| Summary: | 碩士 === 高雄醫學大學 === 生物醫學暨環境生物學系碩士班 === 104 === Gaseous molecules, such as nitric oxide (NO) and hydrogen sulfide (H2S), are important signal transmitters in the endothelial cells (ECs). In the past era, researchers can only detect the key enzymes involved in the gaseous molecular biosynthesis to reflect the innate production of gaseous molecule. For detecting NO, griess reagent and biotin switch are the main method. However, due to the poor specificity, the reagent remains unsatisfied. Biotin switch estimates the levels of NO by detecting amount of proteins S-nitrosylation. With the defect of indirect and complicated procedure, this also unfeasible to the researcher. With the advance in fluorescent techniques, we develop gaseous molecular detecting platform. The gaseous NO can be detected by two new-designed probes, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid methyl ester (FA-OMe) and Rhodamine B hydrazide (RH). Cysteine/ homocysteine, the precursors of H2S that represents the levels of H2S can be observed by using 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) fluorescent probe. With gaseous molecular detecting platform, we identified three different gaseous molecular relative experiments. First, low concentration of arsenic can induce NO production in ECs. Secondly, the capability of Ni-SOD compound in preventing NO generation in the impaired rat brain was investigated. In the last result, the induction of endothelial H2S under mechanical shear flow was observed by NBD-SCN. In addition, we set up mitochondrial functional assessment platform; there have four items to evaluate the mitochondrial functions. The first one is the use of MitoSOX probe to measure mitochondrial ROS production. Secondly, SIRT3 activity, which is the key enzyme of mitophagy pathway, were detecting by SIRT3 activity kit. In the third part, we used the JC-1 probe to quantify the mitochondrial membrane potential. In the final part, the mitochondria were stained by MitoTracker and then the fusion/fission ratio of mitochondria were subsequently estimated by MicroP software. With this platform, we found that low concentration of camptothecin (CPT) can reduce mitochondrial ROS level, recover SIRT3 activity impaired and prevent mitochondria to become swollen under oxidative stress. But, CPT cannot significantly prevent membrane potential dysfunction from ROS stress. Establishing the platforms to detect cellular NO and H2S molecules, and also the mitochondrial function, the study of endothelial physiology would be more feasible than the past.
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