【摘要】 目的:筛选出适合于肌腱玻璃化冷冻保存的溶液及保存程序,并通过细胞培养来进一步检测玻璃化肌腱的活性。方法:实验于2003-11/2005-02在解放军总医院骨科研究所完成。实验包括①筛选与检测:取来亨鸡30只,切取屈趾深肌腱,按随机数字法分为2组,新鲜鸡肌腱组,玻璃化组。运用正交设计和肌腱活性快速检测技术,筛选出适合鸡屈趾肌腱玻璃化保存程序,再从6种玻璃化溶液中(二甲基亚砜+乙酰胺+丙二醇+聚乙二醇;乙二醇+二甲基亚砜+蔗糖;乙二醇+二甲基亚砜+丁二醇;丙二醇+二甲基亚砜+蔗糖;二甲基亚砜+乙酰胺+丙二醇;二甲基亚砜+丙二醇)筛选出适于肌腱玻璃化保存的配比方案。②细胞培养:将玻璃化法保存鸡屈趾深肌腱进行细胞培养,观察培养后细胞的存活情况,以了解玻璃化法保存肌腱的细胞活性。结果:①通过统计学分析,肌腱玻璃化保存的最佳程序为平衡处理选择4℃3个浓度梯度、平衡时间选择4℃30min、洗脱处理选择4℃3个浓度梯度、洗脱时间选择4℃20min。玻璃化溶液二甲基亚砜+丙二醇为的最佳配比方案。②应用酶消法测得新鲜肌腱活性为89.26%,玻璃化法优化组合所保存肌腱的活性为78.49%。③将玻璃化组肌腱进行细胞培养,细胞第8天自组织块周边长出,第21天后传代,培养3代后出现明显的退化现象,玻璃化组肌腱细胞的生物学特性与新鲜肌腱组相似。④将两组肌腱所培养的细胞分别进行免疫组织化学染色,经鉴定均为肌腱细胞。结论:玻璃化法冷冻保存的肌腱具有良好的细胞活性,经细胞培养获得成功,其生物学特性无明显变异。更多还原

【Abstract】 AIM: To investigate the appropriate solution and program for cryopreservation of flexor tendon in chicken by vitrification, and detect the cellular viability of vitrified tendons though cell culture. METHODS: The Experiment was conducted in the Department of Orthopedics, China PLA General Hospital from November 2003 to December 2005.①Screening and Detection: flexor tendons were obtained from the toes of 30 chickens, which were divided into two groups, fresh tendon group and vitrification group. Orthogonal design and rapid detection technique of tendon viability were adopted to find out the appropriate preserved program of flexor tendon in toes of chickens with vitrification. The matching plans of tendon preservation with vitrification were screened from 6 kinds of vitrified solution [dimethyl sulfoxide (DMSO) + acetamide + propylene glycol (PF) + polyethylene; ethylene glycol (EG) + DMSO + cane sugar; EG + DMSO + butanediol (BG); PF + DMSO + cane sugar; DMSO + acetamide + PF; DMSO + PF].②Cell culture: vitrification-preserved flexor tendons were done under the cell culture, and the survival status of cells after culture was observed so as to acknowledge the cellular viability of preserved tendons with vitrification. RESULTS: ①Statistical analysis showed that the best program to preserve tendon by vitrification was three concentration gradient by equilibrium at 4 ℃, and time was 30 minutes 4 ℃. The elution process was three gradient concentration at 4 ℃, and the time was 20 minutes 4 ℃. The best matching program was vitrified solution of DMSO + PF.②Enzyme digestion was used to detect the viability of fresh tendon, which was 89.26%, and that of vitrification-preserved tendons was 78.49%.③The vitrified tendons were cultivated, and the cells were grew out from the periphery of tissues on the 8th day, which were transferred of culture on the 21st day, however, there was obvious degeneration found in cells after the third generation. The biological characteristics of tendons in the vitrification group were similar to those of fresh tendons.④The cultivated cells of both groups that were identified to be the tendon cells through immunohistochemiscal dyeing. CONCLUSION: Cryopreserved tendons with vitrification are good in cellular viability, and no obvious variation is found in its biological characteristics after successful cultivation.更多还原