EphB4信号通路调节人胚胎神经干细胞增殖、分化、凋亡的作用机制研究

发布时间：2018-04-22 04:28

本文选题：EphB4信号通路 + 神经干细胞　；参考：《首都医科大学》2017年博士论文

【摘要】：目的缺血性脑卒中是一种严重损害神经功能的疾病,其具有很高的发病率和死亡率。缺血性脑损伤后期的神经再生与修复是脑卒中治疗的关键环节。脑卒中后可以诱发内源性神经干细胞增殖、分化并向损伤区域迁移,其中多条信号通路参与调节神经干细胞的活动。通过调节特异性的分子机制,可以增强脑损伤诱导的内源性神经干细胞的增殖与分化过程。目前,参与调节神经干细胞增殖与分化的经典信号通路有四条,即Wnt信号通路、Notch信号通路、Shh(Sonic Hedgehog,Shh)信号通路与骨形态发生蛋白(bone morphogenetic protein,BMP)信号通路。在前期我们研究Wnt信号通路时发现,β-catenin做为Wnt信号通路的关键调节因子,同时也是Eph信号通路的下游蛋白之一。因此,我们关注到了Eph/ephrin这条信号通路。Eph受体是具有最多成员的受体酪氨酸激酶家族,其与配体ephrin结合后可以诱发双向传导过程。近年来研究表明Eph受体与ephrin配体参与调节神经干细胞的增殖、分化、存活与迁移活动。但对于EphB4的研究都集中在肿瘤方面,其对神经干细胞的调节作用却未见报道。而肿瘤细胞中存在少部分肿瘤激活细胞,这些细胞与干细胞有着共同的特性,肿瘤可以从正常组织干细胞转化而来。我们猜想EphB4是否在神经干细胞的增殖与分化方面也起到重要作用。本实验旨在研究EphB4是否参与调节了人胚胎神经干细胞的增殖、分化、凋亡活动,并探索其下游信号通路与调节机制,为脑卒中治疗提供新的靶点。方法(1)人胚胎神经干细胞的鉴定:培养原代人胚胎神经干细胞,正常培养3天后进行Nestin免疫荧光染色标记巢蛋白;分化培养10天后进行βⅢ-tubulin与胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫荧光染色分别标记神经元与星形胶质细胞;分化培养16天后进行半乳糖脑苷脂(galactocerebroside,Galc)免疫荧光染色标记少突胶质细胞,进行人胚胎神经干细胞鉴定。(2)EphB4信号通路的机制研究:培养原代人胚胎神经干细胞,使用沉默慢病毒与过表达慢病毒转染细胞,分别下调和上调EphB4基因水平及其蛋白表达水平,分为EphB4基因沉默对照组(Scrambled-sh RNA)、EphB4基因沉默组(EphB4-sh RNA)、EphB4基因过表达对照组(EphB4-control)、EphB4基因过表达组(EphB4-OE)。然后通过实时荧光定量聚合酶链反应(real-time reverse transcription-polymerase chain reaction,RT-PCR)检测EphB4的基因表达水平,Western blot检测EphB4的蛋白表达水平来确定沉默及过表达效率。通过克隆分析、Brd U免疫荧光染色观察EphB4对细胞自我更新与增殖的影响;通过Dcx、βⅢ-tubulin、GFAP、NG2免疫荧光染色及RT-PCR检测Dcx、βⅢ-tubulin、GFAP、NG2基因水平,Western blot检测Mash1蛋白表达水平观察EphB4对人胚胎神经干细胞分化的影响;通过Cleaved-caspase3、caspase8免疫荧光染色,Western blot检测活性caspase8的表达水平、AnnexinⅤ-PE/7-AAD流式试剂盒分析观察EphB4对人胚胎神经干细胞凋亡的影响;通过细胞周期分析及使用Cyclin D1/CDK4抑制剂FAS、Abl抑制剂Gleevec、Abl激动剂DPH抑制和激活蛋白表达水平探索EphB4下游信号通路。结果(1)我们培养的人胚胎神经干细胞,Nestin~+细胞的比例为77.37%,并且可以成功分化为βⅢ-tubulin~+神经元、GFAP~+星形胶质细胞与Galc~+少突胶质细胞。(2)与对照组相比,转染EphB4沉默慢病毒可以显著降低EphB4的m RNA水平与蛋白表达水平,转染EphB4过表达慢病毒可以显著提高EphB4的m RNA水平与蛋白表达水平,人胚胎神经干细胞EphB4基因沉默及过表达模型成功建立。(3)克隆分析与免疫荧光染色结果显示,与对照组相比,EphB4基因沉默显著降低了神经球直径、神经球个数与Brd U阳性细胞数,抑制了细胞增殖;EphB4过表达显著增加了神经球的直径、神经球个数与Brd U阳性细胞数,促进了细胞增殖。(4)免疫荧光染色、RT-PCR分析与Western blot结果显示,与对照组相比,EphB4基因沉默显著降低了Dcx、βⅢ-tubulin阳性细胞数及其m RNA水平、Mash1蛋白表达水平,显著增加了GFAP、NG2阳性细胞数及其m RNA水平,抑制细胞向神经元分化,促进细胞向胶质细胞分化;EphB4基因过表达显著增加了Dcx、βⅢ-tubulin阳性细胞数及其m RNA水平、Mash1蛋白表达水平,显著降低了GFAP、NG2阳性细胞数及其m RNA水平,促进细胞向神经元分化,抑制细胞向胶质细胞分化。(5)免疫荧光染色、Western blot及细胞流式结果显示,与对照组相比,EphB4沉默组与过表达组Cleaved-caspase3、caspase8阳性细胞数、活性caspase8蛋白表达水平及AnnexinⅤ-PE~+/7-AAD-标记的细胞数量无显著变化,EphB4基因沉默及过表达对细胞凋亡无影响。(6)细胞周期检测结果显示,与对照组相比,EphB4基因沉默可以显著增加G0/G1期细胞数量,相对的减少S期与G2/M期的细胞数量;EphB4基因过表达可以显著减少G0/G1期细胞数量,相对的增加S期与G2/M期的细胞数量。EphB4基因沉默及过表达可以影响G1期调节蛋白Cyclin D1、CDK4的表达水平,使用Cyclin D1/CDK4抑制剂FAS抑制蛋白活性后消除了EphB4过表达促进细胞增殖的作用。结果表明Cyclin D1/CDK4介导了EphB4蛋白对人胚胎神经干细胞增殖的调节作用。(7)Western blot结果显示,EphB4基因沉默及过表达可以影响Abl的表达水平,使用Abl抑制剂Gleevec与激动剂DPH抑制与激活Abl蛋白可以影响Cyclin D1、CDK4的表达水平,并且在Abl蛋白活性被抑制后可以消除EphB4过表达促进细胞增殖的作用。结果表明,Abl作为EphB4的下游蛋白介导了EphB4调控Cyclin D1/CDK4影响细胞增殖的作用。(8)使用Abl抑制剂Gleevec与Cyclin D1/CDK4抑制剂FAS抑制蛋白活性并未影响EphB4过表达促进细胞向神经元方向分化,抑制细胞向胶质细胞分化的作用。结果表明,Abl-Cyclin D1/CDK4信号通路并未参与调节EphB4对细胞分化的作用。结论:EphB4基因沉默后,抑制体外培养人胚胎神经干细胞的增殖及细胞向神经元分化,促进细胞向胶质细胞分化,对细胞凋亡无影响。EphB4基因过表达后,促进体外培养人胚胎神经干细胞的增殖及细胞向神经元的分化,抑制细胞向胶质细胞分化,对细胞凋亡无影响。EphB4是通过下游信号通路Abl-Cyclin D1调节细胞增殖,此信号通路不参与EphB4在细胞分化方面的调节。EphB4参与调节神经干细胞增殖,而且是决定神经干细胞向神经元分化还是向胶质细胞分化的开关,其很可能成为脑损伤后神经元修复的有效治疗靶点。
