本文選題：EphB4信號通路 + 神經干細胞��； 參考：《首都醫(yī)科大學》2017年博士論文

【摘要】：目的缺血性腦卒中是一種嚴重損害神經功能的疾病,其具有很高的發(fā)病率和死亡率。缺血性腦損傷后期的神經再生與修復是腦卒中治療的關鍵環(huán)節(jié)。腦卒中后可以誘發(fā)內源性神經干細胞增殖、分化并向損傷區(qū)域遷移,其中多條信號通路參與調節(jié)神經干細胞的活動。通過調節(jié)特異性的分子機制,可以增強腦損傷誘導的內源性神經干細胞的增殖與分化過程。目前,參與調節(jié)神經干細胞增殖與分化的經典信號通路有四條,即Wnt信號通路、Notch信號通路、Shh(Sonic Hedgehog,Shh)信號通路與骨形態(tài)發(fā)生蛋白(bone morphogenetic protein,BMP)信號通路。在前期我們研究Wnt信號通路時發(fā)現(xiàn),β-catenin做為Wnt信號通路的關鍵調節(jié)因子,同時也是Eph信號通路的下游蛋白之一。因此,我們關注到了Eph/ephrin這條信號通路。Eph受體是具有最多成員的受體酪氨酸激酶家族,其與配體ephrin結合后可以誘發(fā)雙向傳導過程。近年來研究表明Eph受體與ephrin配體參與調節(jié)神經干細胞的增殖、分化、存活與遷移活動。但對于EphB4的研究都集中在腫瘤方面,其對神經干細胞的調節(jié)作用卻未見報道。而腫瘤細胞中存在少部分腫瘤激活細胞,這些細胞與干細胞有著共同的特性,腫瘤可以從正常組織干細胞轉化而來。我們猜想EphB4是否在神經干細胞的增殖與分化方面也起到重要作用。本實驗旨在研究EphB4是否參與調節(jié)了人胚胎神經干細胞的增殖、分化、凋亡活動,并探索其下游信號通路與調節(jié)機制,為腦卒中治療提供新的靶點。方法(1)人胚胎神經干細胞的鑒定:培養(yǎng)原代人胚胎神經干細胞,正常培養(yǎng)3天后進行Nestin免疫熒光染色標記巢蛋白;分化培養(yǎng)10天后進行βⅢ-tubulin與膠質纖維酸性蛋白(glial fibrillary acidic protein,GFAP)免疫熒光染色分別標記神經元與星形膠質細胞;分化培養(yǎng)16天后進行半乳糖腦苷脂(galactocerebroside,Galc)免疫熒光染色標記少突膠質細胞,進行人胚胎神經干細胞鑒定。(2)EphB4信號通路的機制研究:培養(yǎng)原代人胚胎神經干細胞,使用沉默慢病毒與過表達慢病毒轉染細胞,分別下調和上調EphB4基因水平及其蛋白表達水平,分為EphB4基因沉默對照組(Scrambled-sh RNA)、EphB4基因沉默組(EphB4-sh RNA)、EphB4基因過表達對照組(EphB4-control)、EphB4基因過表達組(EphB4-OE)。然后通過實時熒光定量聚合酶鏈反應(real-time reverse transcription-polymerase chain reaction,RT-PCR)檢測EphB4的基因表達水平,Western blot檢測EphB4的蛋白表達水平來確定沉默及過表達效率。通過克隆分析、Brd U免疫熒光染色觀察EphB4對細胞自我更新與增殖的影響;通過Dcx、βⅢ-tubulin、GFAP、NG2免疫熒光染色及RT-PCR檢測Dcx、βⅢ-tubulin、GFAP、NG2基因水平,Western blot檢測Mash1蛋白表達水平觀察EphB4對人胚胎神經干細胞分化的影響;通過Cleaved-caspase3、caspase8免疫熒光染色,Western blot檢測活性caspase8的表達水平、AnnexinⅤ-PE/7-AAD流式試劑盒分析觀察EphB4對人胚胎神經干細胞凋亡的影響;通過細胞周期分析及使用Cyclin D1/CDK4抑制劑FAS、Abl抑制劑Gleevec、Abl激動劑DPH抑制和激活蛋白表達水平探索EphB4下游信號通路。