NF-κB信号通路对波动性高糖诱导的人脐静脉内皮细胞增殖和凋亡的影响
发布时间：2019-06-28 08:52
【摘要】： 目的:以核因子κB (NF-κB) p65基因特异的RNA干扰(RNAi)腺病毒表达载体作为研究工具,初步探讨NF-κB信号通路对波动性高糖(含5.5或30.5 mM D-葡萄糖培养液,每日交替)下人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell, HUVEC)增殖的影响,同时检测细胞凋亡相关分子bcl-2表达情况。 方法:利用体外同源重组技术构建NF-κB p65基因特异的siRNA腺病毒表达载体,在HEK293A细胞中包装并扩增病毒、空斑实验法测定病毒滴度;p65特异的siRNA腺病毒感染HUVEC,同时予波动性高糖干预,Western印记法测定p65蛋白表达和NF-κB核转录的影响;以AlamarBlue法检测细胞增殖;Western印记法检测bcl-2表达。 结果:1.成功构建了针对NF-κBp65特异的RNAi腺病毒表达载体;2.NF-κBp65特异的RNAi腺病毒感染HUVEC后可使p65表达量明显下降; 3.波动性高糖下,RNAi腺病毒感染HUVEC后,亦可检测到p65表达量下降,且随时间延长p65表达量进一步减少,抑制效应至少持续至干预后第七天;4.NF-κBp65特异腺病毒感染HUVEC,可以明显抑制波动性高糖刺激的NF-κBp65核蛋白转录,使核内p65蛋白表达处于基础水平; 5.AlamarBlue结果分析,NF-κBp65特异腺病毒感染HUVEC可以使波动性高糖诱导的细胞增殖抑制下降(P0.01),但比正常葡萄糖浓度(5.5 mmol/L)下的细胞增殖率低(P0.01); 6.NF-κBp65特异腺病毒可以上调波动性高糖下HUVEC bcl-2的表达,有可能减少细胞凋亡。 结论:构建NF-κB p65特异的RNAi腺病毒表达载体能有效抑制HUVEC p65基因的表达;腺病毒感染HUVEC,可以明显抑制波动性高糖刺激的NF-κB p65核转录,从而保护波动性高糖下的HUVEC增殖抑制,且有可能减少波动性高糖诱导的HUVEC凋亡。
[Abstract]:Aim: to investigate the effect of nuclear factor kappa B (NF- kappa B) p65 gene specific RNA interfering (RNAi) adenoviral expression vector on the proliferation of human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cell, HUVEC) in fluctuating high glucose (containing 5.5 or 30.5 mM D-glucose medium, alternately daily), and to detect the expression of apoptosis related molecule bcl-2 in human umbilical vein endothelial cells (umbilical vein endothelial cells). Methods: NF- 魏 B p65 gene specific siRNA adenoviral expression vector was constructed by homologous recombination technique in vitro, and the virus was packaged and amplified in HEK293A cells. The virus titer was determined by plaque assay. P65 specific siRNA adenovirus infected HUVEC, was interfered with volatile high glucose, Western printing was used to detect the expression of p65 protein and NF- kappa B nuclear transcription, AlamarBlue assay was used to detect cell proliferation, and Western printing method was used to detect the expression of bcl-2. Result: 1. The RNAi adenoviral expression vector specific to NF- 魏 Bp65 was successfully constructed. 2. The expression of p65 was significantly decreased after infection with RNAi adenoviruses. Under fluctuating high glucose, p65 expression decreased after HUVEC infection with RNAi adenoviral infection, and the expression of p65 decreased further with the prolongation of time, and the inhibitory effect lasted at least until the seventh day after intervention. 4. NF-魏 Bp65 specific adenoviral infection HUVEC, could significantly inhibit the transcription of NF- 魏 Bp65 nucleoprotein stimulated by fluctuating high glucose, making the expression of p65 protein in the nucleus at the basic level. 5.AlamarBlue results showed that HUVEC infection with HUVEC could decrease the cell proliferation inhibition induced by fluctuating high glucose (P01), but lower the cell proliferation rate (P01) than that at normal glucose concentration (5.5Bp65). 6. NF-魏 Bp65 specific adenovirus could up-regulate the expression of HUVEC bcl-2 under fluctuating high glucose, which might reduce apoptosis. Conclusion: the construction of NF- 魏 B p65 specific RNAi adenoviral expression vector can effectively inhibit the expression of HUVEC p65 gene, and adenoviral infection with HUVEC, can significantly inhibit the nuclear transcription of NF- 魏 B p65 stimulated by volatile high glucose, thus protecting the proliferation inhibition of HUVEC under volatile high glucose, and may reduce the apoptosis of HUVEC induced by volatile high glucose.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
本文编号：2507167
[Abstract]:Aim: to investigate the effect of nuclear factor kappa B (NF- kappa B) p65 gene specific RNA interfering (RNAi) adenoviral expression vector on the proliferation of human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cell, HUVEC) in fluctuating high glucose (containing 5.5 or 30.5 mM D-glucose medium, alternately daily), and to detect the expression of apoptosis related molecule bcl-2 in human umbilical vein endothelial cells (umbilical vein endothelial cells). Methods: NF- 魏 B p65 gene specific siRNA adenoviral expression vector was constructed by homologous recombination technique in vitro, and the virus was packaged and amplified in HEK293A cells. The virus titer was determined by plaque assay. P65 specific siRNA adenovirus infected HUVEC, was interfered with volatile high glucose, Western printing was used to detect the expression of p65 protein and NF- kappa B nuclear transcription, AlamarBlue assay was used to detect cell proliferation, and Western printing method was used to detect the expression of bcl-2. Result: 1. The RNAi adenoviral expression vector specific to NF- 魏 Bp65 was successfully constructed. 2. The expression of p65 was significantly decreased after infection with RNAi adenoviruses. Under fluctuating high glucose, p65 expression decreased after HUVEC infection with RNAi adenoviral infection, and the expression of p65 decreased further with the prolongation of time, and the inhibitory effect lasted at least until the seventh day after intervention. 4. NF-魏 Bp65 specific adenoviral infection HUVEC, could significantly inhibit the transcription of NF- 魏 Bp65 nucleoprotein stimulated by fluctuating high glucose, making the expression of p65 protein in the nucleus at the basic level. 5.AlamarBlue results showed that HUVEC infection with HUVEC could decrease the cell proliferation inhibition induced by fluctuating high glucose (P01), but lower the cell proliferation rate (P01) than that at normal glucose concentration (5.5Bp65). 6. NF-魏 Bp65 specific adenovirus could up-regulate the expression of HUVEC bcl-2 under fluctuating high glucose, which might reduce apoptosis. Conclusion: the construction of NF- 魏 B p65 specific RNAi adenoviral expression vector can effectively inhibit the expression of HUVEC p65 gene, and adenoviral infection with HUVEC, can significantly inhibit the nuclear transcription of NF- 魏 B p65 stimulated by volatile high glucose, thus protecting the proliferation inhibition of HUVEC under volatile high glucose, and may reduce the apoptosis of HUVEC induced by volatile high glucose.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363
【共引文献】
中国硕士学位论文全文数据库 前2条
1 乔玉芳;NF-κB特异的RNAi腺病毒载体的构建及其对血管内皮细胞增殖凋亡的影响[D];福建医科大学;2007年
2 孔永;腺病毒介导CTLA4Ig和CD40Ig基因转染对人皮肤成纤维细胞免疫性能影响的体外研究[D];西南大学;2008年
，本文编号：2507167
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2507167.html
最近更新
教材专著
