MONITORING OF INTRACELLULAR OXYGEN -CONCENTRATION DURING HYPOXIA INDUCIBLE FACTOR ACTIVATION USING OXYGEN PHOSPHORECSENCE QUENCHING

哺乳动物细胞的生存严重依赖氧气。细胞感知和响应氧气供应减少的分子机制尚不清楚，尽管一些作者已经报道了信号转导途径的发现。一个典型的例子是，缺氧刺激了一些对缺氧状态下生存至关重要的基因的转录，以及细胞凋亡。缺氧对某些基因转录的增强主要是由缺氧诱导因子-1 （hif-1）介导的。然而，目前尚不清楚该转录因子是通过缺氧诱导的能量代谢变化激活的，还是通过氧传感器直接激活的。为了回答这个问题，我们试图将基因表达与细胞内氧合状态联系起来。摘要绿色荧光蛋白（GFP）是一种功能强大的荧光团，由65-67位ser - tyr1 - gly序列的内部环化和氧化而形成。继克隆gfp cdna之后，一些研究者对gfp和gfp融合蛋白的表达产生了极大的兴趣，这些蛋白作为一种独特的荧光团来监测一系列细胞内过程，特别是蛋白质的定位。Hif - 1是一种转录调节蛋白，在缺氧时从细胞质转移到细胞核。我们观察了模拟缺氧的钴处理下标记hif-1的GFP的核转运。磷光的氧依赖性猝灭是磷光体与分子氧碰撞频率和碰撞过程中能量传递效率的函数。研制了一种共聚焦显微镜下卟啉钯磷光寿命检测系统。利用先前报道的磷光衰减常数，估计培养基中氧浓度在常氧条件下为194微摩尔，在缺氧条件下为29微摩尔。我们计划测定细胞质溶胶和培养基之间的氧梯度。少

MAMMALIAN CELLS ARE CRITICALLY DEPENDENT ON OXYGEN FOR SURVIVAL. THE MOLECULAR MECHANISMS BY WHICH CELLS SENSE AND RESPOND TO A REDUCTION IN OXYGEN SUPPLY ARE NOT KNOWN ALTHOUGH FINDINGS ON THE SIGNAL TRANSDUCTION PATHWAY HAVE BEEN REPORTED BY SEVERAL AUTHORS. A TYPICAL EXAMPLE IS THAT HYPOXIA STIMULATES TRANSCRIPTION OF A NUMBER OF GENES CRUCIAL TO SURVIVAL IN THE HYPOXIC STATE, AS WELL AS APOPTOSIS. THE ENHANCEMENT OF TRANSCRIPTION OF SOME GENES BY HYPOXIA IS MAINLY MEDIATED BY HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1). HOWEVER, IT IS NOT CLEAR WHETHER THIS TRANSCRIPTION FACTOR ARE ACTIVATED BY HYPOXIA-INDUCED CHANGES IN ENERGY METABOLISM OR BY OXYGEN SHORTAGE DIRECTLY THROUGH AN OXYGEN SENSOR. TO ANSWER THIS, WE ATTEMPTED TO CORRELATE THE GENE-EXPRESSION AND INTRACELLULAR OXYGENATION STATE.GREEN FLUORESCENT PROTEIN (GFP) FROM AEQUOREA VICTORIA IS A POWERFUL FLUOROPHORE FORMED AUTOCATALYTICALLY BY INTERNAL CYCLIZATION AND OXIDATION OF THE SER-TYR-GLY SEQUENCE AT POSITIONS 65-67 WITHIN THE 2 … More 38 AMINO ACID. FOLLOWING THE CLONING OF THE CDNA FOR GFP, SEVERAL INVESTIGATORS TOOK A GREAT INTEREST IN THE EXPRESSION OF GFP AND GFP-FUSION PROTEINS AS A UNIQUE FLUOROPHORE TO MONITOR A RANGE OF INTRACELLULAR PROCESSES, ESPECIALLY THE LOCALIZATION OF PROTEINS. HIF- 1 IS THE TRANSCRIPTION REGULATORY PROTEIN AND TRANSLOCATE FROM THE CYTOPLASM TO THE NUCLEUS DURING HYPOXIA. WE OBSERVED THE NUCLEAR TRANSLAOCATION OF GFP LABELED HIF-1 BY COBALT TREATMENT WHICH MIMICS HYPOXIA.OXYGEN-DEPENDENT QUENCHING OF PHOSPHORESCENCE IS A FUNCTION OF THE FREQUENCY OF COLLISION BETWEEN THE PHOSPHOR AND MOLECULAR OXYGEN AND OF THE EFFICIENCY OF ENERGY TRANSFER DURING THESE COLLISMNS. WE DEVELOPED A DETECTION SYSYTE FOR PALLADIUM PORPHYRIN PHOSPHORESCENCE LIFETIME USING CONFOCAL MICROSCOPE. USING PHOSPHORESCENCE DECAY CONSTANT PREVIOUS REPORTED, THE OXYGEN CONCENTRATIONS IN THE MEDIUM WERE ESTIMATED AS 194 MICROMOLAR UNDER NORMOXIA AND 29 MICROMOLAR UNDER ANOXIA, RESPECTIVELY. WE PLAN TO DETERMINE OXYGEN GRADIENT BETWEEN CYTOSOL AND MEDIUM. Less