結果(1)我們培養(yǎng)的人胚胎神經干細胞,Nestin~+細胞的比例為77.37%,并且可以成功分化為βⅢ-tubulin~+神經元、GFAP~+星形膠質細胞與Galc~+少突膠質細胞。(2)與對照組相比,轉染EphB4沉默慢病毒可以顯著降低EphB4的m RNA水平與蛋白表達水平,轉染EphB4過表達慢病毒可以顯著提高EphB4的m RNA水平與蛋白表達水平,人胚胎神經干細胞EphB4基因沉默及過表達模型成功建立。(3)克隆分析與免疫熒光染色結果顯示,與對照組相比,EphB4基因沉默顯著降低了神經球直徑、神經球個數與Brd U陽性細胞數,抑制了細胞增殖;EphB4過表達顯著增加了神經球的直徑、神經球個數與Brd U陽性細胞數,促進了細胞增殖。(4)免疫熒光染色、RT-PCR分析與Western blot結果顯示,與對照組相比,EphB4基因沉默顯著降低了Dcx、βⅢ-tubulin陽性細胞數及其m RNA水平、Mash1蛋白表達水平,顯著增加了GFAP、NG2陽性細胞數及其m RNA水平,抑制細胞向神經元分化,促進細胞向膠質細胞分化;EphB4基因過表達顯著增加了Dcx、βⅢ-tubulin陽性細胞數及其m RNA水平、Mash1蛋白表達水平,顯著降低了GFAP、NG2陽性細胞數及其m RNA水平,促進細胞向神經元分化,抑制細胞向膠質細胞分化。(5)免疫熒光染色、Western blot及細胞流式結果顯示,與對照組相比,EphB4沉默組與過表達組Cleaved-caspase3、caspase8陽性細胞數、活性caspase8蛋白表達水平及AnnexinⅤ-PE~+/7-AAD-標記的細胞數量無顯著變化,EphB4基因沉默及過表達對細胞凋亡無影響。(6)細胞周期檢測結果顯示,與對照組相比,EphB4基因沉默可以顯著增加G0/G1期細胞數量,相對的減少S期與G2/M期的細胞數量;EphB4基因過表達可以顯著減少G0/G1期細胞數量,相對的增加S期與G2/M期的細胞數量。EphB4基因沉默及過表達可以影響G1期調節(jié)蛋白Cyclin D1、CDK4的表達水平,使用Cyclin D1/CDK4抑制劑FAS抑制蛋白活性后消除了EphB4過表達促進細胞增殖的作用。結果表明Cyclin D1/CDK4介導了EphB4蛋白對人胚胎神經干細胞增殖的調節(jié)作用。(7)Western blot結果顯示,EphB4基因沉默及過表達可以影響Abl的表達水平,使用Abl抑制劑Gleevec與激動劑DPH抑制與激活Abl蛋白可以影響Cyclin D1、CDK4的表達水平,并且在Abl蛋白活性被抑制后可以消除EphB4過表達促進細胞增殖的作用。結果表明,Abl作為EphB4的下游蛋白介導了EphB4調控Cyclin D1/CDK4影響細胞增殖的作用。(8)使用Abl抑制劑Gleevec與Cyclin D1/CDK4抑制劑FAS抑制蛋白活性并未影響EphB4過表達促進細胞向神經元方向分化,抑制細胞向膠質細胞分化的作用。結果表明,Abl-Cyclin D1/CDK4信號通路并未參與調節(jié)EphB4對細胞分化的作用。結論:EphB4基因沉默后,抑制體外培養(yǎng)人胚胎神經干細胞的增殖及細胞向神經元分化,促進細胞向膠質細胞分化,對細胞凋亡無影響。EphB4基因過表達后,促進體外培養(yǎng)人胚胎神經干細胞的增殖及細胞向神經元的分化,抑制細胞向膠質細胞分化,對細胞凋亡無影響。EphB4是通過下游信號通路Abl-Cyclin D1調節(jié)細胞增殖,此信號通路不參與EphB4在細胞分化方面的調節(jié)。EphB4參與調節(jié)神經干細胞增殖,而且是決定神經干細胞向神經元分化還是向膠質細胞分化的開關,其很可能成為腦損傷后神經元修復的有效治療靶點。
[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.

【學位授予單位】：首都醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R743.3

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