NF-κB信号传导通路和自噬在神经干细胞分化中的作用
发布时间：2018-07-29 11:22
【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(P0.05、P0.01)。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(p0.05, 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(p0.001)。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。
[Abstract]:Objective: neural stem cell research is one of the hotspots in life sciences, which is derived from its important developmental differentiation mechanism and potential clinical application value, but the understanding of complex regulation mechanism is not clear enough. It limits the wide application of neural stem cells. Our research project will look at the effects of NF- 魏 B transduction pathway on the proliferation and differentiation of neural stem cells and the role of autophagy in it. We try to prove that autophagy activation and blocking NF- 魏 B pathway is an effective way to induce neural stem cell differentiation. Methods: NF- 魏 B inhibitor SN50 was added to cultured neural stem cells in vitro. The proliferation of neural stem cells was observed under phase contrast microscope at different doses (6.25-25 渭 g/ml) and time points (2-24 h). The effect of SN50 on the nuclear metastasis of NF- 魏 B was detected by immunofluorescence assay with NF- 魏 B p65. The proliferation rate of NSCs was detected by MTT method at different doses and time points. The cytotoxicity of SN50 to neural stem cells was detected by LDH leakage rate, and the cytotoxicity of PCNA protein expressed by proliferating cell nuclear antigen (PCNA) was detected by Western blot. Neural stem cells were detected by immunofluorescence and Western blot. The expression of GAP-43, cyclin D1 and Cdk4 in neurons and glial cells were detected by Western blot fluorescence staining and LC3 immunofluorescence staining. The expression of LC3-I and LC3-II was detected by Western blot. Results: after adding NF- 魏 B inhibitor SN50 to the cultured neural stem cells in vitro, the results showed that at 12 h and 24 h after addition of 1: SN50 (6.25-50 渭 g/ml), the neural stem cells (NSCs) could be inhibited significantly. Compared with the negative control group, there were significant or highly significant differences (P0.05, P0.01) .SN50 had an inhibitory effect on the proliferation of neural stem cells, and the effect was time-dependent and dose-dependent, with the prolongation of administration time and the increase of drug concentration. There was no significant difference in the leakage rate of LDH between the SN50 group and the control group (p0.05, Figure 6). The expression of cyclin D1 and Cdk 4 in neural stem cells treated with SN50 decreased significantly after 24 hours of treatment with SN50. The results of Western blot assay showed that the expression of cyclin D1 and Cdk 4 in neural stem cells treated with SN50 decreased significantly with time. On the other hand, the expression of GAP-43 increased significantly with time, and the expression of MAP-2Neun increased with the prolongation of differentiation time and the increase of drug dosage. In a dose-dependent manner, the inhibition of NF- 魏 B activity by SN50 increased the proportion of neural stem cells differentiated into neurons. The number of acidic vesicles labeled by MDC increased the ratio of LC3-IIR LC3-I and the expression of Beclin 1. The inhibitory effect of autophagy inhibitor 3-MA (3-methyladenine) on the differentiation of neural stem cells induced by SN50 after autophagy was significantly weakened. These results suggest that SN50 promotes neural stem cell differentiation by autophagy activation. Conclusion: after inhibiting NF- 魏 B nuclear metastasis, the proliferation of neural stem cells is inhibited, but the mechanism of promoting the differentiation of neural stem cells. NF- 魏 B inhibitors trigger the differentiation of neural stem cells may be related to the inhibition of cell cycle and activation of autophagy. These results suggest that NF- 魏 B inhibitors not only inhibit neuronal death, but also promote stem cell differentiation and induce stem cell migration in neurodegenerative diseases.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329
本文编号：2152525
[Abstract]:Objective: neural stem cell research is one of the hotspots in life sciences, which is derived from its important developmental differentiation mechanism and potential clinical application value, but the understanding of complex regulation mechanism is not clear enough. It limits the wide application of neural stem cells. Our research project will look at the effects of NF- 魏 B transduction pathway on the proliferation and differentiation of neural stem cells and the role of autophagy in it. We try to prove that autophagy activation and blocking NF- 魏 B pathway is an effective way to induce neural stem cell differentiation. Methods: NF- 魏 B inhibitor SN50 was added to cultured neural stem cells in vitro. The proliferation of neural stem cells was observed under phase contrast microscope at different doses (6.25-25 渭 g/ml) and time points (2-24 h). The effect of SN50 on the nuclear metastasis of NF- 魏 B was detected by immunofluorescence assay with NF- 魏 B p65. The proliferation rate of NSCs was detected by MTT method at different doses and time points. The cytotoxicity of SN50 to neural stem cells was detected by LDH leakage rate, and the cytotoxicity of PCNA protein expressed by proliferating cell nuclear antigen (PCNA) was detected by Western blot. Neural stem cells were detected by immunofluorescence and Western blot. The expression of GAP-43, cyclin D1 and Cdk4 in neurons and glial cells were detected by Western blot fluorescence staining and LC3 immunofluorescence staining. The expression of LC3-I and LC3-II was detected by Western blot. Results: after adding NF- 魏 B inhibitor SN50 to the cultured neural stem cells in vitro, the results showed that at 12 h and 24 h after addition of 1: SN50 (6.25-50 渭 g/ml), the neural stem cells (NSCs) could be inhibited significantly. Compared with the negative control group, there were significant or highly significant differences (P0.05, P0.01) .SN50 had an inhibitory effect on the proliferation of neural stem cells, and the effect was time-dependent and dose-dependent, with the prolongation of administration time and the increase of drug concentration. There was no significant difference in the leakage rate of LDH between the SN50 group and the control group (p0.05, Figure 6). The expression of cyclin D1 and Cdk 4 in neural stem cells treated with SN50 decreased significantly after 24 hours of treatment with SN50. The results of Western blot assay showed that the expression of cyclin D1 and Cdk 4 in neural stem cells treated with SN50 decreased significantly with time. On the other hand, the expression of GAP-43 increased significantly with time, and the expression of MAP-2Neun increased with the prolongation of differentiation time and the increase of drug dosage. In a dose-dependent manner, the inhibition of NF- 魏 B activity by SN50 increased the proportion of neural stem cells differentiated into neurons. The number of acidic vesicles labeled by MDC increased the ratio of LC3-IIR LC3-I and the expression of Beclin 1. The inhibitory effect of autophagy inhibitor 3-MA (3-methyladenine) on the differentiation of neural stem cells induced by SN50 after autophagy was significantly weakened. These results suggest that SN50 promotes neural stem cell differentiation by autophagy activation. Conclusion: after inhibiting NF- 魏 B nuclear metastasis, the proliferation of neural stem cells is inhibited, but the mechanism of promoting the differentiation of neural stem cells. NF- 魏 B inhibitors trigger the differentiation of neural stem cells may be related to the inhibition of cell cycle and activation of autophagy. These results suggest that NF- 魏 B inhibitors not only inhibit neuronal death, but also promote stem cell differentiation and induce stem cell migration in neurodegenerative diseases.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329
【引证文献】
相关博士学位论文 前2条
1 胡玉萍;补肾化痰益智法治疗AD的理论探讨及其抗炎作用研究[D];湖北中医药大学;2011年
2 莫镇涛;β-细辛醚对缺糖缺氧PC12细胞自噬的影响[D];广州中医药大学;2012年
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