Calu-3细胞内琥珀酸脱氢酶活性及表达的双重染色法研究
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摘要
目的:利用Calu-3细胞探讨肿瘤细胞琥珀酸脱氢酶活性及表达双重染色技术。方法:体外培养Calu-3细胞,将细胞爬片分为三部分,第一部分采用氯化硝基四唑氮蓝法对细胞进行琥珀酸脱氢酶活性染色,并分别孵育40 min、1 h和2 h。第二部分进行该酶的免疫组织化学染色。第三部分进行琥珀酸脱氢酶的组织化学和免疫组织化学的双重染色。在光学显微镜下观察并比较琥珀酸脱氢酶活性及表达染色结果的差异。结果:(1)相比于孵育40 min和1h的细胞,孵育2 h的细胞则反应更加充分,蓝紫色双甲臜颗粒明显增多。(2)免疫组织化学染色的细胞内出现黄棕色DAB颗粒。(3)在活性和表达双重染色中,孵育1h时蓝紫色双甲臜颗粒和DAB黄棕色颗粒较其他孵育时间的更加明显。结论:在Calu-3细胞中,琥珀酸脱氢酶活性染色时孵育2h效果较好,而在活性和表达双重染色中,孵育1h时染色效果最好。
Objective To investigate the activity and expression of succinate dehydrogenase in Calu-3 tumor cells by a the double staining method. Method Calu-3 cells were plated on coverslips and divided into three groups. The sections in first group were incubated in Nitrotetrazolium blue chloride for 40 min,1 h and 2 h respectively to observe succinate dehydrogenase activity. Histochemical and immunohistochemical double staining were carried out in the third groups,and cells in the second group was Immunohistochemical stained only. Observation and comparison of the stained results were done in the optical microscope. Results(1)There were more abundant and stronger expression blue and purple formazan particles in cells of with Nitrotetrazolium blue chloridev incubation for 2 h,compared to 40 min and 1 h incubation.(2) Immunohistochemical staining of the cells appeared yellow brown particles.(3)In double staining for the activity and expression of succinate dehydrogenase,blue and purple formazan particles and yellow brown particles were more obvious in cells incubated for 1 h. Conclusion In Calu-3cells,the staining effect of succinate dehydrogenase activity was the best in cells with 2 h incubation,and that of double staining for activity and expression of succinate dehydrogenase was best in cells with 1hr incubation.
Objective To investigate the activity and expression of succinate dehydrogenase in Calu-3 tumor cells by a the double staining method. Method Calu-3 cells were plated on coverslips and divided into three groups. The sections in first group were incubated in Nitrotetrazolium blue chloride for 40 min,1 h and 2 h respectively to observe succinate dehydrogenase activity. Histochemical and immunohistochemical double staining were carried out in the third groups,and cells in the second group was Immunohistochemical stained only. Observation and comparison of the stained results were done in the optical microscope. Results(1)There were more abundant and stronger expression blue and purple formazan particles in cells of with Nitrotetrazolium blue chloridev incubation for 2 h,compared to 40 min and 1 h incubation.(2) Immunohistochemical staining of the cells appeared yellow brown particles.(3)In double staining for the activity and expression of succinate dehydrogenase,blue and purple formazan particles and yellow brown particles were more obvious in cells incubated for 1 h. Conclusion In Calu-3cells,the staining effect of succinate dehydrogenase activity was the best in cells with 2 h incubation,and that of double staining for activity and expression of succinate dehydrogenase was best in cells with 1hr incubation.
引文
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[5]Tan AS,Baty JW,Dong LF,et al.Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA[J].Cell Metab,2015,21(1):81.
[6]Wolf DA.Is reliance on mitochondrial respiration a"chink in the armor"of therapy-resistant cancer?[J].Cancer Cell,2014,26(6):788.
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[10]Gyedotun KS,Yau PF,Lemire BD.Identification of the heme axial ligands in the cytochrome b562 of the Saccharomyces cerevisiae succcinate dehydrogenase[J].J Biol Chem,2004,279(10):9432.
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