Tissue Culture and Molecular Biology

大量流行病学研究表明，适量饮酒和红酒可降低罹患糖尿病的风险 冠心病和与心肌梗死相关的死亡率。我们的初步研究已经 证明酒精饮料的心脏保护作用来自酒精和非酒精 成分(红酒多酚)。然而，酒精和多酚的确切分子机制 对心脏的保护作用尚未完全阐明。本计划项目的总体纲领主题 GRANT的应用是进一步确定/定义支撑这种低乙醇的分子调控机制-或 多酚诱导的心脏保护作用。组织培养和质粒制备的总体目标 B)是为了更好地确保高质量的研究、一致性、促进最佳运作和协作 个人计划项目资助研究项目与研究人员之间的互动。核心B将提供 计划项目调查人员培养的哺乳动物细胞类型和相应的个体质粒DNA 以高成本效益、可重复性和高效率的方式开展研究项目。这些核心单元支持功能 将减轻计划项目拨款调查员的后勤责任，劳动密集型 哺乳动物细胞类型的培养/维持及相关质粒DNA的扩增和纯化 每个项目都需要。核心单元B将常规提供培养的人脐静脉内皮细胞 人脐静脉内皮细胞、人主动脉内皮细胞、人冠状动脉内皮细胞和培养的小鼠 内皮细胞参与项目1(《乙醇和多酚诱导的内皮纤维蛋白溶解》，Boyse，1年- 5)、2(《乙醇和多酚对PAI-1基因表达的调节》，Griett，1-5年)和3； (“乙醇和多酚对心血管的保护”，Parks，1-5年)；人主动脉内皮细胞 (HAECs)投射3(1-5岁)和成年小鼠心肌细胞投射到投射4(“线粒体和 乙醇和多酚对心脏的保护“，Darley-Usmar，第1-5年)。核心B也将例行扩大 并提纯相关的质粒DNA，提供计划项目资助项目(项目1[1-5年]；项目2 [第1-5年]；项目3[第1-5年]和项目4[第1-2年])原位杂交(t-PA、u-PA、PAI-1、u-PAR、 ANN-II、肉桂醇酶eNOS、诱导型一氧化氮合酶、铜锌超氧化物歧化酶、锰超氧化物歧化酶)；实时聚合酶链式反应(PAS、PARS、PAL1、Vitronectin、eNOS、 INOS，MnSOD，CuZnSOD)；核连续分析(u-PAR，ANN II，_enolase，e-NOS，iNOS，MnSOD， 荧光素酶(t-PA、u-PA、PAI-1)；荧光素酶诱变 转录因子的构建(PAI-1)和表达载体。

Numerous epidemiological studies indicate that moderate alcohol and red wine consumption reduces the risk for coronary heart disease and the mortality associated with myocardial infarction. Our preliminary studies have demonstrated that the cardioprotective effects of alcoholic beverages result from both alcohol and non-alcoholic components (red wine polyphenols). However, the exact molecular mechanisms of how alcohol and polyphenols result in cardioprotection have not been fully elucidated. The overall programmatic theme of this Program Project Grant application is to further identify/define the molecular regulatory mechanisms that underlie this low ethanol- or polyphenol-induced cardioprotection. The overall goal of Tissue Culture and Plasmid Preparation Core Unit (Core B) is to better ensure high quality research, consistency, facilitate the optimal functioning and collaborative interaction between individual Program Project Grant research projects and investigators. Core B will provide Program Project investigators with cultured mammalian cell types and relevant plasmid DNA for their individual research projects in a highly cost effective, reproducible and efficient manner. These core unit support functions will alleviate Program Project Grant investigators of the logistic responsibilities, labor-intensive culture/maintenance of mammalian cell types and expansion and purification of the relevant plasmid DNA required by the each project. Core Unit B will routinely provide cultured human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), human coronary endothelial cells and cultured mouse endothelial cells to Projects 1 ("Ethanol and Polyphenolic Induced Endothelial Fibrinolysis", Booyse, years 1- 5), 2 ("Regulation of PAI-1 Gene Expression by Ethanol and Polyphenols", Grenett, years 1-5) and 3; ("Cardiovascular Protection by Ethanol and Polyphenols", Parks, years 1-5); human aortic endothelial cells (HAECs) to project 3 (years 1-5) and adult mouse heart muscle cells to Project 4 ("Mitochondria and Cardioprotection by Ethanol and Polyphenols", Darley-Usmar, years 1-5). Core B will also routinely expand and purify relevant plasmid DNA to provide Program Project Grant projects (Projects 1 [years 1-5]; Project 2 [years 1-5]; Project 3 [years 1-5] and Project 4 [years 1-2]) for in-situ hybridizations (t-PA, u-PA, PAI-1, u-PAR, Ann-II, ocenolase eNOS, iNOS, CuZnSOD, MnSOD); Real-Time PCR (PAs, PARs, PAL1, vitronectin, eNOS, iNOS, MnSOD, CuZnSOD); nuclear run-on assays (u-PAR, Ann II, _ enolase, e-NOS, iNOS, MnSOD, CuZnSOD); luciferase constructs for transient transfection assays (t-PA, u-PA, PAI-1); luciferase mutagenesis constructs (PAI-1) and expression vectors for transcription factors.