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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。
Background: The molecular mechanism of combining biological and optical techniques to study cell proliferation and apoptosis has become a hot topic in the field of bioengineering. OBJECTIVE: To observe the molecular mechanism of HCC-induced apoptosis in live cells by confocal fluorescence microscopy and fluorescence resonance energy transfer (FTIR). Design: observe the contrast experiment. Unit: South China Normal University Key Laboratory of Laser Life Science and Laser Life Science Institute. MATERIALS: Experiments were performed at the Laser Life Science Laboratory and Laser Life Science Institute of South China Normal University from October 2006 to March 2007. Human lung adenocarcinoma cells are cultured in our laboratory. Xiao-ping injection Tonghua God source Pharmaceutical Co., Ltd. production (Zhunzi Z20025869), G418 purchased from China and the United States biological companies. The plasmid SCAT3 was provided by Professor Miura, an infinite M200, Tecan, Austria, and a mitochondria-targeting fluorescent probe was purchased from Molecular Probe. Methods: ① The cell counting kit was used to detect the inhibitory effect of different dosage of Xiaochangping on the activity of human lung adenocarcinoma cells. ② Real-time detection of dynamic changes of caspase-3 induced caspase-3 activation in most of the living cells by confocal fluorescence microscopy and fluorescence resonance energy transfer. (3) Fluorescence detection technology was used to detect the fluorescence emission spectra of SCAT3 at different time points after the action of carboplatin in living cells. MAIN OUTCOME MEASURES: ①Detection of cell activity after eliminating cancer cells. (2) Real-time detection of the dynamic changes of SCAT3 fluorescence resonance energy transfer efficiency after anticancer effect. (3) The dynamic changes of mitochondrial morphology after the action of Carcinolide. Results: ① Xiaowanping dose-dependent inhibition of tumor cell activity. ② Xiaochangping induced intracellular caspase 3 activation. ③ eliminate cancer-induced mitochondrial rounding and rupture cells. Conclusion: Xiaochangping can induce the apoptosis of human lung adenocarcinoma cells, and caspase 3 is involved in its regulation.