免疫球蛋白κJ区重组信号结合蛋白对CD133阳性室管膜细胞增殖与分化的影响

doi:10.16098/j.issn.0529-1356.2022.02.002

解剖学报 ›› 2022, Vol. 53 ›› Issue (2): 144-154.doi: 10.16098/j.issn.0529-1356.2022.02.002

免疫球蛋白κJ区重组信号结合蛋白对CD133阳性室管膜细胞增殖与分化的影响

叶鑫^1,2 边维^1,2 李梦一^1,2 吴安婷^1,2 张庭³ 李军伟^1,2 周鹏^1,2 崔怀瑞¹ 孙臣友^1,2*

1.温州医科大学基础医学院解剖学教研室; 2.神经科学研究所; 3.基础医学院，浙江温州 325035

收稿日期:2020-08-20 修回日期:2020-11-08 出版日期:2022-04-06 发布日期:2022-04-06
通讯作者: 孙臣友 E-mail:sunchenyou1972@aliyun.com
基金资助:
别孕烯醇酮促进PD小鼠多巴胺能神经元新生的作用及机制研究;激活 mTORC2/Akt 信号通路在改善 6-羟基多巴胺模型小鼠多巴胺能神经元数目和行为学缺陷中的作用及机制研究

Effect of recombination signal binding protein for immunoglobulin Kappa J region on the proliferation and differentiation of CD133-positive ependymal cells

YE Xin^1,2 BIAN Wei^1,2 LI Meng-yi^1,2 WU An-ting^1,2 ZHANG Ting³ LI Jun-wei^1,2 ZHOU Peng^1,2 CUI Huai-rui¹ SUN Chen-you^1,2*

1.Department of Anatomy, School of Basic Medical Sciences; 2.Institution of Neuroscience, School of Basic Medical Sciences; 3.School of Basic Medical Sciences, Zhejiang Wenzhou 325035, China

Received:2020-08-20 Revised:2020-11-08 Online:2022-04-06 Published:2022-04-06
Contact: SUN Chen-you E-mail:sunchenyou1972@aliyun.com

摘要/Abstract

摘要：

目的探讨免疫球蛋白κJ区的重组信号结合蛋白（RBP-Jκ）对CD133阳性室管膜细胞增殖与分化的影响及可能的机制。方法分离孕12 d的美国癌症研究所（ICR）胚胎小鼠（3只）侧脑室室管膜细胞进行原代培养，并使用RBP-Jκ-siRNA干扰RBP-Jκ，以及选用2~3月龄体重为20~25 g的CD133-CreERTM::ROSA26-LacZ::RBP-Jκflox/flox小鼠（3只），通过腹腔注射他莫昔芬（TAM）敲除RBP-Jκ，通过免疫荧光双标染色检测CD133阳性室管膜细胞的增殖和分化变化的情况，最后通过Real-time PCR、Western blotting检测RBP-Jκ及其上下游相关分子表达情况。结果干扰或敲除RBP-Jκ后，无论是细胞还是动物水平CD133或β-半乳糖苷酶（β-GAL）/双肾上腺皮质激素（DCX）/β-微管蛋白Ⅲ（β-tubulinⅢ）、CD133或β-GAL/微管相关蛋白2（MAP-2）或神经元核抗原（NeuN）、CD133或β-GAL/增殖细胞核抗原（PCNA）双阳性细胞数目均明显增加。同时Real-time PCR和Western blotting结果表明，在干扰或敲除RBP-Jκ后RBP-Jκ-和Splity多毛增强子1（Hes1）mRNA及蛋白表达量均显著下降，而Notch1 mRNA及蛋白的表达量反而显著增加。结论干扰或敲除RBP-Jκ，通过Notch1表达上调、Hes1表达下调，最终引起CD133阳性室管膜细胞的增殖与分化增加。

关键词: CD133阳性室管膜细胞, Notch信号通路, 免疫球蛋白κJ区重组信号结合蛋白, 免疫荧光, 小鼠

Abstract:

Objective To explore the effect of recombination signal binding protein for immunoglobulin Kappa J region (RBP-Jκ) on the proliferation and differentiation of CD133-positive ependymal cells and its possible mechanism. Methods RBP-Jκ in CD133-positive ependymal cells of lateral ventricle was interfered with siRNA in the fetuses of embryos which were isolated from 12-day pregnant Institute of Cancer Research(ICR) mouse (3 mice) and knocked out in CD133-CreERTM: :ROSA26-LacZ: :RBP-Jκflox/floxmouse(3 mice) treated with tamoxifen (TAM) intraperitoneally. The proliferation and differentiation of CD133-positive ependymal cells, as well as the related molecules in RBP-Jκ upstream or downstream were evaluated and measured by immunofluorescent staining, Real-time PCR and Western blotting. ResultsBy interfering or knocking out RBP-Jκ, the number of CD133 or β-galactosidase(β-GAL)/doublecortin(DCX) β-tubulin Ⅲ or CD133 β-GAL/microtubule associated protein-2 (MAP-2) neuronal nuclear antigen(NeuN) or CD133 β-GAL/proliferating cell nuclear antigen(PCNA) double-positive cells were significantly increased in vitro or in vivo. Meanwhile, the result from Real-time PCR and Western blotting showed that RBP-Jκ and hairy and enhancer of split1(Hes1) mRNA and their protein expression levels decreased significantly, while Notch signaling pathway receptor 1(Notch1) mRNA and its protein expression levels increased significantly. Conclusion The interference or knockout of RBP-Jκ will lead to upregulateing Notch1 and downregulate Hes1 expression levels, which eventually increase the proliferation and differentiation of CD133-positive ependymal cells.

Key words: CD133-positive ependymal cell, Notch signaling pathway, Recombination signal binding protein for immunoglobulin Kappa J region, Immunofluorescentce, Mouse

中图分类号:

Q189

叶鑫边维李梦一吴安婷张庭李军伟周鹏崔怀瑞孙臣友. 免疫球蛋白κJ区重组信号结合蛋白对CD133阳性室管膜细胞增殖与分化的影响[J]. 解剖学报, 2022, 53(2): 144-154.

YE Xin BIAN Wei LI Meng-yi WU An-ting ZHANG Ting LI Jun-wei ZHOU Peng CUI Huai-rui SUN Chen-you. Effect of recombination signal binding protein for immunoglobulin Kappa J region on the proliferation and differentiation of CD133-positive ependymal cells[J]. Acta Anatomica Sinica, 2022, 53(2): 144-154.

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免疫球蛋白κJ区重组信号结合蛋白对CD133阳性室管膜细胞增殖与分化的影响

Effect of recombination signal binding protein for immunoglobulin Kappa J region on the proliferation and differentiation of CD133-positive ependymal cells

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