jneumeth.2006.05.030.
[2]Schneider S,Wright CM,Heuckeroth RO.Unexpected roles for the second brain:enteric nervous system as master regulator of bowel function[J].Annu Rev Physiol,2019,81.DOI:10.1146/annurev-physiol-021317-121515.
[3]Muller PA,Koscso B,Rajani GM,et al.Crosstalk between muscularis macrophages and enteric neurons regulates gastrointestinal motility[J].cell,2014,158(2):300-313.DOI:10.1016/j.cell.2014.04.050.
[4]Bassotti G,Villanacci V,Maurer CA,et al.The role of glial cells and apoptosis of enteric neurons in the neuropathology of intractable slow transit constipation[J].Gut,2006,55(1):41-46.DOI:10.1136/gut.2005.073197.
[5]Desmet A,Cirillo C,Tack J,et al.Live calcium and mitochondrial imaging in the enteric nervous system of Parkinson patients and controls[J].e Life,2017,6.pii:e26850.DOI:10.7554/e Life.26850.
[6]Bessac A,Cani PD,Meunier E,et al.Inflammation and gut-brain axis during type 2 Diabetes:focus on the crosstalk between intestinal immune cells and enteric nervous system[J].Front Neurosci,2018,12:725.DOI:10.3389/fnins.2018.00725.
[7]Guerrero-Alba R,Valdez-Morales E,Jimenez-Vargas N,et al.Stress activates pronociceptive endogenous opioid signalling in DRGneurons during chronic colitis[J].Gut,2017,66(12):2121-2131.DOI:10.1136/gutjnl-2016-311456.
[8]Gracie DJ,Guthrie EA,Hamlin PJ,et al.Bi-directionality of braingut interactions in patients with inflammatory bowel disease[J].Gastroenterology,2018,154(6):1635-1646.DOI:10.1053/j.gastro.2018.01.027.
[9]Filpa V,Moro E,Protasoni M,et al.Role of glutamatergic neurotransmission in the enteric nervous system and brain-gut axis in health and disease[J].Neuropharmacology,2016,111:14-33.DOI:10.1016/j.neuropharm.2016.08.024.
[10]De Vadder F,Grasset E,Manners Holm L,et al.Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks[J].Proc Natl Acad Sci USA,2018,115(25):6458-6463.DOI:10.1073/pnas.1720017115.
[11]杨小薇,刘安军,任安经,等.小鼠子宫内胚胎电转技术方法的改良[J].解剖学报,2018:391-394.DOI:10.16098/j.issn.0529-1356.2018.03.020.
[12]赵又谊,张力,杨吉平,等.Wnt/β-catenin信号参与慢性痛引起的小鼠海马神经元发生减少[J].神经解剖学杂志,2017,33(6):652-658.DOI:10.16557/j.cnki.1000-7547.2017.06.002.
[13]Day JS,O’Neill E,Cawley C,et al.Noradrenaline acting on astrocyticβ2-adrenoceptors induces neurite outgrowth in primary cortical neurons[J].Neuropharmacology,2014,77:234-248.DOI:10.1016/j.neuropharm.2013.09.027.
[14]Sarnat HB.Immunocytochemical markers of neuronal maturation in human diagnostic neuropathology[J].Cell Tissue Res,2015,359(1):279-294.DOI:10.1007/s00441-014-1988-4.
[15]Thiere M,Kliche S,Müller B,et al.Integrin activation through the hematopoietic adapter molecule ADAP regulates dendritic development of hippocampal neurons[J].Front Mol Neurosci,2016,9:91.DOI:10.3389/fnmol.2016.00091.
[16]Bardy C,van den Hurk M,Eames T,et al.Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro[J].Proc Natl Acad Sci USA,2015,112(25):E2725-E2734.DOI:10.1073/pnas.1509741112.
[17]Hu W,He Z,Yang L,et al.The Ca2+channel inhibitor 2-APBreversesβ-amyloid-induced LTP deficit in hippocampus by blocking BAX and caspase-3 hyperactivation[J].Br J Pharmacol.2015,172(9):2273-2285.DOI:10.1111/bph.13048.
[18]刘检,王宇红,徐雅岚,等.改良的胎鼠海马神经元原代培养及模拟糖尿病并发抑郁环境对其的损伤作用[J].神经解剖学杂志,2016,32(4):459-465.DOI:10.16557/j.cnki.1000-7547.2016.04.007.
[19]Nolan AM,Collins LM,Wyatt SL,et al.The neurite growth inhibitory effects of soluble TNFαon developing sympathetic neurons are dependent on developmental age[J].Differentiation,2014,88(4-5):124-130.DOI:10.1016/j.diff.2014.12.006.
[20]Ristanovi'c D,Milo2evi'c N T,2tuli'c V.Application of modified Sholl analysis to neuronal dendritic arborization of the cat spinal cord[J].J Neurosci Methods,2006,158(2):212-218.DOI:10.1016/j.jneumeth.2006.05.030.