發(fā)布時(shí)間：2018-02-26 09:56

  本文關(guān)鍵詞： Caspr4 LNX2 神經(jīng)前體細(xì)胞 神經(jīng)分化　出處：《蘇州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：神經(jīng)前體細(xì)胞（NPCs）是存在于中樞神經(jīng)系統(tǒng)，具有自我更新和多種分化潛能的始祖細(xì)胞。在一定條件下，神經(jīng)前體細(xì)胞可以保持增殖能力，并能夠向神經(jīng)元、少突膠質(zhì)細(xì)胞和星型膠質(zhì)細(xì)胞分化。本課題研究Caspr（Contactin associatedprotein）家族中的Caspr4與LNX2（Ligand of Numb protein X2）之間的相互關(guān)系及對神經(jīng)前體細(xì)胞的增殖和分化的影響。 方法：利用免疫熒光染色（Immunofluorescence staining）、PCR（Polymerase ChainReaction）及蛋白免疫印跡（Western blot）等技術(shù)檢測Caspr4及LNX2在小鼠神經(jīng)前體細(xì)胞上的表達(dá)；分離、培養(yǎng)來源于C57BL/6小鼠胚胎發(fā)育期14天的神經(jīng)前體細(xì)胞，利用電轉(zhuǎn)的方法，在神經(jīng)前體細(xì)胞上電轉(zhuǎn)SiRNA，并用BrdU嵌合實(shí)驗(yàn)分析Caspr4或LNX2對神經(jīng)前體細(xì)胞增殖的影響；在離體培養(yǎng)的神經(jīng)前體細(xì)胞上電轉(zhuǎn)Caspr4、Caspr4sh、LNX2、LNX2sh及C4ICD等質(zhì)粒，并誘導(dǎo)神經(jīng)前體細(xì)胞的分化，，分析Caspr4及LNX2對神經(jīng)前體細(xì)胞分化的影響以及兩者之間的相互聯(lián)系；利用免疫熒光染色觀察Caspr4與LNX2在神經(jīng)前體細(xì)胞中的共定位情況；在COS7細(xì)胞中轉(zhuǎn)染質(zhì)粒Caspr4和myc-LNX2，并利用免疫共沉淀法（Co-Immunoprecipitation, Co-IP）研究Caspr4和LNX2的相互作用關(guān)系。 結(jié)果：1）免疫熒光染色顯示：Caspr4在神經(jīng)前體細(xì)胞中表達(dá)； 2）Caspr4能夠抑制離體培養(yǎng)的神經(jīng)前體細(xì)胞的增殖并能促進(jìn)神經(jīng)前體細(xì)胞向神經(jīng)元分化； 3）Caspr4通過其胞內(nèi)段C4ICD促進(jìn)離體培養(yǎng)的神經(jīng)前體細(xì)胞向神經(jīng)元分化； 4）免疫熒光染色顯示：在細(xì)胞系中，Caspr4和LNX2共定位于細(xì)胞膜上，并且Co-IP證實(shí)它們是有相互作用的； 5）免疫熒光染色顯示，LNX2在神經(jīng)前體細(xì)胞中是表達(dá)的，并且與Caspr4共定位在細(xì)胞膜上； 6）體外實(shí)驗(yàn)顯示，LNX2同樣能夠抑制神經(jīng)前體細(xì)胞增殖并能促進(jìn)神經(jīng)前體細(xì)胞向神經(jīng)元分化； 7）電轉(zhuǎn)質(zhì)粒LNX2sh使LNX2干擾后，C4ICD不能促進(jìn)神經(jīng)前體細(xì)胞向神經(jīng)元分化； 8）電轉(zhuǎn)質(zhì)粒Caspr4sh使Caspr4被干擾后，LNX2仍然可以促進(jìn)神經(jīng)前體細(xì)胞向神經(jīng)元分化。 結(jié)論：在神經(jīng)發(fā)生的早期，Caspr4通過其下游LNX2抑制神經(jīng)前體細(xì)胞增殖并促進(jìn)神經(jīng)前體細(xì)胞向神經(jīng)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R744

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 林艷青;耿建國;楊雪松;王麗京;;雞胚早期發(fā)育過程中細(xì)胞周期調(diào)控的研究進(jìn)展[J];廣東藥學(xué)院學(xué)報(bào);2010年06期

2 熊永潔;肖連臣;閔U

本文編號：1537534