CN109679913A - 嗅觉干细胞三维培养方法 - Google Patents

嗅觉干细胞三维培养方法 Download PDF

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CN109679913A
CN109679913A CN201910143242.9A CN201910143242A CN109679913A CN 109679913 A CN109679913 A CN 109679913A CN 201910143242 A CN201910143242 A CN 201910143242A CN 109679913 A CN109679913 A CN 109679913A
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余逸群
张秀娟
任雯雯
王丽
余洪猛
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Eye and ENT Hospital of Fudan University
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Abstract

本发明公开了一种嗅觉干细胞三维培养的方法。该方法是从小鼠嗅上皮直接分离的嗅觉干细胞，并在体外三维培养，获得成熟的嗅感觉神经元。体外培养嗅觉干细胞可以解决嗅觉受损、嗅觉缺失等嗅觉相关疾病，为嗅觉疾病的临床治疗提供了思路。

Description

嗅觉干细胞三维培养方法
技术领域
本发明涉及生物技术领域，特别涉及一种嗅觉干细胞三维培养的方法。
背景技术
嗅觉是哺乳动物的重要感觉系统。哺乳动物的嗅觉系统包括三个部分:鼻腔和嗅上皮、嗅球以及嗅觉中枢。嗅上皮神经元直接暴露于外界环境,易因物理、化学、生物等各种因素引起损伤,导致嗅觉功能减退。嗅上皮中除了有神经元外，还有支持细胞、微绒毛细胞、鲍曼氏腺和基底细胞。其中，支持细胞特异性表达生物转化酶和细胞角蛋白8和角蛋白18，SUS4可特异性标记支持细胞；基底细胞包括球形基底细胞和水平基底细胞，前者特异性标记物为Lgr5，后者表达角蛋白5和角蛋白14。嗅上皮干细胞能自我更新和定向分化，产生嗅上皮内几乎所有的细胞类型。嗅神经元的寿命为30～90天。新生神经元由基底细胞分化而来,补充衰老死亡的神经元,从而维持嗅上皮细胞的内稳态。OMP可特异性标记成熟的嗅觉感受器神经元。球形基底细胞从嗅上皮基底部向顶部迁移,神经树突逐渐向嗅上皮鼻腔面生长,在黏膜表面形成树突小结；同时神经轴突穿过基底膜进入黏膜下层,向嗅球方向生长,在嗅球的嗅小球层与僧帽细胞的树突形成突触连接,进一步分化为成熟嗅神经元。水平基底细胞在正常嗅上皮中保持静止状态,一般只在嗅上皮受到一定程度损伤情况下被激活。
发明内容
本发明通过体外分离后小鼠嗅上皮的嗅觉干细胞，将其分离消化成单个细胞，并培养在含有基质胶的培养基中，7～14天以后，嗅觉干细胞在三维培养系统中分化出成熟嗅感觉神经元。
附图说明
通过结合附图对本公开示例性实施方式进行更详细的描述，本公开的上述以及其它目的、特征和优势将变得更加明显，其中，在本公开示例性实施方式中，相同的参考标号通常代表相同部件。
图1示出了根据本发明一个实施例的嗅觉干细胞的示意图。
图2A示出了根据本发明一个实施例的Ki67标记的增殖细胞的示意图。
图2B示出了根据本发明一个实施例的p63标记的嗅觉干细胞的示意图。
图3A示出了根据本发明一个实施例的Tuj1标记非成熟神经元的示意图。
图3B示出了根据本发明一个实施例的PGP9.5标记嗅神经元的示意图。
图3C示出了根据本发明一个实施例的OMP标记成熟嗅感觉神经元的示意图。
图3D示出了根据本发明一个实施例的SUS4标记支持细胞的示意图。
具体实施方式
一种适用于嗅觉干细胞三维培养的方法，将单个嗅觉干细胞培养在含有基质胶的完全培养基中，其中所述完全培养基为基础培养基DMEM/F12中添加了各种成分以刺激嗅觉干细胞的增殖与分化。参见图1示出的嗅觉干细胞的示意图。所述的基质胶为干细胞三维培养提供营养基础及固定支持细胞形态。其中所述的完全培养基中各成分含量如下：
具体培养步骤如下：
1)麻醉小鼠，脱颈处死，小鼠断头，剥去皮肤，分离下颚和眼眶，在嗅球与大脑分界处剪断，游离出小鼠鼻腔，解剖出嗅区粘膜，置于冰上预冷的PBS中。用冰冷的PBS洗两次，置于Tyrode’s缓冲液中。
2)用显微剪将取下的组织剪碎成尽量小的组织块。
3)0.25％胰酶37℃消化组织15分钟。
4)加入含FBS的DMEM培养基终止消化。
5)1200rpm离心5分钟，使组织沉淀。
6)去除胰酶，加入1ml完全培养基，用玻璃吸管吹打20次，获得细胞悬液。
7)将细胞悬液分别通过70μm和40μm的细胞筛网，获得单细胞悬液。
8)在单细胞悬液中加入基质胶，将细胞接种于低吸附孔板上，置于37℃培养箱中培养。
9)每隔三天对细胞进行换液，24小时左右开始长出嗅觉细胞；至第5天，细胞增殖明显；至第8天左右嗅觉细胞成熟并有活性(一般范围为7-14天)，培养至2周时，可进行传代，可传数十次，培养90天，而不影响细胞活性。
上述步骤1)中所述Tyrode’s缓冲液配方为：145mM NaCl,5mM KCl,10mM Hepes,5mM NaHCO3,10mM pyruvate,10mM glucose，pH 7.4。
上述步骤8)总所述基质胶占培养基体积的5％。所述低吸附孔板为低吸附能力的24孔板。
哺乳动物嗅上皮具有持续更新的能力。本发明通过体外分离获得小鼠的嗅觉干细胞，具有体外增殖和分化能力，并且在没有神经支持的情况下可以分化为具有活性的嗅觉细胞。此方法模拟了体内嗅觉细胞生成与发育的过程，阐述了从嗅觉干细胞到嗅觉细胞的分化过程，用此方法体外获得嗅觉细胞，可以对嗅觉缺失、嗅觉受损等嗅觉相关疾病提供研究思路与依据，可作为嗅觉研究的一项里程碑发明。
Ki67普遍存在于细胞核内，在细胞周期S、G2、M期均有表达，在G0期不表达，与细胞增生密切相关，常作为细胞增殖标记物。图2A示出了根据本发明一个实施例的Ki67标记的增殖细胞的示意图。P63蛋白主要表达与上皮组织的基底层中具有高度增殖能力和分化潜能的细胞和祖细胞中，可用作干细胞的标记物，用来标记嗅觉干细胞。图2B示出了根据本发明一个实施例的p63标记的嗅觉干细胞的示意图。
TuJ1是一种被认为参与神经元细胞类型特异性分化的微管蛋白，存在于未成熟的神经元胞体，树突，轴突和轴突末端。图3A示出了根据本发明一个实施例的Tuj1标记非成熟神经元的示意图。PGP9.5是一种神经纤维中的特异性泛素羟基水解酶，作为一种神经轴突标记物，抗PGP9.5抗体，可标记出组织中PGP9.5阳性的神经纤维。图3B示出了根据本发明一个实施例的PGP9.5标记嗅神经元的示意图。OMP存在于嗅觉感受神经元树突和树突末梢的整个细胞质内以及成熟的嗅觉感受细胞的树突末梢的纤毛中。图3C示出了根据本发明一个实施例的OMP标记成熟嗅感觉神经元的示意图。SUS4可特异性与上皮组织的支持细胞结合，主要用于标记支持细胞。图3D示出了根据本发明一个实施例的SUS4标记支持细胞的示意图。

