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荧光共振能量转移技术对肺腺癌活细胞中消癌平诱导半胱氨酸天冬氨酸酶3活化过程的实时监测

ISSN号：2095-4344
期刊名称：《中国组织工程研究》
时间：0
分类：R318[医药卫生—生物医学工程;医药卫生—基础医学]
作者机构：[1]华南师范大学激光生命科学教育部重点实验室暨激光生命科学研究所,广东省广州市510631
相关基金：中国自然科学基金资助项目（30670507,60378043,30470494）

关键词：荧光共聚焦显微成像, 荧光共振能量转移, 消癌平, 人类肺腺癌细胞, 凋亡, 生物医学工程

中文摘要：

背景：将生物学技术和光学技术结合起来研究细胞增殖和凋亡的分子机制已成为生物工程学领域的研究热点。目的：应用共聚焦荧光显微成像技术和荧光共振能量转移技术在活细胞中观察消癌平诱导肿瘤细胞凋亡的分子机制。设计：观察对比实验。单位：华南师范大学激光生命科学教育部重点实验室暨激光生命科学研究所。材料：实验于2006-10/2007-03在华南师范大学激光生命科学重点实验室暨激光生命科学研究所进行。人类肺腺癌细胞为本实验室培养。消癌平注射液为通化神源药业有限公司生产（国药准字Z20025869）,G418购自华美生物公司。质粒SCAT3由Miura教授提供,酶标仪（infinite M200,Tecan,Austria）,线粒体定位荧光探针购于Molecular Probe公司。方法：①利用细胞计数试剂盒技术检测不同剂量的消癌平对人类肺腺癌细胞活性的抑制作用。②利用共聚焦荧光显微成像技术和荧光共振能量转移技术在大半个活细胞中实时检测消癌平诱导半胱氨酸天冬氨酸酶3活化的动态过程。③利用荧光检测技术在活细胞中检测消癌平作用后不同时间点的SCAT3的荧光发射谱。主要观察指标：①消癌平作用细胞后检测细胞的活性。②消癌平作用后实时检测SCAT3荧光共振能量转移效率的动态变化。③消癌平作用后线粒体形态的动态变化。结果：①消癌平剂量依赖性抑制肿瘤细胞的活性。②消癌平诱导细胞内半胱氨酸天冬氨酸酶3的活化。③消癌平诱导细胞中线粒体变圆和破裂。结论：消癌平可以诱导人类肺腺癌细胞的凋亡,半胱氨酸天冬氨酸酶3参与了其调控过程。

英文摘要：

BACKGROUND：Combination of biological and optical technique is used to study the molecular mechanism of cel proliferation and apoptosis, which has become the study hotspot in the filed of bioengineering. OBJECTIVE： The goal of this study was to study the molecular mechanism of YAP-induced apoptosis in single living human lung adenocarcinoma （ASTC-a-1） cells by using confocal fluorescence microscopy imaging and fluorescence resonance energy transfer （FRET） techniques. DESIGN ： A controlled observation SETTIGN： Institute of Laser Life Science （Key Laboratory of Education Department）, South China Normal University. MARERIALS： This experiment was carried out in the Institute of Laser Life Science （Key Laboratory of Education Ministry）, South China Normal University between October 2006 and March 2007. Human lung adenocarcinoma （ASTC-a-1） cells were cultured in our laboratory. Xiaoaiping （YAP） parenteral solution was purchased from Tonghua Shenyuan Pharmaceutical Co.,Ltd （No. Z20025869）, and G418 was purchased from Huamei Biological Company. SCAT3 was provided by Professor Masayuki Miura. Auto microplate reader （infinite M200, Tecan, Austria）and mitochondrial location fluorescent probe （Mitertracker Red）was purchased from Molecular Probe Company. METHODS ： ① The inhibition of YAP at different doses to ASTC-a-1 cell viability was detected by CCK-8. ② Dynamical change of caspase 3-induced by YAP was detected by confocal fluorescence microscopy imaging and fluorescence resonance energy transfer （FRET） techniques. ③The fluorescence emission spectrum of SCAT3 was detected by confocal fluorescence microscopy imaging at different time points after YAP treatment. MAIN OUTCOME MEASURES： ①Cell viability after YAP treatment.② Dynamic change of fluorescence resonance energy transfer efficiency of SCAT3 detected after YAP treatment. ③Dynamical change of mitochondrial morphology after YAP treatment. RESULTS： ①XAP inhibited the viability of tumor cell

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