• ISSN 1000-0615
  • CN 31-1283/S
Article Contents
Turn off MathJax

Citation:

Primary Cell Culture and Identification of brain neurons in Chinese perch (Siniperca chuatsi)

  • The diversity of species makes the research on cell level of Chinese perch extremely limited. Establishing a simple and feasible method for primary culture of brain neurons from Chinese perch (Siniperca chuatsi) in vitro is beneficial to further study on fish nervous system. Our laboratory combined with the usual method of cell culture, Chinese perch brain neruons (born 3 months) were isolated by collagenase digestion and mechanical blow. L15+20%FBS suspended brain neruons were inoculated on the cell culture vessels, in 28 °C without CO2 incubator. The medium was replaced after 3 days. Subculture was carried out after the cells covered with the bottom of the cell culture vessels. The morphological changes of the neurons were observed under inverted phase-contrast microscope; Immunofluorescence staining for NeuN or β-tubulin was performed to identify the purity of neurons. The results showed that the cells began to adhere to the culture bottle and develop small neurites and form network gradually after Primary culture for 2 days. Up to the 5th day, many neurites extended to form dense network and Soma of neurons became well. Fluorescence staining with NeuN or β-tubulin showed that the purity of neurons can reach above 95%. The present protocol is a simple and efficient method for culturing brain neurons of Chinese perch with high purity, Which is of great significance for the fine study of fish growth and development, expression regulation of various receptors and proteins, cell apoptosis and cell signal transduction.
  • 加载中
  • [1] Giordano G, Costa L G. Primary neurons in culture and neuronal cell lines for in vitro neurotoxicological studies[M]//Costa L G, Giordano G, Guizzetti M. In Vitro Neurotoxicology: Methods and Protocols. New York: Humana Press, 2011: 13-27.
    [2] 申军现, 金卫林, 鞠躬. 低密度体外培养大鼠海马神经元的形态学观察[J]. 第四军医大学学报, 2003, 24(6): 489-491. doi: 10.3321/j.issn:1000-2790.2003.06.003Shen J X, Jin W L, Ju G. Morphological observation of rat hippocampal neuron in low-density culture in vitro[J]. Journal of the Fourth Military Medical University, 2003, 24(6): 489-491(in Chinese). doi: 10.3321/j.issn:1000-2790.2003.06.003
    [3] Augusti-Tocco G, Sato G. Establishment of functional clonal lines of neurons from mouse neuroblastoma[J]. Proceedings of the National Academy of Sciences of the United States of America, 1969, 64(1): 311-315. doi: 10.1073/pnas.64.1.311
    [4] Betz R, Sandhoff K, Fischer K D, et al. Detection and identification of Vav1 protein in primary cultured murine cerebellar neurons and in neuroblastoma cells (SH-SY5Y and Neuro-2a)[J]. Neuroscience Letters, 2003, 339(1): 37-40. doi: 10.1016/S0304-3940(02)01457-X
    [5] 万荣, 丁健, 周振明, 等. 成年鲁西牛肌内前脂肪细胞的分离培养[J]. 农业生物技术学报, 2007, 15(3): 419-423. doi: 10.3969/j.issn.1674-7968.2007.03.011Wan R, Ding J, Zhou Z M, et al. Isolation and in vitro culture of intramuscular preadipocyte from Luxi adult yellow cattle[J]. Journal of Agricultural Biotechnology, 2007, 15(3): 419-423(in Chinese). doi: 10.3969/j.issn.1674-7968.2007.03.011
    [6] Krämer D, Minichiello L. Cell culture of primary cerebellar granule cells[M]//Ward A, Tosh D. Mouse Cell Culture: Methods and Protocols. New York: Humana Press, 2010: 233-239.
    [7] Fu X, Li N, Lai Y, et al. A novel fish cell line derived from the brain of Chinese perch Siniperca chuatsi: Development and characterization[J]. Journal of Fish Biology, 2015, 86(1): 32-45. doi: 10.1111/jfb.12540
