HIF-1α基因转染对β-淀粉样蛋白25-35诱导原代培养海马神经细胞凋亡的影响  

Effect of human hypoxia inducible factor-1α gene transfection on apoptosis of Aβ25-35 induced hippocampal neurons of primary culture

作　　者：刘凌云[1] 柴锡庆[2] 李清君[1] 程桂花[2] 

机构地区：[1]河北医科大学基础医学院病理生理学教研室,石家庄050017 [2]河北化工医药职业技术学院药学系,石家庄050017

出　　处：《第三军医大学学报》2007年第7期595-598,共4页Journal of Third Military Medical University

摘　　要：目的构建人类缺氧诱导因子-1α(human hypoxia inducible factor-1α,HIF-1α)基因真核表达载体pSNAV-HIF-1α,并观察其对β-淀粉样蛋白25-35(Aβ25-35)诱导原代培养海马神经细胞凋亡及细胞内游离钙离子浓度的影响。方法采用基因重组技术,将pBSKhHIF1αT7原核表达载体上HIF-1α基因序列克隆至真核表达载体pSNAV2.0,磷酸钙共沉淀法介导转染pSNAV-HIF-1α,Aβ25-35诱导原代培养海马神经细胞凋亡。Western blot检测HIF-1α蛋白表达,流式细胞术分析细胞凋亡百分率,激光共聚焦显微镜测定细胞内游离钙离子浓度。结果酶切、PCR及DNA测序结果均表明成功构建了重组质粒pSNAV-HIF-1α,磷酸钙共沉淀法介导的pSNAV-HIF-1α质粒转染显著增加了海马神经细胞HIF-1α蛋白表达(P<0.05),同时可显著降低Aβ25-35诱导海马神经细胞凋亡百分率及细胞内游离钙离子浓度上调(P<0.05)。结论缺氧诱导因子-1α基因转染可能通过抑制细胞内游离钙离子浓度上调使海马神经细胞对Aβ25-35产生抗凋亡作用。Objective To construct the eukaryotic expression vector for human hypoxia inducible factor-1α gene （pSNAV-HIF-1α）, and to investigate the apoptosis and intracellular calcium concentration of the transfected Aβ25-35 induced hippocampal neurons of primary culture. Methods Human hypoxia inducible factor-1α gene from pBSKhHIF1αT7 was inserted into pSNAV2.0 by the method of gene recombination, then the constructed vector was transfected into hippocampal neurons of primary culture and followed by Aβ25-35 for treatment. Empty pSNAV2.0 vector was treated the same way as pSNAV-HIF-1α as a control. The expression level of HIF-1α protein was assayed by Western blotting. Apoptosis was detected by flow cytometry. Intracellular calcium concentration was determined by laser scanning confocal microscopy with Fluo-3/AM as the fluorescent dye. Results It was shown that pSNAV-HIF-1α was successfully constructed by restriction enzyme digestion, PCR and DNA sequencing. The expression level of HIF-1α protein was significantly increased in transfected hippocampal neurons of primary culture （P 〈 0. 05 ）. The up-regulation of apoptosis and intracellular calcium concentration induced by Aβ25-35 were significantly decreased in transfected hippocampal neurons of primary culture （P 〈 0.05 ）. Conclusion Human hypoxia inducible factor-1α gene transfection could inhibit Aβ25-35 induced apoptosis of hippocampal neurons of primary culture by suppressing the up-regulation of intracellular calcium concentration.

关 键 词：缺氧诱导因子-1Α 原代培养 海马 转染 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]