Page 45 - 南京医科大学自然版

第46卷第9期 南京医科大学学报（自然科学版）
2026年9月 Journal of Nanjing Medical University（Natural Sciences） ·1315 ·

·基础研究·

Pin1负向调控Notch1信号通路抑制神经干细胞增殖

潘 婷 ，汪 静 ，孙晓琦 ，陆文晗 ，李 琳 ，戴一凡 ，王 盈 ，杨海元 1，2*
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南京医科大学医学遗传学系，江苏省器官衰竭生物治疗重点实验室，江苏 南京 211166
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［摘 要］ 目的：利用CRISPR/Cas9技术构建肽基脯氨酰顺反式异构酶永不分裂基因A相互作用蛋白（peptidyl⁃prolyl cis⁃trans
isomerase never in mitosis A⁃interacting 1，Pin1）基因敲除的C17.2神经干细胞，揭示Pin1缺失对胚胎神经干细胞增殖能力的影
响，并基于转录组测序探究其潜在的分子机制。方法：针对小鼠 Pin1 外显子 2 设计特异性小向导 RNA（single guide RNA，
sgRNA），构建靶向Pin1的CRISPR/Cas9打靶载体，转染C17.2神经干细胞并经G418筛选鉴定获得Pin1敲除的单细胞克隆。利
用免疫荧光染色和Western blot验证Pin1敲除效率及干性标志物Nestin的表达；通过BrdU掺入实验和Ki67免疫荧光染色评估
细胞增殖能力的改变。通过RNA⁃Seq筛选差异表达基因并进行通路富集分析，通过qRT⁃PCR和Western blot验证差异基因的
表达；最后利用γ⁃分泌酶抑制剂DAPT阻断Notch通路，验证该通路在Pin1调控细胞增殖中的作用。结果：成功获得3个Pin1
双等位基因敲除的C17.2细胞克隆。与野生型细胞相比，Pin1敲除细胞维持Nestin阳性表达，BrdU掺入率和Ki67阳性率显著
升高（P < 0.05），呈过度增殖状态。RNA⁃Seq及验证实验表明，Pin1缺失导致Notch信号通路显著激活，表现为Notch1受体蛋白
及其下游靶基因Hes1表达水平显著上调（P < 0.05）。DAPT处理不仅有效抑制了Pin1敲除细胞中Notch1通路的过度激活，而
且显著降低了细胞的增殖速率。结论：Pin1在C17.2神经干细胞中作为一种负向调控因子，通过抑制Notch1信号通路来限制
细胞的过度增殖。这一发现揭示了Pin1在胚胎神经干细胞发育阶段维持增殖稳态的新机制。
［关键词］ Pin1；Notch1；CRISPR/Cas9；神经干细胞；增殖
［中图分类号］ R329.2 ［文献标志码］ A ［文章编号］ 1007⁃4368（2026）09⁃1315⁃09
doi：10.7655/NYDXBNSN251472

Pin1 negatively regulates the Notch1 signaling pathway to restrict the proliferation of
neural stem cells
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PAN Ting ，WANG Jing ，SUN Xiaoqi ，LU Wenhan ，LI Lin ，DAI Yifan ，WANG Ying ，YANG Haiyuan 1，2*
Department of Medical Genetics，Jiangsu Provincial Key Laboratory of Biological Therapy for Organ Failure，
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Nanjing Medical University，Nanjing 211166，China
［Abstract］ Objective：To construct peptidyl⁃prolyl cis⁃trans isomerase never in mitosis A⁃interacting 1（Pin1）knockout C17.2
neural stem cell（NSC）lines using CRISPR/Cas9 technology，elucidate the impact of Pin1 deficiency on the proliferation capacity of
embryonic NSCs，and explore the underlying molecular mechanisms based on transcriptome sequencing. Methods：Specific single
guide RNAs（sgRNAs）targeting exon 2 of the mouse Pin1 gene were designed to construct the CRISPR/Cas9 targeting vectors. These
vectors were transfected into C17.2 NSCs，followed by G418 selection to obtain Pin1 knockout single⁃cell colonies. The knockout
efficiency and stemness maintenance（Nestin expression）were validated by immunofluorescence and Western blot analyses. Cell
proliferation was evaluated using BrdU incorporation assays and Ki67 immunofluorescence staining. Differentially expressed genes
（DEGs）were screened via RNA⁃Seq and subjected to pathway enrichment analysis. The expression of DEGs was further validated by
quantitative real⁃time PCR（qRT⁃PCR）and Western blot. Finally，DAPT，a γ⁃secretase inhibitor，was employed to block the Notch
signaling pathway to verify its role in Pin1⁃mediated cell proliferation. Results：Three C17.2 cell clones with biallelic Pin1 knockout
were successfully generated. Compared to wild⁃type cells，Pin1 knockout cells maintained positive Nestin expression，but exhibited
significantly increased BrdU incorporation and Ki67⁃positive rates（P < 0.05），indicating a state of hyperproliferation. RNA⁃Seq and
subsequent validation experiments demonstrated that Pin1 deletion resulted in the significant activation of the Notch signaling

［基金项目］ 国家自然科学基金（81970164）
通信作者（Corresponding author），E⁃mail：hyyang@njmu.edu.cn（ORCID：0000⁃0001⁃5253⁃0079）
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