[Abstract]:Objective ischemic stroke is a serious damage to nerve function. It has a high incidence and mortality. Neural regeneration and repair in the later stage of ischemic brain injury is the key link in the treatment of stroke. After stroke, it can induce endogenous neural stem cells to proliferate, differentiate and migrate to the injured area, including multiple signal pathways. By regulating the activity of neural stem cells, the proliferation and differentiation process of endogenous neural stem cells induced by brain damage can be enhanced by regulating specific molecular mechanisms. There are four classical signaling pathways involved in regulating the proliferation and differentiation of neural stem cells, namely, Wnt signal pathway, Notch signaling pathway, and Shh (Sonic Hedgehog, Shh) signal The pathway and the bone morphogenetic protein (BMP) signaling pathway. In our earlier study of the Wnt signaling pathway, we found that beta -catenin is a key regulator of the Wnt signaling pathway, and is also one of the downstream proteins of the Eph signaling pathway. Therefore, we have noticed that Eph/ephrin, a signal pathway, is the most important of the.Eph receptors. Many members of the receptor tyrosine kinase family, which bind to the ligand ephrin, can induce two-way conduction process. In recent years, studies have shown that Eph receptors and ephrin ligands are involved in regulating the proliferation, differentiation, survival and migration of neural stem cells. However, the study of EphB4 is concentrated in the swelling of the tumor and its regulation on neural stem cells is not There are few tumor activated cells in the tumor cells. These cells have a common characteristic with the stem cells, and the tumor can be transformed from normal tissue stem cells. We guess whether EphB4 plays an important role in the proliferation and differentiation of neural stem cells. This test is to investigate whether EphB4 participates in the regulation of human embryos. The proliferation, differentiation and apoptosis of fetal neural stem cells, and explore the downstream signal pathway and regulation mechanism to provide new targets for the treatment of stroke. Method (1) identification of human embryonic neural stem cells: culture of human embryonic neural stem cells, Nestin immunofluorescence staining for 3 days after normal culture and 10 days after differentiation and culture. The neurons and astrocytes were labeled with beta III -tubulin and glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP), and 16 days after differentiation and culture, galactosida (galactocerebroside, Galc) immunofluorescence staining was used to mark oligodendrocytes, and human embryonic neural stem cells were identified. (2) EphB4 The mechanism of signal pathway: the culture of human embryonic neural stem cells, the use of silent lentivirus and overexpression of lentivirus transfected cells, down and up regulation of EphB4 gene level and protein expression level, divided into EphB4 