Skuk Daniel, Caron Nicolas J, Goulet Marlyne, Roy Brigitte, Tremblay Jacques P
Unité de recherche en Génétique humaine, Centre de Recherche du Centre Hospitalier de l'Université Laval, CHUL du CHUQ, Ste-Foy, Québec, Canada.
J Neuropathol Exp Neurol. 2003 Sep;62(9):951-67. doi: 10.1093/jnen/62.9.951.
We conducted a study in mice to reevaluate and clarify many aspects of the early survival of muscle cells following transplantation. Male mouse muscle cells (primary-cultures and T-antigen-immortalized clones) labeled with [14C]thymidine and beta-galactosidase were injected into female muscles. Each label was detected in the muscles after different time periods. TUNEL, alizarin red, and immunodetection of active caspase-3 were done in muscle sections. The donor cell labels disappeared from the muscles following donor cell death, but this was not instantaneous and even if the donor cells were killed before transplantation, the first 6 hours were not enough to clear [14C]thymidine and Y chromosome. Using the cell pellet before injection as the 100% baseline for cells injected to evaluate cell death can lead to misinterpretations: the Y-chromosome band was 5-fold stronger than that of a muscle injected with cells, irrespective of whether the cells were previously killed or not. There was no evidence of an immediate massive donor cell death. Necrosis (detected by alizarin red) and apoptosis (detected by active caspase-3) were present among the donor myoblasts following transplantation. Necrosis seemed to be the most important mechanism during the first hours. T-antigen immortalized cells died earlier and more massively than primary-cultured cells, but the surviving cells proliferated more. Indeed, they seemed to exhibit more apoptosis and they triggered a more rapid CD8+ cell infiltration. As a result of our findings, many concepts concerning the early donor cell death following myoblast transplantation must be reconsidered.
我们在小鼠中进行了一项研究，以重新评估并阐明移植后肌肉细胞早期存活的诸多方面。将用[14C]胸腺嘧啶核苷和β-半乳糖苷酶标记的雄性小鼠肌肉细胞（原代培养物和T抗原永生化克隆）注射到雌性肌肉中。在不同时间段后在肌肉中检测每种标记物。对肌肉切片进行TUNEL、茜素红染色以及活性半胱天冬酶-3的免疫检测。供体细胞死亡后，供体细胞标记物从肌肉中消失，但这并非瞬间发生，而且即使供体细胞在移植前被杀死，最初的6小时也不足以清除[14C]胸腺嘧啶核苷和Y染色体。以注射前的细胞沉淀作为注射细胞的100%基线来评估细胞死亡可能会导致误解：无论细胞之前是否被杀死，Y染色体条带都比注射细胞的肌肉中的条带强5倍。没有证据表明供体细胞会立即大量死亡。移植后供体成肌细胞中存在坏死（通过茜素红检测）和凋亡（通过活性半胱天冬酶-3检测）。在最初的几个小时内，坏死似乎是最重要的机制。T抗原永生化细胞比原代培养细胞更早且更大量地死亡，但存活的细胞增殖更多。实际上，它们似乎表现出更多的凋亡，并且引发了更快的CD8+细胞浸润。基于我们的研究结果，许多关于成肌细胞移植后早期供体细胞死亡的概念必须重新考虑。
