【作者】 张丽；

【导师】 秦正红； 顾振纶； 周文轩； 郭次仪；

【作者基本信息】 苏州大学 ， 药理学， 2008， 博士

【摘要】 目的:神经干细胞研究是当今生命科学研究的热点之一,源于其重要的发育分化机制和潜在的临床应用价值,但对复杂的调控机制的了解还不够清楚,限制了神经干细胞的广泛应用。我们的研究计划将从一个全新的角度:从NF-κB传导通路入手研究其对神经干细胞增殖分化的影响及自噬在其中发挥的作用,试图证明自噬激活和阻断NF-κB传导通路是诱导神经干细胞分化的有效手段。方法:体外培养的神经干细胞中加入NF-κB的抑制剂SN50,取不同的剂量(6.25-25μg/ml)和时间点(2-24 h)相差显微镜下观察神经干细胞的增殖、分化情况;SN50阻断NF-κB核转移的作用用NF-κB p65的免疫荧光检测,用激光共聚焦显微镜作定性分析;采用MTT法取不同的剂量和时间点检测细胞的增殖率;Western blot检测增殖细胞核抗原PCNA蛋白表达;SN50对神经干细胞的细胞毒性采用LDH漏出率来检测;采用免疫荧光法、Western blot法检测神经干细胞、神经元及神经胶质细胞的标志性蛋白的表达(Nestin、MAP-2、GFAP);生长相关蛋白(GAP-43)、细胞周期调节蛋白cyclin D1和细胞周期调节蛋白激酶Cdk4的表达均用Western blot法;MDC荧光染色及LC3免疫荧光检测自噬体形成;Western blot检测LC3-I和LC3-II的生成,Beclin 1蛋白的表达变化。结果:体外培养的神经干细胞中加入NF-κB的抑制剂SN50后,结果显示:SN50(6.25-50μg/ml)在各时间段12 h、24 h,对神经干细胞s有明显的抑制作用,与阴性对照组相比均有显著或高度显著性差异(P<0.05、P<0.01)。SN50给药组对神经干细胞细胞的增殖有抑制作用,且此作用呈明显的时间、剂量依赖性,随着给药时间的延长和给药浓度的增加,抑制增殖的作用越显著;SN50各剂量组和对照组相比,LDH漏出率变化没有显著差异(p>0.05, Figure 6)。正常对照组PCNA呈高水平表达,SN50各剂量组作用24小时后,PCNA蛋白表达均显著降低(p<0.001)。Western blot实验结果显示cyclin D1和Cdk 4在SN50处理后的神经干细胞中的表达随时间呈显著下降的趋势;而GAP-43的表达则相反,随时间成显著增高的趋势;MAP-2、NeuN的蛋白表达随着诱导分化时间的延长和用药剂量的增加,其表达增强,呈现出明显的时间、剂量依赖性;SN50抑制NF-κB的活性促使神经干细胞分化为神经元的比例增加。MDC标记的酸性囊泡增多,LC3-II/LC3-I比值及Beclin 1表达增加;用自噬抑制剂3-MA(3-methyladenine)阻断自噬后,SN50诱导的神经干细胞分化的作用显著减弱。这些结果表明SN50促进神经干细胞分化与自噬激活有关。结论:在抑制NF-κB核转移后,神经干细胞的增殖被抑制,但促进了神经干细胞的分化。NF-κB抑制剂触发神经干细胞分化的机制可能和抑制细胞周期,激活自噬有关。本研究结果提示NF-κB抑制剂在神经退行性疾病中的作用不仅仅是抑制神经元的死亡,而且它在促进干细胞分化、诱导干细胞迁移方面也起着重要作用。更多还原

【Abstract】 Aim: A number of experimental methods have been developed to promote the differentiation of neural stem cells (NSCs) toward specific lineages. Neuronal differentiation is often associated with cell cycle arrest, altered rate of macromolecule synthesis and degradation as well as remodeling of cytoskeletal structures. NF-κB plays important roles in proliferation of lymphocytes, tumor cells and NSCs, however, its possible role in NSCs differentiation has nerve been explored. In this study, we studied if inhibiting NF-κB signaling pathway is involved in NSCs differentiation in vitro.Method: After treatment with SN50, the growth inhibition of NSCs was assessed by MTT colorimetric assay and the cytotoxicity of SN50 was measured by lactose dehydrogenase (LDH) leakage. Immunohistochemistry and Western blot analysis were used to study the mechanisms involved in NSCs differentiation in vitro by inhibiting NF-κB signaling pathway. MDC staining was used to examine autophagy induced by SN50 in NSCs. To examine the involvement of autophagy in SN50-induced differentiation of NSCs, the autophagy specific inhibitor 3-methyadenine (3-MA) was added with SN50. Immunohistochemistry and Western blot analysis were used to study the autophagic mechanisms involved in differentiation of NSCs.Results: NSCs cultured from mouse embryonic brains exposed to SN50, a recombinant cell-permeable peptide blocking nuclear translocation of NF-κB, resulted in the decrease in proliferation of NSCs. Meanwhile, SN50 promoted migration of NSCs away from neurospheres and growth of neuritis in a dose- and time-dependent fashion. Biochemical analysis revealed increases in the expression of protein levels of MAP-2, NeuN and GAP-43, indicating enhanced differentiation of NSCs towards neuronal lineage. Activation of autophagy during SN50-induced differentiation of NSCs was evidenced by presence of increased number of acidic vacuoles in the cytoplasm and increased expression of membrane form of microtubule associate protein light chain 3 (LC3-II) and becllin 1. Inhibition of autophagy by 3-methyladenine (3-MA) abrogated SN50-induced NSCs differentiation.Conclusion: These results suggest that blocking NF-κB transcriptional activation activity arrests cell cycle and promotes stem cell differentiation. Autophagy activation is associated with NF-κB inhibition-evoked stem cell differentiation.更多还原