Deming Paula, Kornbluth Sally
Department of Pharmacology and Cancer Biology, Duke University, Durham, NC, USA.
Methods Mol Biol. 2006;322:379-93. doi: 10.1007/978-1-59745-000-3_27.
It was first shown by Newmeyer and colleagues in the 1990s that the molecular events of apoptosis could be reconstituted in vitro using Xenopus egg extracts. When the egg extract is allowed to incubate at room temperature for an extended time, the biochemical events of apoptosis are activated spontaneously. The features of apoptosis in the Xenopus reconstitution system mimic those that occur in mammalian cells: Cytochrome c is released from the mitochondria, caspases are activated, cellular substrates are cleaved, and added nuclei are fragmented. Moreover, these apoptotic events can be inhibited by addition of antiapoptotic proteins such as Bcl-2 and conventional caspase inhibitors (e.g., ZVAD-fmk). The mitochondria, which are triggered to release cytochrome c by as-yet-unknown signals in the extract, are essential for induction of the spontaneous apoptotic program in these extracts. However, one can study the apoptotic events that occur downstream of mitochondrial cytochrome c release by preparing extracts devoid of membrane components (including mitochondria). When purified cytochrome c is added to such cytosolic extracts, biochemical markers of apoptosis (e.g., activation of caspases) can be monitored. The egg extract therefore offers a tractable system for studying either separately or together the events of apoptosis occurring upstream and downstream of the mitochondria.
20世纪90年代，纽迈耶及其同事首次证明，利用非洲爪蟾卵提取物可在体外重建细胞凋亡的分子事件。当卵提取物在室温下长时间孵育时，细胞凋亡的生化事件会自发激活。非洲爪蟾重建系统中细胞凋亡的特征与哺乳动物细胞中发生的特征相似：细胞色素c从线粒体释放，半胱天冬酶被激活，细胞底物被切割，添加的细胞核碎片化。此外，添加抗凋亡蛋白如Bcl-2和传统的半胱天冬酶抑制剂（如ZVAD-fmk）可抑制这些凋亡事件。提取物中尚不清楚的信号触发线粒体释放细胞色素c，线粒体对于诱导这些提取物中的自发凋亡程序至关重要。然而，通过制备不含膜成分（包括线粒体）的提取物，可以研究线粒体细胞色素c释放下游发生的凋亡事件。当将纯化的细胞色素c添加到这种胞质提取物中时，可以监测凋亡的生化标志物（如半胱天冬酶的激活）。因此，卵提取物为分别或共同研究线粒体上游和下游发生的凋亡事件提供了一个易于处理的系统。