    [8] 肖兴莉, 杨朝鲜, 高小青, 等. 海马神经元原代培养方法[J]. 泸州医学院学报, 2014, 37(2): 175-178. doi: 10.3969/j.issn.1000-2669.2014.02.014Xiao X L, Yang C X, Gao X Q, et al. Primary culture method of hippocampal neurons[J]. Journal of Luzhou Medical College, 2014, 37(2): 175-178(in Chinese). doi: 10.3969/j.issn.1000-2669.2014.02.014
    [9] 杨秀平, 高秀勤, 黄祥柱. 鳜脑及脑神经形态的研究[J]. 华中农业大学学报, 1993, 12(6): 617-623.Yang X P, Gao X Q, Huang X Z. Studies on morphology of the branin and cranial nervers for the mandarin fish Siniperca chuatsi (Basilewsky)[J]. Journal of Huazhong Agricultural University, 1993, 12(6): 617-623(in Chinese).
    [10] 杨胜, 刘振伟, 万勤, 等. 大鼠海马神经元膜离子通道随培养时间变化的特点[J]. 中国应用生理学杂志, 2004, 20(2): 151-155. doi: 10.3969/j.issn.1000-6834.2004.02.010Yang S, Liu Z W, Wan Q, et al. Changes of ion channel in rat hippocampal neurons during primary culturne[J]. Chinese Journal of Applied Physiology, 2004, 20(2): 151-155(in Chinese). doi: 10.3969/j.issn.1000-6834.2004.02.010
    [11] 许熊飞, 李润平, 李泉, 等. 大鼠脑微血管内皮细胞的分离与原代培养[J]. 细胞生物学杂志, 2005, 27(1): 84-88.Xu X F, Li R P, Li Q, et al. Isolation and primary culture of rat cerebral microvascular endothelial cells[J]. Chinese Journal of Cell Biology, 2005, 27(1): 84-88(in Chinese).
    [12] 李振, 刘云会, 薛一雪, 等. 大鼠原代脑微血管内皮细胞体外分离与培养的实验研究[J]. 中国医科大学学报, 2012, 41(10): 873-876. doi: 10.3969/j.issn.0258-4646.2012.10.003Li Z, Liu Y H, Xue Y X, et al. In vitro isolation and culture of primary rat brain microvascular endothelial cells[J]. Journal of China Medical University, 2012, 41(10): 873-876(in Chinese). doi: 10.3969/j.issn.0258-4646.2012.10.003
    [13] 马华根, 唐元瑜, 纪立金. 原代大鼠脑微血管内皮细胞的培养及鉴定[J]. 解剖学杂志, 2018, 41(3): 276-279. doi: 10.3969/j.issn.1001-1633.2018.03.008Ma H G, Tang Y Y, Ji L J. Primary culture and identification of microvascular endothelial cells of rat brains[J]. Chinese Journal of Anatomy, 2018, 41(3): 276-279. doi: 10.3969/j.issn.1001-1633.2018.03.008
    [14] Selvakumar T, Kilpatrick D L. Culturing mouse cerebellar granule neurons[M]//Zhou R P, Mei L. Neural Development: Methods and Protocols. Totowa, NJ: Humana Press, 2013: 49-59.
    [15] 杨银芬. 消化酶、血清和催乳素对奶牛乳腺上皮细胞培养的影响[D]. 呼和浩特: 内蒙古农业大学, 2012.Yang Y F. Influence of digestive enzymes、FBS and prolactin on cuituring the bovine mammary epithelial cell[D]. Hohhot: Inner Mongolia Agricultural University, 2012(in Chinese).
    [16] Ichikawa N, Naora K, Hirano H, et al. Isolation and primary culture of rat cerebral microvascular endothelial cells for studying drug transport in vitro[J]. Journal of Pharmacological and Toxicological Methods, 1996, 36(1): 45-52. doi: 10.1016/1056-8719(96)00072-X
    [17] 邓莉, 代荣阳, 廖彦生, 等. 新生大鼠大脑皮层神经元原代培养与鉴定[J]. 现代医药卫生, 2011, 27(5): 646-647.Deng L, Dai R Y, Liao Y S, et al. Primary culture and identification of neurons from cerebral cortex of newborn rats[J]. Modern Medicine & Health, 2011, 27(5): 646-647(in Chinese).
    [18] Lind D, Franken S, Kappler J, et al. Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization[J]. Journal of Neuroscience Research, 2005, 79(3): 295-302. doi: 10.1002/jnr.20354
    [19] Mullen R J, Buck C R, Smith A M. NeuN, a neuronal specific nuclear protein in vertebrates[J]. Development, 1992, 116(1): 201-211.
    [20] Soylemezoglu F, Onder S, Tezel G G, et al. Neuronal nuclear antigen (NeuN): A new tool in the diagnosis of central neurocytoma[J]. Pathology-Research and Practice, 2003, 199(7): 463-468. doi: 10.1078/0344-0338-00446
    [21] Darnell R B. RNA protein interaction in neurons[J]. Annual Review of Neuroscience, 2013, 36: 243-270. doi: 10.1146/annurev-neuro-062912-114322
    [22] Reynolds B A, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[J]. Science, 1992, 255(5052): 1707-1710. doi: 10.1126/science.1553558
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(2)

