CAJ | 학술논문

目的主要观察液氮（-196℃）保存1～24 mo不同时间段的瓣膜组织细胞活力及组织培养细胞生长情况的变化,为临床适用的人同种主动脉瓣的适宜保存期限提供实验依据. 方法选取（18～35）岁健康成年男性脑死亡30 min内取材的主动脉瓣膜20个. 依据保存时间分为10组，Ⅰ组为新鲜对照组, 取材后直接供实验用，Ⅱ～Ⅹ组为冷冻保存组,分别为冷冻保存1,3,6,9,12,15,18,21,24 mo的瓣膜，每组2个瓣膜共6个瓣叶. 冷冻保存组各组瓣膜缓慢降温（1℃*min-1）,液氮中保存，实验时进行快速复温. 瓣叶剪为细小组织块,一半置入培养瓶中做组织培养,相差显微镜下观察其组织细胞生长情况；另一半组织块用胰酶消化,光镜下做细胞计数并计算其细胞活力. 结果Ⅰ组的组织细胞活力最高(93.8%),Ⅱ～Ⅹ组细胞活力均较Ⅰ组明显减低(67.3% vs 93.8% P<0.05)；Ⅷ, Ⅸ,Ⅹ组细胞活力较Ⅱ～Ⅶ组瓣膜各组明显减低(52.4% vs 76.5% P<0.05). Ⅰ组组织细胞1 wk镜下可见,生长较快,4 wk时镜下细胞数较多；Ⅱ～Ⅶ组组织细胞2 wk镜下可见, 生长较Ⅰ组稍缓慢,4 wk时镜下细胞数减少；Ⅷ, Ⅸ, Ⅹ组细胞3 wk镜下可见,生长较慢,4 wk时镜下细胞数较Ⅰ组明显减少. 结论液氮保存对人同种瓣膜组织细胞活力有显著影响,其程度随保存时间增长而加重；液氮保存1～15 mo瓣膜组织细胞生长较新鲜瓣膜组稍晚,但组织细胞活力仍较高；液氮保存18～24 mo瓣膜组织细胞活力明显减低,组织细胞生长缓慢，因此瓣膜组织活性及瓣膜耐久性可能减低. 据此可认为, 人同种主动脉瓣的适宜保存期限为15 mo以内.
목적주요관찰액담（-196℃）보존1～24 mo불동시간단적판막조직세포활력급조직배양세포생장정황적변화,위림상괄용적인동충주동맥판적괄의보존기한제공실험의거. 방법선취（18～35）세건강성년남성뇌사망30 min내취재적주동맥판막20개. 의거보존시간분위10조，Ⅰ조위신선대조조, 취재후직접공실험용，Ⅱ～Ⅹ조위냉동보존조,분별위냉동보존1,3,6,9,12,15,18,21,24 mo적판막，매조2개판막공6개판협. 냉동보존조각조판막완만강온（1℃*min-1）,액담중보존，실험시진행쾌속복온. 판협전위세소조직괴,일반치입배양병중주조직배양,상차현미경하관찰기조직세포생장정황；령일반조직괴용이매소화,광경하주세포계수병계산기세포활력. 결과Ⅰ조적조직세포활력최고(93.8%),Ⅱ～Ⅹ조세포활력균교Ⅰ조명현감저(67.3% vs 93.8% P<0.05)；Ⅷ, Ⅸ,Ⅹ조세포활력교Ⅱ～Ⅶ조판막각조명현감저(52.4% vs 76.5% P<0.05). Ⅰ조조직세포1 wk경하가견,생장교쾌,4 wk시경하세포수교다；Ⅱ～Ⅶ조조직세포2 wk경하가견, 생장교Ⅰ조초완만,4 wk시경하세포수감소；Ⅷ, Ⅸ, Ⅹ조세포3 wk경하가견,생장교만,4 wk시경하세포수교Ⅰ조명현감소. 결론액담보존대인동충판막조직세포활력유현저영향,기정도수보존시간증장이가중；액담보존1～15 mo판막조직세포생장교신선판막조초만,단조직세포활력잉교고；액담보존18～24 mo판막조직세포활력명현감저,조직세포생장완만，인차판막조직활성급판막내구성가능감저. 거차가인위, 인동충주동맥판적괄의보존기한위15 mo이내.
AIM　To observe the cellular viability and cell proliferative capacity of human aortic valves cryopreserved in liquid nitrogen at -196℃. METHODS　Twenty valves, procured from 18 to 35 years old healthy adult hearts within 30 min after the determination of brain death, were divided into 10 groups. Group Ⅰ, the control group, is of fresh valves. Valves from Group Ⅱ to Group Ⅹ were those preserved in liquid nitrogen for 1, 3, 6, 9, 12, 15, 18, 21, 24 mo respectively. The fresh valves were examined immediately after procurement. All the cryopreserved valves were thawed in 37℃ saline pool. Each specimen was cut into pieces of 1 mm×1 mm×1 mm. Half of the tissues were reduced with 2.5 g*L-1 trypsin and dyed with trypan blue, then the number of viable and nonviable cells were counted. The remaining tissues were incubated in RPMI1640 medium containing 100 mL*L-1 fetal calf serum at 37℃ to observe the capacity of cell proliferation on the 7th and 28th day. RESULTS　Fresh valves seemed to have the highest cell survival rate of all the groups (93.8%). In cryopreservation groups, there was a tendency that the longer the duration of preservation was, the lower the survival rate was. Valves from Group Ⅱ to Group Ⅹ showed a significant decrease in cell survival rate compared with those from Group Ⅰ(67.3% vs 93.8% P<0.05). Valves from Group Ⅷ, Ⅸ and Ⅹ showed a significant decrease in cell survival rate compared with those from Group Ⅰ to Group Ⅶ (52.4% vs 76.5% P<0.05). Cells in Group Ⅰ grew out of the tissue pieces during the first week and fully covered the bottom of culture container during the fourth week. In the middle of the second week, however, cells of Group Ⅱ to Group Ⅶ started to appear at the edge of the tissues and occupied the most part of the container bottom. In Group Ⅷ, Ⅸ and Ⅹ, cells were scarcely seen during the first week and the cell number was markedly decreased during the fourth week compared with that of the fresh valves. CONCLUSION　Cryopreservation in liquid nitrogen has significant effects on the cellular viability and cell proliferative capacity of human aortic valve tissues. Valves that are preserved for 18 or more months have lower cellular survival rates and show a dramatic decrease in cellular capacity of proliferation. According to the results of our experiments, the durability of valves cryopreserved 18 or more months would decrease correspondingly.