ALLOGRAFT HEART VALVES--CELL VIABILITY ASSESSMENT BY CONFOCAL MICROSCOPY

用激光扫描共聚焦荧光显微镜检测细胞 同种异体心脏瓣膜的存活能力。石蜡切片 福尔马林固定的瓣膜组织同时染色 DNA(DAPI；蓝色荧光)、细胞凋亡(掺入 末端脱氧核苷酸对荧光素标记脱氧核苷酸的研究 转移酶；绿色荧光)和任一因子的免疫染色 (血管内皮细胞或增殖细胞核抗原)(德克萨斯州 红色标记抗体；红色荧光)。天然羊主动脉瓣 同种异体羊移植(n=4)(n=8)(植入胸主动脉 羊的带瓣管道)和人类同种异体瓣膜移植是因为 肺移植术后5年零9个月钙化 对闭锁进行评估。研究结果表明：1)有显著的 小叶细胞有丝分裂潜能在两天内降低 2)植入后内皮细胞进行性丢失 植入，以及3)植入两天后发生细胞凋亡 结缔组织细胞，并导致形成众多 小叶细胞凋亡小体和进行性减少。通过 植入20周后，植入同种异体瓣叶。 基本上是无细胞的，除了罕见的肌成纤维细胞和 中等数量的巨噬细胞(被认为是宿主来源)；内部没有 或细胞外有凋亡小体，未见增殖细胞核抗原阳性细胞 在这个时候观察到的。然而，此时尖端已经经历了 由于广泛形成的明显增厚和收缩 由宿主组织形成的纤维鞘。类似的发现也出现在 人类同种异体移植。因此，细胞凋亡是细胞丢失的主要原因。 移植的同种异体移植物的存活率。这些方法的进一步应用 这项研究中使用的方法应该会增加我们对这些变化的理解。

Laser scanning confocal fluorescence microscopy was used to assess cell viability in allograft heart valves. Paraffin sections of formalin-fixed valve tissues were stained for the simultaneous demonstration of DNA (DAPI; blue fluorescence), apoptosis (incorporation of fluorescein-labeled deoxynucleotides by terminal deoxynucleotidyl transferase; green fluorescence) and immunostaining for either Factor VIII for endothelium or proliferating cell nuclear antigen (PCNA) (Texas red-labeled antibodies; red fluorescence). Native ovine aortic valves (n=4), explanted ovine allografts (n=8) (implanted as thoracic aortic valved conduits in sheep) and a human allograft valve explanted because of calcification 5 years and 9 months after implantation for pulmonary atresia were evaluated. Results obtained indicated that: 1) a marked reduction in mitotic potential of leaflet cells occurs within two days of implantation; 2) endothelial cells are progressively lost after implantation, and 3) apoptosis occurs after two days of implantation in connective tissue cells, and leads to the formation of numerous apoptotic bodies and progressive decrease in leaflet cellularity. By 20 weeks after implantation, the allograft valve leaflets were essentially acellular with the exception of rare myofibroblasts and a moderate number of macrophages (thought to be of host origin); no intra or extracellular apoptotic bodies and no PCNA-positive cells were observed at this time. However, at this time the cusps had undergone marked thickening and contraction due to the formation of extensive fibrous sheathing by host tissue. Similar findings were present in the human allograft. Thus, apoptosis is a major cause of loss of cell viability in implanted allografts. Further application of the methods used in this study should increase our understanding of these changes.