gene silencing control group (Scrambled-sh RNA), EphB4 gene silencing group (EphB4-sh RNA), EphB4 gene overexpression control group ( EphB4-control), EphB4 gene overexpression group (EphB4-OE). Then the gene expression level of EphB4 was detected by real-time quantitative polymerase chain reaction (real-time reverse transcription-polymerase chain reaction, RT-PCR). Western blot detected the protein expression to determine the silencing and overexpression efficiency. Immunofluorescence staining was used to observe the effect of EphB4 on cell self renewal and proliferation. The effects of Dcx, beta III -tubulin, GFAP, NG2 immunofluorescence staining and RT-PCR on the detection of Dcx, beta III -tubulin, GFAP, NG2 gene level, and Western blot test protein expression level on the differentiation of human embryonic neural stem cells were observed. The expression level of active caspase8 was detected by immunofluorescence staining and Western blot. The effect of EphB4 on the apoptosis of human embryonic neural stem cells was analyzed by Annexin V -PE/7-AAD flow reagent box, and the expression level of Cyclin D1/CDK4 inhibitor FAS, Abl inhibitor Gleevec, Abl agonist inhibition and activation protein expression level were explored. 4 downstream signal pathway. Results (1) we cultured human embryonic neural stem cells, the proportion of Nestin~+ cells is 77.37%, and can successfully differentiate into beta III -tubulin~+ neurons, GFAP~+ astrocytes and Galc~+ oligodendrocytes. (2) compared with the control group, EphB4 silencing lentivirus can significantly reduce the m RNA level of EphB4 and eggs. In white expression level, transfection of EphB4 over expression of lentivirus could significantly increase the level of M RNA and protein expression of EphB4, and the EphB4 gene silencing and overexpression model of human embryonic neural stem cells were successfully established. (3) cloning analysis and immunofluorescence staining showed that the EphB4 gene silencing significantly reduced the diameter of the nerve bulb compared with the control group. The number of balls and the number of Brd U positive cells inhibited the proliferation of cells; EphB4 overexpression significantly increased the diameter of the nerve bulb, the number of nerve spheres and the number of Brd U positive cells, and promoted the cell proliferation. (4) immunofluorescence staining, RT-PCR analysis and Western blot results showed that the EphB4 gene silencing significantly decreased Dcx, beta III -tubulin Yang compared with the control group. The number of sex cells, the level of M RNA and the expression level of Mash1 protein significantly increased the number of GFAP, the number of NG2 positive cells and the level of M RNA, inhibited the differentiation of cells into neurons and promoted the differentiation of cells into glial cells, and the overexpression of EphB4 gene significantly increased the Dcx, the number of positive cells of beta III -tubulin and the m RNA level, and the level of protein expression decreased significantly. GFAP, NG2 positive cells and their m RNA levels, promote cells to