LIU Miaoqing,WANG Yongbiao,HUANG Xiaozhong, et al. Comparison of biological properties of enteric neural stem cells in two ways of culturing in vitro[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2015, 45(8): 566-.
摘要目的：对纤维连接蛋白（FN）包被和未包被两种方式培养的肠神经干细胞进行生物学特性比较，探索肠神经干细胞体外扩增合适的培养方式。方法：从孕15 d SD胎鼠肠管分离出肠神经干细胞，用FN包被和不包被两种方式原代培养第5天的神经球，通过形态学、免疫荧光细胞化学及流式细胞仪检测对其生长状态、增殖及分化能力进行比较。结果：与包被组相比，未包被组的原代培养第5天的神经球呈半贴壁生长，球体形态较规则，球体中央开始变黑，神经球周边未形成大量放射状的神经丝；荧光镜下发现2组原代神经球的Nestin、BrdU都显阳性；包被组的球体周围放射状神经丝Tuj-1显阳性。流式细胞仪检测结果显示，与包被组的（12.67±2.16）%相比，未包被组的Nestin阳性细胞百分比显著增高，为（20.69±2.15）%（P＜0.05）；与包被组的（12.69±1.13）%相比，未包被组的BrdU标记的增殖细胞百分比显著增加，为（35.82±2.11）%（P＜0.05）；与包被组的（21.77±2.53）%相比，未包被组的Tuj-1阳性细胞百分比显著减少，为（10.67±1.14）%（P＜0.05）。结论：未包被FN的体外培养加快了形成神经球的时间，且形态较规则，能较好地维持干细胞自我增殖，减少分化，适宜作为肠神经干细胞体外增殖培养方式。
Abstract：Objective: To find a suitable proliferation way of culturing enteric neural stem cells in vitro, and to compare the biological characteristics of stem cells which were cultured in dishes coated and uncoated with fibronectin (FN). Methods: Enteric neural stem cells were isloated from gut of 15 days rat embryos, the neurospheres of fifth days were extracted from two groups. Growth status, proliferation and differentiation capacity of cells were compared through morphological observation, immunofluorescence cytochemistry and flow cytometer analysis between the two groups. Results: Compared with the FN coated group, the neurospheres of fifth days were the state of semi-adherent growth, the neurospheral morphology was regular, and were beginning to get dark in the center of them, and the spheres did not form a large amount of nerve fiber. Under fluorescence microscope, the typical neurospheres which were shown positive to BrdU in two groups were found. Radial nerve fiber of the neurospheres were positive to Tuj-1 (neuronal axons). After culturing for 5 days, compared with treatment with FN group (12.67±2.16)%, flow cytometer analysis showed positive to Nestin of the FN uncoated group had inacresed significantly (20.69±2.15)% (P<0.05); BrdU which mark proliferative cell increased significantly (35.82±2.11)% vs FN group (12.69±1.13)% (P<0.05); The percentage of Tuj-1 positive to neuron decreased significantly (10.67±1.14)% vs FN group (21.77±2.53)% (P<0.05). Conclusion: The way of uncoating culture reduces the time of forming neurospheres which were regular shape; maintains stem cell self-proliferation, reduce enteric neural stem cell differentiation and is suitable for stem cell self-proliferation in vitro.
[1] Bondurand N, Natarajan D, Thapar N, et al. Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures[J]Development, 2003, 130(25): 6387-6400.
[2] 朱利斌, 刘征吉, 李仲荣, 等. 大鼠肠神经干细胞分离和体外培养的初步研究[J]. 温州医学院学报, 2007, 37(1): 5-8.
[3] 朱利斌, 刘征吉, 王爱和, 等. 大鼠肠神经干细胞体外培养和鉴定[J]. 中华实验外科杂志, 2007, 24(1): 122.
[4] De Giorgio R, Camilleri M. Human enteric neuropathies:morphology and molecular pathology[J]. Neurogastroenterol Motil, 2004, 16(5): 515-531.
[5] Metzger M, Bareiss PM, Danker T, et al. Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system[J]. Gastroenterology, 2009, 137(6): 2063-2073.
[6] 许汉鹏, 苟琳, 杨浩, 等. 中枢神经系统不同部位来源的神经干细胞在体外生长特性的比较[J]. 解剖学报, 2004, 35 (4): 358-362.
[7] 王永飚, 朱利斌, 刘征吉, 等. 肠与脑来源神经干细胞的增殖和分化特性比较[J]. 医学研究杂志, 2011, 40(3): 71-74.
[8] Mokry J, Subrtova D, Nemecek S. Differentiation of epidermal growth factor-responsive neural precursor cells with-in neurospheres[J]. Acta Medica (Hradec Kralove), 1996, 39(1): 7-20.
[9] 肖莉, 高亚, 刘勇. 比较胚胎和新生小鼠肠神经嵴干细胞体外增殖分化的实验研究[J]. 中华小儿外科杂志, 2007, 28(2): 73-77.
[10] Micci MA, Pasricha PJ. Neural stem cells for the treatment of disorders of the enteric nervous system: strategies and challenges[J]. Dev Dyn, 2007, 236(1): 33-43.
[11] Nakamura S, Takeda Y, Kanno M, et al. Application of bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody for the in vivo analysis of proliferative characteristics of human leukemia cells in bone marrows[J]. Oncology,1991, 48(4): 285-289.