Article views(1518) PDF downloads(4) Cited by()

Proportional views

Primary Cell Culture and Identification of brain neurons in Chinese perch (Siniperca chuatsi)

    Corresponding author: Xufang LIANG, xufang_liang@hotmail.com
  • 1. Chinese Perch Research Center, College of Fisheries, Huazhong Agricultural University, Wuhan    430070, China
  • 2. Innovation Base for Chinese Perch Breeding, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, Wuhan    430070, China

Abstract: The diversity of species makes the research on cell level of Chinese perch extremely limited. Establishing a simple and feasible method for primary culture of brain neurons from Chinese perch (Siniperca chuatsi) in vitro is beneficial to further study on fish nervous system. Our laboratory combined with the usual method of cell culture, Chinese perch brain neruons (born 3 months) were isolated by collagenase digestion and mechanical blow. L15+20%FBS suspended brain neruons were inoculated on the cell culture vessels, in 28 °C without CO2 incubator. The medium was replaced after 3 days. Subculture was carried out after the cells covered with the bottom of the cell culture vessels. The morphological changes of the neurons were observed under inverted phase-contrast microscope; Immunofluorescence staining for NeuN or β-tubulin was performed to identify the purity of neurons. The results showed that the cells began to adhere to the culture bottle and develop small neurites and form network gradually after Primary culture for 2 days. Up to the 5th day, many neurites extended to form dense network and Soma of neurons became well. Fluorescence staining with NeuN or β-tubulin showed that the purity of neurons can reach above 95%. The present protocol is a simple and efficient method for culturing brain neurons of Chinese perch with high purity, Which is of great significance for the fine study of fish growth and development, expression regulation of various receptors and proteins, cell apoptosis and cell signal transduction.

  • 体外培养的神经细胞模型是研究神经系统的基本生理过程、分析化学相关的神经毒性效应及其潜在机制的重要模型[1-2]。在过去一个世纪,神经学家和生物学家已投入极大的精力建立体外培养神经细胞的方法[3],目前,一些神经细胞系如PC12、Neuro2a等已被广泛用于神经生物学研究[1, 4],相比于原代细胞,这些细胞系虽然容易大量获取,但是作为已变异细胞,其具备的特征与在体细胞存在差异。已有研究发现体外培养原代细胞6代基本都保留着正常细胞的基本遗传特征[5],原代培养的神经细胞既接近细胞的正常发育状态,在神经细胞体外的生理、生化等功能研究更具有代表性,又可避免机体生理病理状态干扰的影响,可操作性强、条件可控、成本低[6]

