【摘要】： 目的:神經(jīng)干細(xì)胞研究是當(dāng)今生命科學(xué)研究的熱點(diǎn)之一,源于其重要的發(fā)育分化機(jī)制和潛在的臨床應(yīng)用價(jià)值,但對(duì)復(fù)雜的調(diào)控機(jī)制的了解還不夠清楚,限制了神經(jīng)干細(xì)胞的廣泛應(yīng)用。我們的研究計(jì)劃將從一個(gè)全新的角度:從NF-κB傳導(dǎo)通路入手研究其對(duì)神經(jīng)干細(xì)胞增殖分化的影響及自噬在其中發(fā)揮的作用,試圖證明自噬激活和阻斷NF-κB傳導(dǎo)通路是誘導(dǎo)神經(jīng)干細(xì)胞分化的有效手段。 方法:體外培養(yǎng)的神經(jīng)干細(xì)胞中加入NF-κB的抑制劑SN50,取不同的劑量(6.25-25μg/ml)和時(shí)間點(diǎn)(2-24 h)相差顯微鏡下觀察神經(jīng)干細(xì)胞的增殖、分化情況;SN50阻斷NF-κB核轉(zhuǎn)移的作用用NF-κB p65的免疫熒光檢測,用激光共聚焦顯微鏡作定性分析;采用MTT法取不同的劑量和時(shí)間點(diǎn)檢測細(xì)胞的增殖率;Western blot檢測增殖細(xì)胞核抗原PCNA蛋白表達(dá);SN50對(duì)神經(jīng)干細(xì)胞的細(xì)胞毒性采用LDH漏出率來檢測;采用免疫熒光法、Western blot法檢測神經(jīng)干細(xì)胞、神經(jīng)元及神經(jīng)膠質(zhì)細(xì)胞的標(biāo)志性蛋白的表達(dá)(Nestin、MAP-2、GFAP);生長相關(guān)蛋白(GAP-43)、細(xì)胞周期調(diào)節(jié)蛋白cyclin D1和細(xì)胞周期調(diào)節(jié)蛋白激酶Cdk4的表達(dá)均用Western blot法;MDC熒光染色及LC3免疫熒光檢測自噬體形成;Western blot檢測LC3-I和LC3-II的生成,Beclin 1蛋白的表達(dá)變化。 結(jié)果:體外培養(yǎng)的神經(jīng)干細(xì)胞中加入NF-κB的抑制劑SN50后,結(jié)果顯示:SN50(6.25-50μg/ml)在各時(shí)間段12 h、24 h,對(duì)神經(jīng)干細(xì)胞s有明顯的抑制作用,與陰性對(duì)照組相比均有顯著或高度顯著性差異(P0.05、P0.01)。SN50給藥組對(duì)神經(jīng)干細(xì)胞細(xì)胞的增殖有抑制作用,且此作用呈明顯的時(shí)間、劑量依賴性,隨著給藥時(shí)間的延長和給藥濃度的增加,抑制增殖的作用越顯著;SN50各劑量組和對(duì)照組相比,LDH漏出率變化沒有顯著差異(p0.05, Figure 6)。正常對(duì)照組PCNA呈高水平表達(dá),SN50各劑量組作用24小時(shí)后,PCNA蛋白表達(dá)均顯著降低(p0.001)。Western blot實(shí)驗(yàn)結(jié)果顯示cyclin D1和Cdk 4在SN50處理后的神經(jīng)干細(xì)胞中的表達(dá)隨時(shí)間呈顯著下降的趨勢;而GAP-43的表達(dá)則相反,隨時(shí)間成顯著增高的趨勢;MAP-2、NeuN的蛋白表達(dá)隨著誘導(dǎo)分化時(shí)間的延長和用藥劑量的增加,其表達(dá)增強(qiáng),呈現(xiàn)出明顯的時(shí)間、劑量依賴性;SN50抑制NF-κB的活性促使神經(jīng)干細(xì)胞分化為神經(jīng)元的比例增加。MDC標(biāo)記的酸性囊泡增多,LC3-II/LC3-I比值及Beclin 1表達(dá)增加;用自噬抑制劑3-MA(3-methyladenine)阻斷自噬后,SN50誘導(dǎo)的神經(jīng)干細(xì)胞分化的作用顯著減弱。這些結(jié)果表明SN50促進(jìn)神經(jīng)干細(xì)胞分化與自噬激活有關(guān)。 結(jié)論:在抑制NF-κB核轉(zhuǎn)移后,神經(jīng)干細(xì)胞的增殖被抑制,但促進(jìn)了神經(jīng)干細(xì)胞的分化。NF-κB抑制劑觸發(fā)神經(jīng)干細(xì)胞分化的機(jī)制可能和抑制細(xì)胞周期,激活自噬有關(guān)。本研究結(jié)果提示NF-κB抑制劑在神經(jīng)退行性疾病中的作用不僅僅是抑制神經(jīng)元的死亡,而且它在促進(jìn)干細(xì)胞分化、誘導(dǎo)干細(xì)胞遷移方面也起著重要作用。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前2條

1 胡玉萍;補(bǔ)腎化痰益智法治療AD的理論探討及其抗炎作用研究[D];湖北中醫(yī)藥大學(xué);2011年

2 莫鎮(zhèn)濤;β-細(xì)辛醚對(duì)缺糖缺氧PC12細(xì)胞自噬的影響[D];廣州中醫(yī)藥大學(xué);2012年

，

本文編號(hào)：2152525