盐酸氟西汀调控神经干细胞信号通路及下游基因表达的实验研究

【作者】 赵宇；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 目的应用P13K特异性阻滞剂LY294002孵育大鼠神经干细胞,探讨P13K信号转导途径在神经干细胞中的作用。方法体外分离、培养大鼠NSCs,应用不同浓度的LY294002孵育NSCs后进行WTS-8、BrdU和βTubulin-Ⅲ检测。结果低浓度的LY294002对NSCs的存活、增殖和分化没有明显影响,在LY294002较高浓度时,NSCs的存活率明显下降(P＜0.05)、BrdU和βTubulin-Ⅲ的阳性细胞数明显少于正常对照组(P＜0.05)。结论PI3K信号转导途径对大鼠NSCs的存活、增殖、分化起重要作用。目的应用MAPKK/MEK特异性阻滞剂PD98059孵育大鼠神经干细胞,探讨MAPK信号通路在神经干细胞中的作用。方法体外分离、培养大鼠NSCs,应用不同浓度的PD98059孵育NSCs后进行WTS-8、BrdU和βTubulin-Ⅲ检测。结果低浓度的PD98059对NSCs的存活、增殖和分化没有明显影响,在PD98059较高浓度时,NSCs的存活率明显下降(P＜0.05)、BrdU和βTubulin-Ⅲ的阳性细胞数明显少于正常对照组(P＜0.05)。结论MAPK信号通路对大鼠NSCs的存活、增殖、分化起重要作用。目的应用PI3K特异性阻滞剂LY294002、MAPKK/MEK特异性阻滞剂PD98059和选择性5-羟色胺再摄取抑制剂(selective serotoninreuptake inhibitors,SSRIs)类抗抑郁药物盐酸氟西汀(Fluoxetine)孵育大鼠神经干细胞,探讨盐酸氟西汀对神经干细胞的PI3K/AKT和MAPK/ERK信号通路的影响。方法体外分离、培养大鼠NSCs,分别应用LY294002、PD98059和盐酸氟西汀孵育NSCs后进行Western Blotting检测。结果Western Blotting检测显示盐酸氟西汀能上调磷酸化AKT和磷酸化ERK1/2在蛋白水平的表达。结论在大鼠神经干细胞模型中,盐酸氟西汀可以上调磷酸化AKT和磷酸化ERK1/2的蛋白表达水平,在盐酸氟西汀激活AKT和ERK1/2的过程中,PI3K/AKT信号通路与MEK/ERK信号通路之间在AKT-ERK1/2水平存在着“对话”(cross-talk)现象。目的应用PI3K特异性阻滞剂LY294002、MEK特异性阻滞剂PD98059和选择性5-羟色胺再摄取抑制剂(selective serotonin reuptakeinhibitors,SSRIs)类抗抑郁药物盐酸氟西汀(Fluoxetine)孵育大鼠神经干细胞,探讨盐酸氟西汀对神经干细胞下游基因表达的影响。方法体外分离、培养大鼠NSCs,分别应用LY294002、PD98059和盐酸氟西汀孵育NSCs后进行real-time RT-PCR检测。结果real-time RT-PCR检测显示盐酸氟西汀能上调BDNF、GDNF、LIF和BCL-2的基因表达,对CNTF和ILGF1/2的基因表达没有影响。结论在大鼠神经干细胞模型中,盐酸氟西汀可以上调BDNF、GDNF、LIF和BCL-2的基因表达水平,而且是通过PI3K/AKT或(和)MEK/ERK信号转导途径上调上述基因的表达。BDNF、GDNF、LIF和BCL-2是与盐酸氟西汀作用相关的下游信号分子靶点,CNTF和ILGF1/2不是盐酸氟西汀作用的下游信号分子靶点。更多还原

【Abstract】 Objective To apply selective specific inhibitor(LY294002) of PI3K to incubate the rat neural stem cells to research the effect of PI3K signal transduction pathway in neural stem cells.Methods To isolate,culture the NSCs derived from E15-16 rats,carry out WTS-8 assay,BrdU andβTubulin-Ⅲimmunofluorescent staining after incubated NSCs in different concentration of LY294002.Results There was not obvious effect in the survival,proliferation and differentiation of NSCs incubated by low concentration LY294002.The viability of the NSCs decreased significantly than the control(P＜0.05),the amount of BrdU andβTubulin-Ⅲpositive cells were notably fewer than the control(P＜0.05) after they were incubated by the higher concentration LY294002.Conclusion The PI3K signal transduction pathway played a vital role in the survival,proliferation and differentiation of NSCs. Objective To apply selective specific inhibitor(PD98059) of MAPKK/MEK to incubate the rat neural stem cells to research the effect of MAPK signal transduction pathway in neural stem cells.Methods To isolate,culture the NSCs derived from E15-16 rats,carry out WTS-8 assay,BrdU andβTubulin-Ⅲimmunofluorescent staining after incubated NSCs in different concentration of PD98059.Results There was not obvious effect in the survival,proliferation and differentiation of NSCs incubated by low concentration PD98059.The viability of the NSCs decreased significantly than the control(P＜0.05 ),the amount of BrdU andβTubulin-Ⅲpositive cells were notably fewer than the control(P＜0.05) after they were incubated by the higher concentration PD98059.Conclusion The MAPK signal transduction pathway played a vital role in the survival,proliferation and differentiation of NSCs. Objective To apply the phosphatidylinositol-3-kinase(PI3-K) inhibitor(LY294002),the mitogen-activated protein kinase kinase(MEK) inhibitor(PD98059) and antidepressant drugs fluoxetine(the selective serotonin reuptake inhibitors) to incubate the rat neural stem cells to research the effect of the role of PI3K/AKT and MAPK/ERK and signaling pathways in fluoxetine treated neural stem cells.Methods To isolate,culture the NSCs derived from E15-16 rats and carried out the Western Blotting detection after respectively incubated NSCs with the phosphatidylinositol-3-kinase(PI3-K) inhibitor (LY294002),the mitogen-activated protein kinase kinase(MEK) inhibitor (PD98059) and fluoxetine.Results The fluoxetine can activated AKT,ERK1/2 protein expression of rats NSCs.Conclusion The fluoxetine can activated AKT,ERK1/2 protein expression of rats NSCs and there was cross-talk between PI3K/AKT pathway and MAPK/ERK pathway at the level of AKT-ERK1/2 in the process of fluoxetine activated AKT、ERK1/2 protein. Objective To apply the phosphatidylinositol-3-kinase(PI3-K) inhibitor(LY294002),the mitogen-activated protein kinase kinase(MEK) inhibitor(PD98059) and antidepressant drugs fluoxetine(the selective serotonin reuptake inhibitors) to incubate the rat neural stem cells to research the expression of downstream genes in fluoxetine-treated neural stem cells.Methods To isolate,culture the NSCs derived from E15-16 rats and carried out the real-time RT-PCR detection after respectively incubated NSCs with the phosphatidylinositol-3-kinase(PI3-K) inhibitor (LY294002),the mitogen-activated protein kinase kinase(MEK) inhibitor (PD98059) and fluoxetine.Results The fluoxetine can upregulated the mRNA expression of BDNF,GDNF,LIF and BCL-2 in rats NSCs and can not regulated the mRNA expression of CNTF and ILGF1/2.Conclusion The mRNA expression of BDNF,GDNF,LIF and BCL-2 can be up-regulated by fluoxetine through PI3K and(or) MAPK mediated pathway in NSCs.BDNF,GDNF,LIF and BCL-2 not CNTF and ILGF1/2 were the downstream signaling molecule targets of fluoxetine.更多还原