液氮保存对人主动脉瓣组织细胞活力的影响_医学论文.docVIP

液氮保存对人主动脉瓣组织细胞活力的影响_医学论文 液氮保存对人主动脉瓣组织细胞活力的影响_医学论文 【关键词】 氮 　　关键词 :氮低温保藏主动脉瓣细胞存活细胞增殖能力组织培养 摘 要:目的 主要观察液氮（-196℃）保存1～24mo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30min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1，3，6，9，12，15，18，21，24mo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℃#12539min-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%vs93.8%0.05）Ⅷ，Ⅸ，Ⅹ组细胞活力较Ⅱ～Ⅶ组瓣膜各组明显减低（52.4%vs76.5%0.05）.Ⅰ组组织细胞1wk镜下可见，生长较快，4wk时镜下细胞数较多Ⅱ～Ⅶ组组织细胞2wk镜下可见，生长较Ⅰ组稍缓慢，4wk时镜下细胞数减少Ⅷ，Ⅸ，Ⅹ组细胞3wk镜下可见，生长较慢，4wk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1～15mo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18～24mo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15mo以内. 　　Keywords:nitrogencryopreservationaortic valvecell sur-vivalcell proliferative capacitytissue culture 　　Abstract:AIM To observe the cellular viability and cell proliferative capacity of human aortic valves cryopreserved in liquid nitrogen at-196℃.METHODS Twenty valves，procured from18to35years old healthy adult hearts within30min after the determination of brain death，were divided into10groups.GroupⅠ，the control group，is of fresh valves.Valves from GroupⅡto GroupⅩwere those pre-served in liquid nitrogen for1，3，6，9，12，15，18，21，24mo respectively.The fresh valves were examined immediately after procurement.All the cryopreserved valves were thawed in37℃saline pool.Each specimen was cut into pieces of1mm×1mm×1mm.Half of the tissues were reduced with2.5g#12539L-1 trypsin and dyed with trypan blue，then the num-ber of viable and nonviable cells were counted.The remaining tissues were incubated in RPMI1640medium containing100mL#12539L-1 fetal calf serum at37℃to observe the capacity of cell proliferation on the7th and28th 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 preser