    目前,以体外培养的哺乳动物神经细胞模型较多[3-4, 6],鱼类神经细胞模型极少[7],物种间的差异性使鱼类细胞水平上的研究具有极大的局限性,且培养的细胞质量将直接影响到研究进展及实验结果的真实性和可靠性[8]。因此,成功培养成熟稳定的鱼类神经细胞是体外研究鱼类生理调控及病理实验顺利进行的必要基础。鳜(Siniperca chuatsi)是我国重要的名优鱼类,其研究价值很高。脑作为中枢神经系统的主要组成部分,其中大量神经细胞聚集在一起有机地构成网络回路,通过传递、储存和加工信息从而支配控制动物的全部行为[9]。分离培养鳜脑原代细胞有利于在细胞水平研究鳜的相关机理,此外,建立体外鳜脑神经细胞模型对鱼类生理调控、疾病防控及细胞信号传导机制等方面更精细的研究具有重要意义。

    本研究结合既往的细胞培养方法[3-6],以鳜为实验材料,建立了一种简单的鳜脑神经细胞原代培养方法,可分离出高纯度,高活性的鳜脑细胞,为鳜后续研究提供了一种较为理想的细胞模型。

    • 鳜,3月龄,体长(21.22 ± 1.35) cm,来自湖北武汉四汇水产科技开发有限公司。实验用鳜暂养于独立的水族箱(60 cm × 45 cm × 45 cm)中,水温维持在(25 ± 0.4) °C,pH 7.11~7.59,溶氧为7.26 ~7.86 mg/L。

    • L15培养液(Genom生物公司);胎牛血清(Fatal bovine serun,FBS)、含0.25% EDTA的胰蛋白酶、I型胶原酶(Gibco公司);成纤维生长因子(Fibroblast growth factors,FGF)、表皮生长因子(Epidermal Growth Factor,EGF)(ProSpec-Tany TechnoGene Ltd公司);DPBS、两性霉素B、硫酸庆大霉素、青链霉素双抗(HyClone公司);4%多聚甲醛(Sigma公司);一抗(β-tubulin、NeuN)、DAPI染液、二抗(Cy3标记山羊抗兔IgG)以及抗荧光淬灭封片剂(谷歌生物科技有限公司)。

    • 倒置相差显微镜CKX41(Olympus公司);恒温细胞培养箱(Thermo公司);TDZ4AWS低速离心机(湘潭湘仪仪器有限公司);超净工作台(苏州安泰空气技术有限公司);倒置荧光显微镜、成像系统(Nikon公司)。

    • AIM液(Antibiotic initial medium):90 mL L15培养液 + 5 mL青链霉素双抗 + 5 mL两性霉素B(250 μg/mL) + 1 mL硫酸庆大霉素(50 mg/mL)混合,4 °C保存;胶原酶消化液:9 mL L15培养液 + 100 μL青链霉素双抗 + 1 mL I型胶原酶(10×)混合,现配现用(其中10× I型胶原酶母液的配置及保存方法:I型胶原酶和DPBS按比例100 mg:10 mL配制,待完全溶解后,0.22 μm滤膜过滤除菌,−20 °C避光保存);原代培养液:40 mL L15培养液 + 500 μL青链霉素双抗 + 10 mL FBS + 500 μL EGF(1 μg/mL) + 500 μL FGF(1 μg/mL)混合,4 °C保存;传代培养液:45 mL L15培养液 + 500 μL青链霉素双抗 + 5 mL FBS混合,4 °C保存。

    • 取鲜活鳜,用75%的酒精浸泡消毒2 min,用纱布擦除体表粘液,无菌条件下剖取鳜脑组织于预冷的DPBS中清洗,用镊子剥去多余的组织,于盛有AIM液的培养皿内浸泡消毒1.5 h;将脑组织取出置于装有少量AIM液的平皿中,剪成1~2 mm3大小的组织块,放入胶原酶消化液中消化2 h;加入L15培养液吹打使胶质裂解、脑细胞分离,1 000 r/min室温离心5 min后弃上清液,随后用L15培养液漂洗两次(1 000 r/min室温离心5 min),最后将收集的鳜脑细胞加入原代培养液重悬后接种到细胞培养瓶,于28 °C无CO2培养箱中培养,每3~5 d更换原代培养液一次。在倒置相差显微镜下观察脑细胞的生长情况。

