生物学杂志

• 技术方法 • 上一篇    下一篇

海马神经元初级纤毛染色技术探索

  

  1. 1. 唐山市丰润区中医院 中风二科, 唐山 063000; 2. 北京市理化分析测试中心, 北京 100089
  • 出版日期:2016-06-18 发布日期:2016-06-18
  • 通讯作者: 安云鹤,博士,助理研究员,研究方向为分子生物学,E-mail:anyunhe_bio@163.com
  • 作者简介:周晓春,主治医师,从事神经内科研究,E-mail:zhoujingmingzjm@163.com
  • 基金资助:
    北京市科学技术研究院青年骨干项目(201522)

The research of staining technology of primary cilium in hippocampal neurons

  1. 1. Tangshan City Hospital of Traditional Chinese Medicine, Tangshan 063000;
     2. Beijing Center for Physical & Chemical Analysis, Beijing 100089, China
  • Online:2016-06-18 Published:2016-06-18

摘要:

为了建立体外原代培养的大鼠海马神经元初级纤毛的染色标记方法,以体外原代培养的18 d 胎鼠的海马神经元为研究材料,应用荧光免疫细胞化学实验技术,分别在不同的时间点,应用不同的初级纤毛蛋白标记物的抗体[抗ADP核糖基化因子相似蛋白-13B(ARL13B)抗体,抗III型腺苷酸环化酶(ACIII)抗体]对海马神经元初级纤毛进行染色,然后应用倒置荧光显微镜观察记录染色结果。从免疫荧光图像中看出,anti-ARL13B抗体和anti-ACIII抗体都能较好地标记海马神经元初级纤毛,但anti-ARL13B抗体的染色效果更佳。随着体外原代培养天数的增加,初级纤毛长度逐步增加(anti-ACIII抗体标记组:纤毛生长从第1天的3.3 μm到第14天的8.5 μm逐渐增加;anti-ARL13B抗体标记组:纤毛生长从第1天的4.4 μm到第14天的9.1 μm逐渐增加)。对星型胶质细胞的ACIII和ARL13B进行免疫荧光染色,也能较好地标记其初级纤毛。ARL13B和ACIII都能作为大鼠海马神经元初级纤毛的标记物,但ARL13B体现出更好的特异性。初级纤毛会伴随着海马神经元的发育成熟而生长,提示初级纤毛在海马神经元的发育过程中可能发挥着重要作用。ARL13B和ACIII也可以作为星型胶质细胞初级纤毛的染色标志。

关键词: 初级纤毛, 海马神经元, 免疫荧光, ADP核糖基化因子相似蛋白-13B, III型腺苷酸环化酶

Abstract:

To explore the staining methods of primary cilia in cultured rat primary hippocampal neurons, primary culture of 18 day fetal rats′ hippocampal neurons was used in this study. In cellular level,the immunofluorescence staining was used to label the primary cilia of hippocampal neurons at different culture time points and with different antibodies recognizing of different ciliary protein (anti-ADP-ribosylation factor-like protein 13B (ARL13B) antibody,anti-Type III adenylyl cyclase (ACIII) antibody). Fluorescence images were acquired by an inverted fluorescence microscope. From the results, we found that both anti-ARL13B antibodies and anti-ACIII antibodies can label the primary cilia of hippocampal neurons, although anti-ARL13B antibodies with more specific labeling effect. The length of primary cilia increasd during the development of hippocampal neuron cultured in vitro (anti-ACIII antibody groups:the length of primary cilia increases from 21.6 μm at the 1st day to 44.6 μm at the 14th day; anti-ARL13B antibody groups:the length of primary cilia increases from 15.2 μm at the 1st day to 37.6 μm at the 14th day) . The same immunofluorescence experiments
were done in astrocyte with these two antibodies. They can also label the primary cilia of astrocyte. To some
extent, both anti-ARL13B antibodies and anti-ACIII antibodies can act as the marker of the primary cilia in
hippocampal neurons. The primary cilia growed during development of hippocampal neurons cultured in vitro.
Accordingly, primary cilia must play an important role in the development of hippocampal neurons. ARL13B and
ACIII can also act as the marker of the primary cilia of astrocytes.

Key words:

13B; type III adenylyl cyclase