An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins.
An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins.
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DOI：
10.1679/aohc.61.395
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发表时间：
1998-12
影响因子：
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通讯作者：
Y. Ohsawa;K. Isahara;S. Kanamori;M. Shibata;S. Kametaka;T. Gotow;Tsuyoshi Watanabe;E. Kominami;Y. Uchiyama
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文献类型：
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作者：
Y. Ohsawa;K. Isahara;S. Kanamori;M. Shibata;S. Kametaka;T. Gotow;Tsuyoshi Watanabe;E. Kominami;Y. Uchiyama
除了半胱天冬酶家族的蛋白酶，组织蛋白酶D，一种溶酶体天冬氨酸蛋白酶，已被认为是哺乳动物细胞中的促凋亡介质。为了进一步了解组织蛋白酶B和D在细胞凋亡中的作用，我们检查了血清剥夺培养的PC 12细胞中这些酶的超微结构和免疫反应性的精确变化过程。激光扫描显微镜显示，免疫反应组织蛋白酶B和D精细分布在细胞质中的PC 12细胞在血清剥夺下培养的开始。在培养开始后3 h，细胞中组织蛋白酶B的免疫反应性略有下降，而细胞中组织蛋白酶D的免疫沉积物变得更强，尺寸更大。TUNEL阳性染色在细胞核中出现在6小时，但数量较少。与TUNEL阳性细胞数量的增加相对应的是，在12 h和24 h，组织蛋白酶B的免疫反应性在细胞中急剧减少，而组织蛋白酶D的免疫反应性显著增强，尤其是在TUNEL阳性细胞中。电镜下，自噬空泡/autolysosomes出现在细胞的细胞质后3小时开始培养。6 h时，细胞核周边部分出现明显的染色质相对浓缩的变化。细胞核染色质浓缩的PC 12细胞数量在24 h时明显增加，而细胞质和细胞核均出现皱缩。细胞内可见致密体和自噬泡，并有界膜。这些结果显示细胞凋亡过程中自噬的发生和组织蛋白酶B和D之间蛋白量的不平衡可能为我们的假设提供论据，即这些酶部分参与了血清剥夺后PC 12细胞的细胞死亡级联反应。
In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic proteinase, has been suggested to act as a proapoptotic mediator in mammalian cells. To further understand the roles of cathepsins B and D in apoptosis of the cells, we examined the precise alteration processes of ultrastructures and immunoreactivity for these enzymes in PC12 cells cultured under serum deprivation. Laser scanning microscopy showed immunoreactivity for cathepsins B and D to be finely distributed in the cytoplasm of PC12 cells at the onset of culture under serum deprivation. At 3 h after the onset of culture, the immunoreactivity for cathepsin B slightly decreased in the cells, while immunodeposits for cathepsin D in the cells became more intense and larger in size than those at 0 h. Positive staining for TUNEL in nuclei of the cells appeared at 6 h, though fewer in number. Corresponding to the increase in the number of TUNEL-positive cells at 12 h and 24 h, the immunoreactivity for cathepsin B was drastically diminished in the cells, whereas that for cathepsin D was significantly augmented, especially in TUNEL-positive cells. Electron microscopically, autophagic vacuoles/autolysosomes appeared in the cytoplasm of the cells 3 h after the onset of culture. A distinct nuclear change showing relatively condensed chromatin first appeared in the peripheral part of the nuclei at 6 h. The number of PC12 cells having nuclei with chromatin condensation increased especially at 24 h, while these cells showed shrinkage of both their cytoplasm and nuclei. Dense bodies and autophagic vacuoles with limiting membranes were seen in these cells. These results showing the occurrence of autophagy and imbalance of protein amounts between cathepsins B and D during apoptosis may argue for our hypothesis that these enzymes are, in part, involved in the cell death cascade for PC12 cells following serum deprivation.