    • 待鳜脑细胞培养长满T25细胞培养瓶,密度>80%时,吸出培养液,用平衡至室温的DPBS清洗2次。加入1 mL胰蛋白酶溶液室温消化2 min,镜下观察到大部分细胞收缩、聚团或呈圆形时,将胰蛋白酶溶液吸出,加入传代培养液终止消化并进行吹打混匀,随后按1∶2传代(传代密度约为105~106个/mL),放入28 °C培养箱中静置培养,至第3代可得到活性较好且稳定性较高的鳜脑神经细胞。此时可进行后续实验或进行冻存。倒置显微镜下观察细胞生长状况,并拍照记录。

    • 细胞传代培养至3代后,进行细胞爬片的制作。取长满鳜脑细胞的爬片(培养至第5天),吸净孔中原有的培养基,PBS浸洗3次,每次5 min;4%多聚甲醛室温固定30 min;滴加BSA封闭30 min,轻轻甩掉封闭液,在细胞孔板里滴加按一定比例配置的一抗:β-tubulin(1:100)、NeuN(1:200),平放于湿盒内4 °C孵育过夜。一抗孵育结束后将爬片用PBS浸洗3次,每次5 min,去除PBS后在爬片上滴加与一抗相应种属的二抗(Cy3稀释比例为1:300)稀释液覆盖细胞,室温避光孵育50 min;随后,爬片用PBS浸洗3次,每次5 min,去除PBS后在爬片上滴加DAPI染液,避光室温孵育10 min;爬片用PBS浸洗3次,每次5 min,滤纸吸干爬片多余液体后将有细胞的一面朝下,用抗荧光淬灭封片剂进行封片。倒置荧光显微镜下观察并采集图像(紫外光激发波长330~380 nm,发射波长420 nm;Cy3红光激发波长510~560 nm,发射波长590 nm)。荧光显微镜下观察,DAPI染的细胞核呈蓝色,Neu-N和β-tubulin阳性表达呈红色。随机选取5个视野(200×),计算阳性细胞个数,换算成百分比,重复5次,取其均值作为阳性神经元的纯度(神经元纯度=阳性细胞数/视野细胞总数×100%)。

    2.   结果
    • 在倒置显微镜下观察,鳜脑原代细胞贴壁较慢,起始细胞密度约为1.29 × 106个/mL,培养2 d细胞贴壁量较少,细胞多呈梭形或三角形,长出突起(图版Ⅰ-1),培养3 d后,细胞数量明显增多,具有典型的神经元形态,胞体饱满,突起间连接成网络(图版Ⅰ-2),培养至5 d神经元胞体成熟饱满,多成梭状,锥状,胞浆丰富,突起进一步增粗增长,许多分支互相连接,形成密集交错的神经纤维网络,此时可进行传代培养(图版Ⅰ-3)。传代培养的细胞贴壁较快,细胞消化后,细胞呈圆形,体积小、透亮呈悬浮状态,单个均匀分布(图版Ⅰ-4),传代细胞密度约为1.48 × 106个/mL,培养24 h后可见大部分细胞已贴壁,培养瓶内可见少部分漂浮的死细胞(图版Ⅰ-5),随后,细胞数量逐渐增多,胞体逐渐增大,突起逐渐延长、增粗,相互连接形成密集交错的神经纤维网络(图版Ⅰ-6)。

      Figure 图版Ⅰ.  Observation of brain neurons derived from Chinese perch cultured at different time

