Matsushita Takakazu, Yagi Toshikazu, Hardin Joseph A, Cragun Jennifer D, Crow Frank W, Bergen H Robert, Gores Gregory J, Nyberg Scott L
Division of Transplantation Surgery, Mayo Clinic, Rochester, MN 55905, USA.
Cell Transplant. 2003;12(2):109-21. doi: 10.3727/000000003108746696.
We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.
我们之前已经表明，冷冻保存会导致用于生物人工肝（BAL）的猪肝细胞凋亡死亡增加。本研究旨在确定一种广谱半胱天冬酶抑制剂IDN - 1965是否能减少冷冻保存的猪肝细胞在静态培养或生物人工肝中的凋亡并增强其功能。使用补充有25微摩尔/升IDN - 1965或赋形剂的培养基，对分离后以及在液氮中冷冻保存2周后的猪肝细胞进行研究。在新鲜和冷冻保存的肝细胞培养物中均观察到凋亡和坏死细胞，但冷冻保存后凋亡细胞的百分比增加。通过TUNEL检测确定，在IDN - 1965中进行冷冻保存可提高肝细胞活力并减少凋亡细胞死亡。在IDN - 1965中冷冻保存肝细胞还与半胱天冬酶3样活性降低、线粒体细胞色素c释放减少以及解冻后线粒体膜电位下降减缓有关。当在培养和冷冻保存培养基中均添加IDN - 1965时，这些凋亡标志物在冷冻保存后最低。在补充有25微摩尔/升IDN - 1965的培养基中对肝细胞进行冷冻保存和培养后，肝细胞活性的功能标志物（白蛋白产生、地西泮代谢、尿素产生）也增加。在半胱天冬酶抑制剂IDN - 1965存在下冷冻保存猪肝细胞与减少凋亡以及改善猪肝细胞在静态培养和灌注生物人工肝中的功能有关。这些数据表明，抑制凋亡也能保留细胞功能。