Claims (9)

1.一种嗅觉干细胞三维培养的方法，将体外分离的小鼠嗅觉干细胞培养在含有基质胶的完全培养基中，培养7-14天以获得具有活性的成熟嗅感觉神经元。
2.根据权利要求1所述一种嗅觉干细胞三维培养的方法，其特征在于：所述的完全培养基为在基础培养基中添加生长因子，细胞凋亡抑制剂，R-spondin、wnt 3a以及Noggin。
3.根据权利要求2所述一种嗅觉干细胞三维培养的方法，其特征在于：所述基础培养基为DMEM/F12培养基(Life Technologies)。
4.根据权利要求2所述一种嗅觉干细胞三维培养的方法，其特征在于：所述培养添加剂为Glutamax(Life Technologies)。
5.根据权利要求2所述一种嗅觉干细胞三维培养的方法，其特征在于：所述生长因子为bFGF(20ng/mL，Life Technologies)，EGF(20ng/mL，Life Technologies)N2(1％，LifeTechnologies)和B27[2％(vol/vol)，Life Technologies]。
6.根据权利要求1所述一种嗅觉干细胞三维培养的方法，其特征在于：所述基质胶为Matrigel(256231，corning)。
7.根据权利要求2所述一种嗅觉干细胞三维培养的方法，其特征在于：所述细胞凋亡抑制剂为Y-27632(10μM，Thermo)。
8.根据权利要求2所述一种嗅觉干细胞三维培养的方法，其特征在于：所述R-spondin(200ng/mL，R&D)用于促进干细胞增殖。
9.根据权利要求2所述一种嗅觉干细胞三维培养的方法，其特征在于：所述Noggin(100ng/mL，PeproTech)用于促进干细胞分化。
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