大鼠肺移植供肺保存过程中细胞死亡类型的动态变化

The dynamic progress of cell death during donor lung preservation

目的通过对供肺保存过程中细胞凋亡和细胞坏死的动态变化的研究,探讨建立一种可以准确评估供肺保存质量的实验模型。方法清洁级健康雄性Sprague-Dawley大鼠42只,体重250～300g,预先分为7组,每组6只。整体获取心肺,置于4℃的新鲜制备的低钾右旋糖酐（LPD）溶液中保存。保存不同的时间（0、2、4、6、12、18、24h）后,通过主肺动脉向肺内灌注20mL的500mmol/L锥虫蓝、20mL生理盐水和10mL4%的多聚甲醛后固定,蜡块包埋后按4μm层厚切片。在光学显微镜下观察锥虫蓝染色情况;另一方面作TUNEL染色和Hoechst染色,在荧光显微镜下观察,严格按照盲法原则由两位研究者对死亡细胞进行分类计数。结果随着供肺低温保存时间的延长,细胞死亡数量上逐渐增加,而且细胞的死亡类型上发生动态变化。在低温保存的条件下,4h组才开始有少量的坏死细胞出现,随着保存时间的逐渐延长,细胞坏死也逐渐增加,在供肺保存18h以上组,坏死细胞显著增加;而细胞的凋亡开始于供肺保存2h组,在供肺保存12h组达到高峰,约占所有细胞的9%,在供肺保存18h组基本消失。结论通过对不同保存时间的供肺进行锥虫蓝、TUNEL、Hoechst三染色的方法,能很好的显示供肺保存后的细胞凋亡和坏死的情况。

Objective To observe the dynamic progress of apoptosis and necrosis of pneumocytes during donor lung preservation, we tried to build a model to accurately assess graft function. Methods Experiments were performed with 42 male inbred （250 to 350 g） Sprague-Dawley rats, which were divided into 7 groups based on preservation times （0,2,4,6,12,18,24 h）. The heart-lung block was then removed and stored in iced low potassium dextran（LPD） solution at 4 ℃ for different times. All lungs were flushed with 20 mL of a 500 mmol/L trypan blue solution through the main pulmonary artery, and then with 20 mL of 0. 9% normal saline and 10 mL of 4% paraformaldehyde. The formalin-fixed lung tissues were embedded in paraffin and cut into 4-μm-thick tissue slices. We observed trypan blue staining with light microscopy. Detection of apoptosis through in situ TUNEL was undertaken, using in situ cell death detection kit according to the manufacturer＇s instructions. Hoechst 33258 was used to show all the nucleated cells. Under fluorescent microscopy, cell counts were done by two researchers in a blinded fashion. Results The number of cell death increased and the type of the cell death changed by prolonged cold preservation time of the donor lungs. After cold ischemic storage, minimal cell death was seen in the 4 h groups. Significant cell death was detected only in lungs after 18 h of cold storage as determined through trypan blue-positive staining. On the contratry, apopotic cell begins in groups, and diminished in 18 h the 2 h groups, reach about 9% of all the nucleated cells in 12 h groups. Conclusions The study demonstrates a significant association among cold preservation time, extent and mode of cell death, suggesting new potential strategies to accurately assess graft function.