【作者】 纪志娴；

【导师】 邢泉生；

【作者基本信息】 青岛大学 ， 胸心外科学， 2005， 硕士

【摘要】 目的:探讨液氮深低温保存对大月龄胎儿带瓣血管细胞活性的影响,对同种大月龄胎儿血管瓣膜组织库的保存效果进行初步评估。 方法:同种大月龄胎儿带瓣主动脉12例,胎龄20～40周,热缺血时间0～12小时。无菌条件下取材、修剪,每份标本均取两块动脉组织设为对照组和实验组,对照组血管直接进行细胞活性检测,实验组血管经程序降温并在-196℃液氮中保存1周后进行活性检测。通过台盼蓝染色、组织培养和四甲基偶氮唑盐比色法(MTT比色法),比较冷冻前后胎儿带瓣血管细胞活性的变化,并对成活细胞进行平滑肌a-actin免疫组化染色。 结果:冷冻前后大月龄胎儿带瓣血管均有细胞生长,台盼蓝染色显示对照组胎儿血管细胞活力为(86.4%±0.8%),实验组血管细胞活力为(79.4%±0.8%),二者之间差异有显著性意义(t=15.825,P<0.001),MTT比色法显示对照组细胞在490nm波长的OD值为(0.442±0.046),实验组细胞的OD值为(0.424±0.041),二者之间差异无显著性意义(t=1.617,P>0.05)。细胞大多数呈梭形、三角形或星性,部分区域平行重叠生长,呈“峰谷”状,平滑肌a-actin免疫组化染色阴性、阳性细胞混合存在。 结论:液氮冷冻保存后的大月龄胎儿带瓣血管有活细胞存在,细胞活力在一定程度上有所降低,但细胞增殖能力保持良好,为建立有活性的大月龄胎儿同种血管和瓣膜组织库并早日应用于临床提供理论依据。更多还原

【Abstract】 Objective: To investigate the influence of cryopreservation on the cellular viability of late-pregnancy fetal valved aortic allografts and preliminary evaluate the preserved efficacy of fetal heart valves and large vascular allografts storaged in liquid nitrogen.Methods: Twelve aortic allografts were sterilely procured from fetuses whose gestational ages ranged from 20 to 40 weeks within 12 hours after brained death. Each sample was bisected into control group and study group.The cellular viability of control group was directly tested and that of study group was examined after being storaged in liquid nitrogen for a week through a programmed frozen procedure. There were three methods to determine cellular viability of fetal aortic allografts ,which were trypan blue stain , tissue culture and Methylthiazol tetrazolium assay (MTT assay). The viable cells were identified by immunohistochemistry and light microscopy.Results: Viable cells might derived from both group. The cell survival rate of fetal aortic allografts was (86.4% ±0.8% )in control group and was (79.4% ±0.8% )in study group showed by trypan blue stain, the difference between them had statistically significance (t=15.825,P<0.001) . The cellular OD valve of control was (0.442 ± 0.046) and that of study was(0.424 ± 0.041)measured by MTT assay ,the difference between them failed to statistically significance(t=1.617,P>0.05). Most cells showed spindles triangle and star morphologic appearance. Some cells developed into overlapping layers of parallel, typically liking "peak and valley" . There were both positive and negative stained cells with the monoclonal antibody of mice anti-human smooth muscle a-actin proteins.Conclusions: There were viable cells in the late-pregnancy fetal aortic allografts after being storaged in the liquid nitrogen. Cryopreservation might harm the cellular viability but had little effect on the cellular proliferative capacity . This study provided the theoretical更多还原