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JoVE Encyclopedia of Experiments
Neuroscience
神经干细胞中 siRNA 介导的基因沉默
神经干细胞中 siRNA 介导的基因沉默
Encyclopedia of Experiments
Neuroscience
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Encyclopedia of Experiments Neuroscience
siRNA-Mediated Gene Silencing in Neural Stem Cells

神经干细胞中 siRNA 介导的基因沉默

Protocol
350 Views
02:35 min
July 8, 2025
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Transcript

以小干扰 RNA 或 siRNA 为例,这是一种参与基因沉默的 RNA。

添加称为脂质体的球形脂质囊泡。

脂质体封装 siRNA,保护其免于降解。

将

siRNA-脂质体复合物添加到含有神经干细胞的培养孔中。保持一个井作为对照,然后孵化。

脂质体与细胞膜融合,释放 siRNA。

siRNA 与 RNA 诱导的沉默复合物 (RISC) 结合,其中一条链被去除。

剩余的链靶向负责细胞分化的 mRNA,导致其降解。

向

孔中添加合适的分化培养基。

在

对照孔中,培养基中的生长因子和其他营养物质促进神经干细胞分化为成骨细胞。

使用合适的染色技术,在细胞上染色成骨细胞特异性标记,并用蓝色荧光染料对细胞核进行染色。

基因沉默细胞中缺乏成骨细胞特异性标记表明它们无法分化。

为了在O9-1细胞中进行siRNA敲低,回收并接种细胞。在适当体积的无血清培养基中稀释脂质体。然后,根据文本方案在无血清培养基中稀释siRNA。之后,根据制造商的说明将稀释的siRNA添加到稀释的脂质体中。通过移液混合溶液并在室温下孵育 5 分钟。

接下来,向细胞中添加适量的siRNA-脂质复合物。然后,将细胞在标准细胞培养箱中孵育 24 小时。O9-1细胞在特定的分化培养条件下可以分化成不同的细胞类型。要将O9-1细胞分化成成骨细胞,请根据文本方案制备成骨分化培养基。最后,通过免疫染色检测分化成骨细胞中的成骨细胞标志物骨钙素。

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