differentiate into neurons and inhibit cell differentiation to glia. (5) immunofluorescence staining, Western blot and cell flow results showed that compared with the control group, EphB4 silencing group and overexpressed group Cleaved-caspase3, the number of caspase8 positive cells, active caspase8 protein expression level and Annexin There was no significant change in the number of cells labeled by V -PE~+/7-AAD-, and the silence and overexpression of EphB4 gene had no effect on cell apoptosis. (6) cell cycle detection showed that EphB4 gene silencing could significantly increase the number of G0/G1 cells and decrease the number of cells in the S phase and G2/M phase compared with the control group, and the EphB4 gene overexpression could significantly reduce G. The number of cells in phase 0/G1, the relative increase of the number of S and G2/M phase.EphB4 gene silencing and overexpression can affect the expression level of Cyclin D1, CDK4, and the use of Cyclin D1/CDK4 inhibitor FAS inhibitory protein activity to eliminate the effect of EphB4 over expression to promote cell proliferation. The regulation of protein on the proliferation of human embryonic neural stem cells. (7) Western blot results show that the silence and overexpression of EphB4 gene can affect the expression level of Abl. The inhibition and activation of Abl protein with the Abl inhibitor Gleevec and activator DPH can affect Cyclin D1, CDK4 expression is flat, and can be eliminated after the activity of Abl protein is inhibited. HB4 overexpression promotes cell proliferation. The results show that Abl as a downstream protein of EphB4 mediates the effect of EphB4 regulating Cyclin D1/CDK4 on cell proliferation. (8) the use of Abl inhibitor Gleevec and Cyclin D1/CDK4 inhibitor FAS inhibitory protein activity does not affect the proliferation of cells to neurons and inhibit cell direction. The effect of glial cell differentiation. The results showed that Abl-Cyclin D1/CDK4 signaling pathway did not regulate the effect of EphB4 on cell differentiation. Conclusion: after the silence of EphB4 gene, the proliferation of human embryonic neural stem cells and the differentiation of cells into neurons in vitro, and the differentiation of cells into glial cells, and no effect of.EphB4 gene on the apoptosis of the cells. After expression, it promotes the proliferation of human embryonic neural stem cells and the differentiation of cells into neurons, inhibits the differentiation of cells into glial cells, and does not affect cell apoptosis by regulating cell proliferation through the downstream signal pathway Abl-Cyclin D1. This signaling pathway does not participate in the regulation of EphB4 in cell differentiation by regulating.EphB4 to regulate the nerve. Stem cells proliferate, and they are the switches that determine whether neural stem cells differentiate into neurons or differentiate into glial cells. It is likely to be an effective target for the repair of neurons after brain damage.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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