    • 为进一步确认所获得的鳜脑细胞的神经元纯度,在细胞培养3代后,取培养5 d的鳜脑细胞进行免疫荧光染色鉴定,观察神经元特异性蛋白NeuN(图版Ⅱ-1, 2, 3)和神经元标志蛋白β-tubulin(图版Ⅱ-4, 5, 6)的表达情况,正置荧光显微镜下采集图像,NeuN和β-tubulin阳性表达呈红色,DAPI染的细胞核呈蓝色。实验结果显示,绝大部分细胞质及突起在荧光显微镜下呈红色,即为NeuN或β-tubulin阳性表达。以随机5个视野中阳性神经元的数目占总细胞的比例为神经元纯度,经计算本实验分离的鳜脑细胞中NeuN的阳性率为(97.01 ± 0.32)%,β-tubulin的阳性率为(96.25 ± 0.15)%,即两种检测方法均得出神经元纯度大于95%。

      Figure 图版Ⅱ.  NeuN and β-tubulin Immunofluorescence staining of brain neurons on the 5th day

    3.   讨论
    • 神经细胞原代培养作为一种研究神经系统基本生理过程的新手段,已被广泛应用于神经生理、药理、病理等领域[10]。由于其培养条件要求高、细胞活性较差以及易受污染等导致神经细胞体外分离培养难度大[11-12]。本实验结合国内外相关研究[13-14],在Fu等[7]建立的鳜脑原代培养方法的基础上略加改动,结合自身对鳜脑原代细胞培养过程中的经验,通过反复试验探索,成功培养出了高活性,可进行传代培养的鳜脑原代细胞,为后续鱼类细胞水平上的研究提供细胞基础。我们发现:采用I型胶原酶消化鳜脑组织可使脑细胞从细胞间质中的胶原纤维释放,且该方式对细胞损伤小[15],可有效提高细胞得率,且获得较高纯度的神经细胞,此外,在消化前对鳜脑组织进行AIM液浸泡处理1.5 h可有效的降低细胞污染概率。相比于CO2培养箱,鳜脑细胞在无CO2的培养箱中生长速度更快,活性更高。

      鳜脑细胞在倒置相差显微镜下观察(图版Ⅰ)发现:培养3 d的鳜脑原代细胞已具有典型神经元的形态特征,胞体饱满,各分支互相连接形成密集交错的神经纤维网络,细胞形态多样,包括梭形、椎体三角形、椭圆形等,培养至5 d的神经元胞体融合度进一步增大,细胞瓶有限的空间已开始限制细胞生长(图版Ⅰ-3)。传代后观察发现,鳜脑传代细胞状态稳定,贴壁及生长速度快。细胞在传代6 h后开始贴壁,24 h后可见大部分细胞已贴壁且培养瓶内仅见少部分漂浮的死细胞(图版Ⅰ-6),其形态和生长特点与大多数文献报道相似[10, 13, 16-17],符合神经元细胞的形态学特征。

      NeuN是一种神经元特有的DNA结合蛋白,分子大小为46~48 ku[18-19],Soylemezoglu等研究证实,NeuN在成熟神经元中表达,是一种神经系统特异性核调节分子[20-21]。本实验用NeuN标记进行免疫荧光检测,结果显示,鳜脑神经细胞NeuN阳性率为(97.01 ± 0.32)%(图版Ⅱ-1, 2, 3)。此外,本实验还采用了微管蛋白(β-tubulin)进行免疫荧光染色检测,结果显示,本实验分离培养的鳜脑细胞β-tubulin阳性率为(96.25 ± 0.15)%(图版Ⅱ-4, 5, 6)。β-tubulin是神经元中表达最丰富的骨架蛋白,也是成熟神经元的标志,主要分布在胞浆和突触中[22]。本实验用NeuN和β-tubulin两种神经元标志蛋白对鳜脑细胞进行鉴定,更具有说服力。

      综上可知,本研究所建立的鳜脑细胞模型状态稳定,纯度高且培养方法简单,不仅为后期鳜细胞水平上的研究奠定了细胞基础,也为其它鱼类原代细胞模型的建立提供参考。

Reference (22)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return