【作者】 余娴；

【导师】 廖玉华；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 第一部分缺氧通过HIF-1α诱导心肌细胞分泌细胞因子TNF-α目的：越来越多证据显示急性心肌梗死中过量肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)表达对心肌细胞不利。心肌缺血时,TNF-α主要来源于巨噬细胞。随着缺血状态的持续,心肌细胞开始分泌TNF-α.我们探讨缺氧是否通过缺氧诱导因子-1α(hypoxia inducible factor 1α, HIF-1α)诱导心肌细胞自分泌细胞因子TNF-α。方法和结果：分离新生大鼠乳鼠心肌细胞,分别给予氧浓度1%缺氧刺激5分钟,15分钟,30分钟,1小时,2小时,4小时,6小时,12小时,24小时。western blot检测细胞HIF-1α蛋白表达水平；免疫荧光检测HIF-1α蛋白表达以及入核情况；实时定量RT-PCR检测TNF-a mRNA表达水平；ELISA检测TNF-α蛋白表达分泌。结果显示TNF-αmRNA从1小时开始表达上调,与空白组相比增加至1.99±0.27倍(p<0.01),12小时及24小时达峰值,分别为4.06±0.84倍和4.09±0.81倍(p<0.01)。TNF-α蛋白分泌表达在6小时可检测到,12小时达峰值为48.12±11.52pg/ml(p＜0.01)。缺氧对HIF-1αmRNA水平无影响,HIF-1α蛋白缺氧30分钟表达明显,1小时达峰值。免疫荧光染色提示缺氧15分钟HIF-1α蛋白开始表达且向核内聚积。核转染法进行心肌细胞HIF-1αRNA干扰,发现siRNA显著抑制HIF-1αmRNA水平的同时,抑制缺氧12小时后TNF-αmRNA水平上调(3.75±0.45 fold vs 1.38±0.76 fold,p<0.01)。HIF-1α抑制剂2-甲氧雌二醇(2-methoxyestradiol,2ME2)能有效抑制缺氧刺激后HIF-1α蛋白表达,也能抑制缺氧12小时后TNF-αmRNA水平上调(2.82±0.41 fold vs 1.31±0.37fold,p<0.01)以及蛋白分泌(48.12±11.52pg/ml vs 23.22±2.62,p<0.05)。结论：缺氧能诱导离体心肌细胞分泌细胞因子TNF-α, HIF-1α在缺氧诱导心肌细胞自分泌TNF-α过程中发挥重要调节作用。第二部分缺氧及模拟缺氧状态下HIF-1α对TNF-α直接启动作用的研究目的：探讨缺氧诱导因子-1α(hypoxia inducible factor 1α, HIF-1α)对于肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)是否有直接启动作用。方法和结果：培养HEK293细胞和HepG2细胞,给予氧浓度1%缺氧刺激及氯化钻模拟缺氧刺激后,western blot检测胞核HIF-1α蛋白表达水平,实时定量RT-PCR检测TNF-αmRNA表达水平。结果显示缺氧6小时及氯化钻刺激3小时可明显观察到HIF-1α蛋白在胞核内稳定表达,缺氧12h及氯化钴刺激12h后与空白组相比TNF-αmRNA水平明显上升。HIF-1α抑制剂2-甲氧雌二醇(2-methoxyestradiol,2ME2)能有效抑制氯化钻模拟缺氧刺激后HIF-1α在核内聚积,也能抑制氯化钴模拟缺氧刺激后TNF-αmRNA水平上调。分析TNF-α转录起始位点上游5000bp的启动子区域,发现七个可能缺氧反应元件(hypoxia response elements, HREs)。5’端续减法构建8个不同长度的报告基因质粒,瞬时转染入HEK293细胞和HepG2细胞,予氧浓度1%缺氧刺激24小时后双荧光素酶报告基因法检测启动子活性。结果显示TNF-α转录起始位点上游-1470bp处可能为缺氧诱导TNF-α启动子的功能位点。结论：我们发现TNF-α是一种缺氧诱导表达基因,它的转录调节可能通过HIF-1与位于TNF-α启动子-1470bp处HRE位点相互作用发挥效应。更多还原

【Abstract】 PartⅠHypoxia induced autocrine of TNF-a by hypoxia-inducible factor-la in cardiomyocytesAims:Growing evidence indicates that excessive tumor necrosis factor-a (TNF-a) expression are detrimental to cardiomyocytes in acute myocardial infarction. During myocardial ischemia, TNF-a is mainly released from macrophages, with persisent ischemia, TNF-a can originate from cardiomyocytes. We hypothesized the direct effects of hypoxia on TNF-a expression by hypoxia-inducible factor-la (HIF-la) in cardiomyocytes.Methods and results:Neonatal rat cardiomyocytes were exposed in a hypoxic incubator containing 1% oxygen for 5 minutes,15 minutes,30 minutes,1 hour,2 hours,4 hours,6 hours,12 hours and 24 hours. TNF-a mRNA increased slightly as early as 30 minutes (1.99±0.27 fold compared to control group,p<0.01), peaked at 12 hours (4.06±0.84 fold,p＜0.01) and 24 hours (4.09±0.81 fold,p＜0.01). TNF-a secretion became detectable