低温冻存同种异体骨膜和骨髓移植免疫原性研究

IMMUNOGENICITY OF ALLOGENEIC FREEZING PERIOSTEUM AND BONE MARROW

目的 研究同种异体关节移植中骨膜和骨髓经低温冻存处理后的免疫原性，探索同种异体关节制备的合适方法。 方法 6月龄新西兰白兔5只，体重2.6～3.0 kg，切取双侧膝关节，分离骨膜及骨髓，梯度降温冻存1个月，经复温后无菌研磨，混合生理盐水制备骨膜、骨髓悬浊液。6月龄青紫蓝兔18只，体重2.1～2.8 kg，随机分成3组（n=6）：A、B、C组分别于腹腔注射生理盐水、骨膜悬浊液、骨髓悬浊液。测定注射前及注射后1、2周血清IL-2、IL-6、TNF-α浓度和外周静脉血CD4＋ T细胞/CD8＋ T细胞比值。 结果 IL-2测定：组内比较注射前后浓度无时间变化趋势（P=0.241）；组间比较差异无统计学意义（P=0.055）。IL-6测定：组内比较注射后浓度出现显著下降趋势（P=0.040）；组间比较差异无统计学意义（P=0.357）。 TNF-α测定：组内比较注射前后浓度无时间变化趋势（P=0.925）；组间比较B组浓度较其余两组显著升高（P 〈 0.05）。 CD4＋ T细胞/CD8＋ T细胞比值测定：组内比较注射前后无时间变化趋势（P=0.248）；组间比较差异无统计学意义（P=0.646）。 结论 经低温冻存处理后的骨膜、骨髓无显著免疫原性；制备同种异体关节时建议保留骨膜，去除骨髓，彻底冲洗松质骨中的红骨髓，降低其免疫原性。

Objective To investigate the immunogenicity of freezing periosteum and bone marrow during allogeneic joint transplantation, and to explore proper pretreatment of allogeneic joint. Methods The allogeneic periosteum and bone marrow were harvested from knee joints of 5 New Zealand white rabbits （aged, 6 months; weighing, 2.6-3.0 kg）. After gradient cooling, the tissue was cryopreserved for 1 month. The freezing periosteum and bone marrow were grinded to pieces after rewarming to prepare the suspension of periosteum and bone marrow. Eighteen Chinchilla rabbits （aged, 6 months; weighing, 2.1-2.8 kg） were divided into 3 groups randomly： normal saline injection group （group A, n=6）, periosteum injection group （group B, n=6）, and bone marrow injection group （group C, n=6）. The normal saline, periosteum suspension, and bone marrow suspension were injected into the peritoneal cavity in groups A, B, and C, respectively. The concentrations of interleukin 2 （IL-2）, IL-6, and tumor necrosis factor α （TNF-α） in serum and the ratio of CD4＋ T cell/CD8＋ T cell in venous blood were measured before injection, at 1 week and 2 weeks after injection. Results There was no significant difference in the concentration of IL-2 between before and after injection in the same group （P=0.241）, and between groups （P=0.055）. The concentration of IL-6 after injection was significantly lower than that before injection in the same group （P=0.040）, but no significant difference was found between groups （P=0.357）. The concentration of TNF-α showed no significant difference between before and after injection in the same group （P=0.925）, but the concentration of TNF-α in group B was significantly higher than that in groups A and C （P 〈 0.05）. The ratio of CD4＋ T cell/CD8＋ T cell of venous blood had no significant difference between before and after operation in the same group （P=0.248）, and between groups （P=0.646）. Conclusion The freezing periosteum and bone marrow are lowly immunogenic. In order to decrease the immunogenicity of the joint, preserving the periosteum and removing the marrow cavity are recommended.