一种肠肌间神经丛胶质细胞无血清原代培养法的建立
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摘要
目的肠胶质细胞(enteric glial cell,EGC)在肠道多种生理及病理过程的调节中发挥重要作用,为了深入研究EGC的功能,排除在体及组织水平中神经体液因素的影响,获得一种简便高效的原代EGC分离培养方法非常必要。方法取小鼠结肠纵行肌-肌间神经丛组织,经消化分离肠胶质细胞,采用含血清和无血清培养基交替培养法去除成纤维细胞后常规传代;运用细胞免疫荧光染色法检测原代EGC纯度;运用胞内钙成像技术实时观察EGC内Ca2+浓度([Ca2+]i)变化以检测细胞活性。结果每次实验获得的EGC至传代前细胞量可达约6. 8×105;无血清培养后成纤维细胞污染显著降低;胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)阳性的EGC占细胞总数的98. 44%±1. 07%,钙结合蛋白S100β阳性EGC占93. 61%±3. 16%,GFAP与S100β双阳性EGC占93. 09%±2. 99%; 96. 00%±4. 00%的细胞在ATP诱导下发生胞内钙瞬变。结论结合无血清培养可有效去除成纤维细胞污染,得到纯度较高、活性较好的肌间神经丛EGC。
Objective Enteric glial cell( EGC) plays an important role in regulation of many physiological and pathological process in the intestine,in order to further study the function of EGC and exclude the influence of neurohumoral factors at the vivo and vitro level,it is necessary to obtain a simple and efficient method for isolation and primary culture of EGC. Methods Colonic myenteric plexus were digested and separated. The fibroblasts were removed by alternating culture with serum-containing and serum-free medium and then subcultured routinely. The purity and activity of primary EGC were detected by immunofluorescence and intracellular Ca2 +imaging.Results The number of EGC was about 6. 8 × 105 on each experiment before passage. After serum-free culture,fibroblasts decreased significantly; the percentage of immunoreactivities of glial fibrillary acidic protein( GFAP),S100β and both GFAP and S100β was98. 44% ± 1. 07%,93. 61% ± 3. 16% and 93. 09% ± 2. 99%,respectively. About 96% EGC manifested intracellular calcium transient when treated with ATP. Conclusion Combined with the serum-free culture,fibroblasts were removed and primary myenteric EGC with higher purity and activity could be obtained.
Objective Enteric glial cell( EGC) plays an important role in regulation of many physiological and pathological process in the intestine,in order to further study the function of EGC and exclude the influence of neurohumoral factors at the vivo and vitro level,it is necessary to obtain a simple and efficient method for isolation and primary culture of EGC. Methods Colonic myenteric plexus were digested and separated. The fibroblasts were removed by alternating culture with serum-containing and serum-free medium and then subcultured routinely. The purity and activity of primary EGC were detected by immunofluorescence and intracellular Ca2 +imaging.Results The number of EGC was about 6. 8 × 105 on each experiment before passage. After serum-free culture,fibroblasts decreased significantly; the percentage of immunoreactivities of glial fibrillary acidic protein( GFAP),S100β and both GFAP and S100β was98. 44% ± 1. 07%,93. 61% ± 3. 16% and 93. 09% ± 2. 99%,respectively. About 96% EGC manifested intracellular calcium transient when treated with ATP. Conclusion Combined with the serum-free culture,fibroblasts were removed and primary myenteric EGC with higher purity and activity could be obtained.
引文
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[14] Gulbransen B D,Sharkey K A. Purinergic neuron-to-glia signaling in the enteric nervous system[J]. Gastroenterology,2009,136(4):1349-1358.
[15] Boesman W,Cirillo C,Van den Abbeel V,et al. Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells[J]. Neurogastroenterol Motil,2013,25(2):e151-160.
[2] von Boyen G B,Steinkamp M, Reinshagen M, et al.Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia[J]. Gut,2004,53(2):222-228.
[3] Gautron L,Rutkowski J M,Burton M D,et al. Neuronal and nonneuronal cholinergic structures in the mouse gastrointestinal tract and spleen[J]. J Comp Neurol,2013,521(16):3741-3767.
[4] McClain J,Grubisic V,Fried D,et al. Ca2+responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice[J]. Gastroenterology,2014,146(2):497-507.
[5] Du F,Wang L,Qian W,et al. Loss of enteric neurons accompanied by decreased expression of GDNF and PI3K/Akt pathway in diabetic rats[J]. Neurogastroenterol Motil,2009,21(11):1229-e114.
[6] Devos D,Lebouvier T,Lardeux B,et al. Colonic inflammation in parkinson's disease[J]. Neurobiol Dis,2013,50:42-48.
[7] Bassotti G,Villanacci V,Cathomas G,et al. Enteric neuropathology of the terminal ileum in patients with intractable slow-transit constipation[J]. Hum Pathol,2006,37(10):1252-1258.
[8] Fujikawa Y,Tominaga K,Tanaka F,et al. Enteric glial cells are associated with stress-induced colonic hyper-contraction in maternally separated rats[J]. Neurogastroenterol Motil,2015,27(7):1010-1023.
[9] Smith T H,Ngwainmbi J,Grider J R,et al. An in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse[J]. J Vis Exp,2013(78).
[10] Ibiza S,Garcia-Cassani B,Ribeiro H,et al. Glial-cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence[J]. Nature,2016,535(7612):440-443.
[11] Sundaresa S,Meininger C A,Kang A J,et al. Gastrin induces nuclear export and proteasome degradation of menin in enteric glial cells[J]. Gastroenterology,2017,153(6):1555-1567.
[12] Komiyama T,Nakao Y,Toyama Y,et al. A novel technique to isolate adult schwann cells for an artificial nerve conduit[J]. J Neurosci Methods,2003,122(2):195-200.
[13]历俊华,万虹,翟晶,等.无血清饥饿法去除神经组织培养中的成纤维细胞[J].首都医科大学学报,2004,25(2):205-207.
[14] Gulbransen B D,Sharkey K A. Purinergic neuron-to-glia signaling in the enteric nervous system[J]. Gastroenterology,2009,136(4):1349-1358.
[15] Boesman W,Cirillo C,Van den Abbeel V,et al. Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells[J]. Neurogastroenterol Motil,2013,25(2):e151-160.