NF-κB在HIF-1α基因修饰的NSCs上调VEGF表达中的桥接作用

发布时间：2018-06-22 03:19

本文选题：神经干细胞 + HIF-1α　；参考：《佳木斯大学》2009年硕士论文

【摘要】： 目的:研究NF-κB在HIF-1α上调VEGF表达通路中的作用。方法:取新生24小时内子鼠海马组织,经机械消化后,在无血清、含生长因子和细胞辅助剂以及低细胞密度的生长环境中培养并传代,单细胞克隆纯化细胞并进行NSCs鉴定。培养HEK293细胞,扩增AdHIF-1α--GFP及空载体Ad-GFP,并进行荧光鉴定、病毒滴度及感染复数测定。通过腺病毒转染法将HIF-1α基因转染到神经干细胞中,并分组为NSCs组,基因转染后NSCs组,空载体转染后NSCs组。免疫荧光观察及免疫组化染色检测及Western blotting法检测转染后NSCs基因表达情况。免疫组化染色检测及Western blotting法检测基因转染后NSCs中VEGF和NF-κB的表达情况,并检测给予梯浓度NF-κB特异性抑制剂PDTC后AdHIF-1α--GFP修饰后NSCs中VEGF的表达情况,并就其结果进行统计学分析。主要结果: 1,从子鼠海马分离出的细胞经原代和传代培养获得高纯度的神经干细胞,可形成细胞克隆,nestin染色呈阳性,证实为具有体外增殖和多向性分化潜能的神经干细胞,并改良NSCs传代时消化方法。 2,AdHIF-1α--GFP及Ad-GFP以HEK293细胞为载体扩增后,荧光显微镜观察大部分细胞有明亮绿色荧光,荧光阳性细胞计数及MTT法测定重组腺病毒滴度为1×108pfu/ml,确定感染复数(MOI)值为50∶1 ,当MOI值超过50∶1时,转染效率升高不明显,与75:1和100:1比较基本达到高峰(P0.01)。 3,AdHIF-1α--GFP及Ad-GFP转染NSCs后荧光显微镜下可直接观察到细胞发出绿色荧光;荧光表达持续30天以上,表明AdHIF-1α--GFP及Ad-GFP有效转染神经干细胞,并能持续表达荧光,NSCs是外源基因的良好载体,并得出基因表达强弱与MOI及转染时间有关。免疫组化、Western blotting显示转染AdHIF-1α--GFP后的NSCs中HIF-1α显著表达,正常NSCs组及空载体组无表达,表明转染成功。与未转染组相比,转染AdHIF-1α--GFP基因的神经干细胞活力增强,且易分化。 4,通过免疫组化、Western blotting显示转染AdHIF-1α--GFP后的神经干细胞中VEGF和NF-κB的表达显著高于正常NSCs组及空载体组(P0.05),且两者表达呈正相关;给予梯浓度NF-κB特异性抑制剂PDTC后□AdHIF-1α--GFP修饰的神经干细胞中VEGF的表达呈浓度依赖性下调,各浓度组之间VEGF表达有显著差异(P0.05)。结论:1、对NSCs传代时细胞消化方法的改良,能增强细胞活力,有利于细胞增殖。 2、HIF-1α基因转染后NSCs中HIF-1α、NF-κB及VEGF表达均上调,三者正相关。 3、给予HIF-1α修饰的NSCs梯浓度NF-κB特异性抑制剂PDTC后,VEGF表达呈梯浓度依赖性下调。 4、NF-κB在HIF-1α上调VEGF表达的信号通路上并起桥接作用。
[Abstract]:Aim: to investigate the role of NF- 魏 B in up-regulation of VEGF expression pathway by HIF-1 伪. Methods: the hippocampal tissue of newborn rat within 24 hours was cultured and subcultured in serum-free environment containing growth factor and cell adjuvant and low cell density after mechanical digestion. The cells were cloned and purified by single cell clone and identified by NSCs. HEK293 cells were cultured, AdHIF-1 伪 -GFP and empty vector Ad-GFPwere amplified. HIF-1 伪 gene was transfected into neural stem cells by adenovirus transfection and divided into NSCs group, NSCs group after gene transfection and NSCs group after empty vector transfection. The expression of NSCs gene after transfection was detected by immunofluorescence, immunohistochemical staining and Western blotting assay. The expression of VEGF and NF- 魏 B in NSCs after transfection was detected by immunohistochemical staining and Western blotting method. The expression of VEGF and NF- 魏 B in NSCs treated with ladder concentration of NF- 魏 B specific inhibitor PDTC was detected, and the expression of VEGF in NSCs modified by AdHIF-1 伪 -GFP was analyzed statistically. The main results were as follows: 1. The high purity neural stem cells were obtained by primary and subculture of the cells isolated from the hippocampus of the offsprings, and the clones were positive for nestin staining. It was proved that NSCs have the potential of proliferation and multidirectional differentiation in vitro, and modified digestion method of NSCs. 2AdHIF-1 伪 -GFP and Ad-GFP were amplified by HEK293 cells. Fluorescence microscopy showed that most of the cells had bright green fluorescence. The fluorescent positive cell count and MTT assay showed that the titer of recombinant adenovirus was 1 脳 10 8 pFu / ml, and the complex number of infection (moi) was 50:1. When the moi value exceeded 50:1, the transfection efficiency was not significantly increased. Compared with 75:1 and 100: 1, the peak was reached (P0.01). After transfection of NSCs with AdHIF-1 伪 -GFP and Ad-GFP, green fluorescence could be observed directly under fluorescence microscope, and the fluorescence expression lasted more than 30 days, indicating that AdHIF-1 伪 -GFP and Ad-GFP could effectively transfect neural stem cells. The expression of NSCs was a good vector of exogenous gene, and the expression of NSCs was related to moi and transfection time. The expression of HIF-1 伪 in NSCs transfected with AdHIF-1 伪-GFP was significantly higher than that in normal NSCs and empty vector group, which indicated that the transfection was successful. The activity of neural stem cells transfected with AdHIF-1 伪 -GFP gene was increased compared with the control group. The expression of VEGF and NF- 魏 B in neural stem cells transfected with AdHIF-1 伪-GFP was significantly higher than that in normal NSCs and empty vector groups (P0.05), and there was a positive correlation between the expression of VEGF and NF- 魏 B in the neural stem cells transfected with AdHIF-1 伪-GFP (P0.05). The expression of VEGF in neural stem cells modified by -AdHIF-1 伪 -GFP was down-regulated in a concentration-dependent manner after treatment with NF- 魏 B specific inhibitor PDTC, and the expression of VEGF was significantly different among different concentration groups (P0.05). Conclusion the modified digestion method for NSCs can enhance cell viability and promote cell proliferation. 2HIF-1 伪 -NF- 魏 B and VEGF expression in NSCs were up-regulated after transfection of HIF-1 伪 gene. The expression of VEGF was down-regulated in a concentration-dependent manner after treatment with HIF-1 伪 modified NF- 魏 B specific inhibitor PDTC. 4NF-kappa B played a bridging role in the signal pathway of HIF-1 伪 upregulation of VEGF expression.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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