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

作　　者：叶鑫 边维 李梦一 吴安婷 张庭 李军伟 周鹏 崔怀瑞 孙臣友 YE Xin;BIAN Wei;LI Meng-yi;WU An-ting;ZHANG Ting;LI Jun-wei;ZHOU Peng;CUI Huai-rui;SUN Chen-you(Department of Anatomy,School of Basic Medical Sciences,Zhejiang Wenzhou 325035,China;Institution of Neuroscience,School of Basic Medical Sciences,Zhejiang Wenzhou 325035,China;School of Basic Medical Sciences,Zhejiang Wenzhou 325035,China)

机构地区：[1]温州医科大学基础医学院解剖学教研室,浙江温州325035 [2]温州医科大学基础医学院神经科学研究所,浙江温州325035 [3]温州医科大学基础医学院基础医学院,浙江温州325035

出　　处：《解剖学报》2022年第2期144-154,共11页Acta Anatomica Sinica

基　　金：国家自然科学基金(81671401,82171577);浙江省自然科学基金(LY22H090004);温州市科技局基础性医疗卫生科技项目(Y20190059)。

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-CreER^(TM)::ROSA26-LacZ::RBP-Jκ^(flox/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。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-CreER^(TM)::ROSA26-LacZ::RBP-Jκ^(flox/flox)mouse(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. Results By 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.

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 

分 类 号：Q189[生物学—神经生物学]