Caspr4通过LNX2调节神经前体细胞的增殖和分化

发布时间：2018-02-26 09:56

本文关键词： Caspr4 LNX2 神经前体细胞神经分化　出处：《苏州大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：神经前体细胞（NPCs）是存在于中枢神经系统，具有自我更新和多种分化潜能的始祖细胞。在一定条件下，神经前体细胞可以保持增殖能力，并能够向神经元、少突胶质细胞和星型胶质细胞分化。本课题研究Caspr（Contactin associatedprotein）家族中的Caspr4与LNX2（Ligand of Numb protein X2）之间的相互关系及对神经前体细胞的增殖和分化的影响。方法：利用免疫荧光染色（Immunofluorescence staining）、PCR（Polymerase ChainReaction）及蛋白免疫印迹（Western blot）等技术检测Caspr4及LNX2在小鼠神经前体细胞上的表达；分离、培养来源于C57BL/6小鼠胚胎发育期14天的神经前体细胞，利用电转的方法，在神经前体细胞上电转SiRNA，并用BrdU嵌合实验分析Caspr4或LNX2对神经前体细胞增殖的影响；在离体培养的神经前体细胞上电转Caspr4、Caspr4sh、LNX2、LNX2sh及C4ICD等质粒，并诱导神经前体细胞的分化，，分析Caspr4及LNX2对神经前体细胞分化的影响以及两者之间的相互联系；利用免疫荧光染色观察Caspr4与LNX2在神经前体细胞中的共定位情况；在COS7细胞中转染质粒Caspr4和myc-LNX2，并利用免疫共沉淀法（Co-Immunoprecipitation, Co-IP）研究Caspr4和LNX2的相互作用关系。结果：1）免疫荧光染色显示：Caspr4在神经前体细胞中表达； 2）Caspr4能够抑制离体培养的神经前体细胞的增殖并能促进神经前体细胞向神经元分化； 3）Caspr4通过其胞内段C4ICD促进离体培养的神经前体细胞向神经元分化； 4）免疫荧光染色显示：在细胞系中，Caspr4和LNX2共定位于细胞膜上，并且Co-IP证实它们是有相互作用的； 5）免疫荧光染色显示，LNX2在神经前体细胞中是表达的，并且与Caspr4共定位在细胞膜上； 6）体外实验显示，LNX2同样能够抑制神经前体细胞增殖并能促进神经前体细胞向神经元分化； 7）电转质粒LNX2sh使LNX2干扰后，C4ICD不能促进神经前体细胞向神经元分化； 8）电转质粒Caspr4sh使Caspr4被干扰后，LNX2仍然可以促进神经前体细胞向神经元分化。结论：在神经发生的早期，Caspr4通过其下游LNX2抑制神经前体细胞增殖并促进神经前体细胞向神经元分化。
[Abstract]:Objective: neural precursor cells (NPCs) are progenitor cells of central nervous system with self-renewal and differentiation potential. Under certain conditions, neural progenitor cells can maintain proliferative ability and become neurons. The relationship between Caspr4 and LNX2(Ligand of Numb protein X2 in the Caspr(Contactin associated protein family and its effect on the proliferation and differentiation of neural progenitor cells were studied. Methods: the expression of Caspr4 and LNX2 in mouse neural progenitor cells was detected by immunofluorescence staining and Western blot, and the cells derived from C57BL / 6 mouse embryonic development were isolated and cultured for 14 days. The effects of Caspr4 or LNX2 on the proliferation of neural precursor cells were analyzed by BrdU chimeric assay, and the plasmids such as Caspr4, Caspr4, Caspr4shl, LNX2, LNX2sh and C4ICD were transferred into neural precursor cells in vitro by electroporation. The effects of Caspr4 and LNX2 on the differentiation of neural precursor cells and the relationship between them were analyzed, and the co-localization of Caspr4 and LNX2 in neural precursor cells was observed by immunofluorescence staining. Plasmid Caspr4 and myc-LNX2 were transfected into COS7 cells and the interaction between Caspr4 and LNX2 was studied by co-immunoprecipitation (Co-IPP). Results: (1) Immunofluorescence staining showed the expression of 1% Caspr4 in neural progenitor cells. (2) Caspr4 can inhibit the proliferation of neural progenitor cells in vitro and promote the differentiation of neural precursor cells into neurons. 3C4ICD of C4ICD promoted the differentiation of neural precursor cells into neurons in vitro. 4) Immunofluorescence staining showed that Caspr4 and LNX2 were co-located on the cell membrane in the cell line, and Co-IP confirmed that they interacted with each other. 5) immunofluorescence staining showed that LNX2 was expressed in neural precursor cells and co-located with Caspr4 on the cell membrane. 6) in vitro experiments showed that LNX2 could also inhibit the proliferation of neural precursor cells and promote the differentiation of neural precursor cells into neurons. 7) after LNX2 interference, C4ICD could not promote the differentiation of neural precursor cells into neurons. 8) after Caspr4 was interfered with by Caspr4sh, LNX2 could still promote neural precursor cells to differentiate into neurons. Conclusion: in the early stage of neurogenesis, Caspr4 inhibits the proliferation of neural precursor cells and promotes the differentiation of neural precursor cells into neurons through its downstream LNX2.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744

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