as early as 6 hours and peaked at 12 hours (48.12±11.52 pg/ml) by ELISA. Hypoxia had no effect on HIF-la mRNA change (p>0.05) by real-time RT-PCR assay. Protein expression of HIF-1αwas detectable at 30 minutes and peaked at 1 hour by western blot. Immunofluorescent staining showed HIF-1αwas obviously observed as early as 15 minutes, meanwhile, it had the trends to translocate into cell nucleus. HIF-1αmRNA was downregulated effectively by siRNA through Nucleofection, meanwhile, TNF-αmRNA elevation after 12-hour hypoxia was obviously inhibited (3.75±0.45 fold vs 1.38±0.76 fold, p<0.01). Expression of HIF-la was inhibited effectively by 2-Methoxyestradiol (2ME2) after hypoxia, and 2ME2 also decreased TNF-αmRNA after 12-hour hypoxia (2.82±0.41 fold vs 1.31±0.37 fold,p＜0.01) and TNF-a secretion (48.12±11.52pg/ml vs 23.22±2.62, p＜0.05).Conclusions:Hypoxia induces autocrine of TNF-αby cardiomyocytes in vitro. HIF-1αplays an important role in hypoxia mediated TNF-αexpression and secretion in cardiomyocytes. PartⅡRegulation of TNF-a promoter by hypoxia inducible factor-la under hypoxia or CoCl2 mimicked hypoxiaAims:We investigated the direct effects of hypoxia on tumor necrosis factor-a (TNF-α) expression, and the role of hypoxia inducible factor-la (HIF-la) in TNF-a regulation.Methods and results:HEK293 and HepG2 cells were exposed in a hypoxia (1% O2) chamber or treated with CoCl2 to mimic hypoxia. HIF-la nuclear protein expression was detected by western blot, TNF-a mRNA levels were assayed by real time PCR. Significant up-regulation of HIF-la nuclear protein could be seen in both cell lines after 6-hour hypoxia and 3-hour CoCl2 mimic hypoxia stimulation. Obvious up-regulation of TNF-αmRNA levels was observed after 12-hour hypoxia and 12-hour CoCl2 mimic hypoxia. Inhibition of HIF-1αby 2-methoxyestradiol (2ME2) inhibited CoCl2 induced HIF-1αnuclear protein accumulation and TNF-a mRNA elevation. We found seven possible hypoxia response elements (HREs) at regions of 5’to the proximal 5000-bp fragment of the TNF-αpromoter relative to the transcription start site. Eight reporter gene plasmids were constructed after promoter truncation analysis, and then transiently transfected into HEK293 and HepG2 cells. Promoter activity was detected by dual-luciferase reporter assay after 24-hour hypoixa treatment. The results showed HIF consensus binding sites spanning bp-1470 relative to the transcription start site were functional for TNF-a promoter activity induction by hypoxia.Conclusions:We demonstrate that TNF-a is a hypoxia-inducible gene, whose transcription is stimulated through HIF-1αinteraction with HRE sites located at-1470 of the TNF-a promoter.更多还原