激光扫描共聚焦显微术在人外周血淋巴细胞线粒体膜电位检测中的应用

Application of laser scanning confocal microscopy to detect mitochondrial membrane potential of human peripheral blood lymphocytes

目的:利用激光扫描共聚焦显微镜检测荧光探针在淋巴细胞中的不同分布形式,研究淋巴细胞在凋亡过程中细胞核形态和线粒体形态的变化。方法:用淋巴细胞分离液体外分离健康成年人外周血淋巴细胞,以5μmol/L地塞米松分别刺激淋巴细胞4h和24h,并设正常对照组。JC-1染色细胞线粒体膜,Hoechst33258染色细胞核,利用激光扫描共聚焦显微镜观察细胞核及线粒体膜的形态学改变。结果:正常人外周血淋巴细胞胞核完整,细胞核呈淡紫色,线粒体呈红色短棒状。地塞米松可诱导淋巴细胞发生凋亡,在凋亡早期(作用时间4h)细胞核完整,呈淡紫色,部分线粒体崩解呈绿色弥散状。在凋亡末期(作用时间24h)细胞核碎裂,呈亮紫色,线粒体完全溶解,呈绿色弥散状。结论:淋巴细胞在地塞米松的作用下发生凋亡,凋亡的淋巴细胞细胞核碎裂、线粒体溶解,线粒体膜电位下降。激光扫描共聚焦显微镜检测淋巴细胞凋亡,观察效果直观,方便实用。

Objective：To look for different styles of fluorescence probe in lymphocytes by confocal microscope, and to study the changes in morphology of the nucleus and mitochondria, as well as mitochondrial membrane potential. Methods：Peripheral blood lymphocytes of healthy adults were separated by Ficoll in vitro and stimulated by 5 μmol/L dexamethasone for 4 and 24 hours respectively. Mitochondrial membrane was stained with JC-1 and nucleus with Hoechst 33258, morphological changes in nucleus and mitochondria were observed under laser scanning confocal microscope. Results： Normal lymphocyte nuclei were stained light violet, and mitochondria in normal human peripheral blood lymphocytes were red and rod-shaped. Dexamethasone caused apoptosis of the lymphocytes. At early stage of apoptosis （4 hours）, some mitochondria were found to be decomposed and appeared green in color, but the nucleus maintained an ordinary state. The nucleus was seen to be fragmentated, and all the mitochondria disrupted into dispersed green particles at the late stage of apoptosis （24 hours）. Conclusion： Dexamethasone provokes apoptosis of the lymphoeytes. In apoptotic lymphocytes, nucleus is broken to pieces and mitochondria decomposed, with depression of mitochondrial membrane potential. It is convenient and efficient to use laser scanning confocal microscopy to investigate apoptosis.